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Plant Protein Secretion Methods and Protocols 2nd
Edition Liwen Jiang Digital Instant Download
Author(s): Liwen Jiang, Jinbo Shen, Caiji Gao, Xiangfeng Wang
ISBN(s): 9781071640586, 1071640585
Edition: 2nd
File Details: PDF, 21.53 MB
Year: 2024
Language: english
Methods in
Molecular Biology 2841
Liwen Jiang
Jinbo Shen · Caiji Gao
Xiangfeng Wang Editors
Plant Protein
Secretion
Methods and Protocols
Second Edition
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, UK
For further volumes:

http://www.springer.com/series/7651
For over 35 years, biological scientists have come to rely on the research protocols and
methodologies in the critically acclaimed Methods in Molecular Biology series. The series was
the first to introduce the step-by-step protocols approach that has become the standard in all
biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by-
step fashion, opening with an introductory overview, a list of the materials and reagents
needed to complete the experiment, and followed by a detailed procedure that is supported
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constitute the key ingredient in each and every volume of the Methods in Molecular Biology
series. Tested and trusted, comprehensive and reliable, all protocols from the series are
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Plant Protein Secretion
Methods and Protocols
Second Edition
Edited by
Liwen Jiang
Centre for Cell and Developmental Biology, State Key Laboratory of Agrobiotechnology, School of Life
Sciences, The Chinese University of Hong Kong, Hong Kong, China
Jinbo Shen
State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou, China
Caiji Gao
School of Life Sciences, South China Normal University, Guangzhou, China
Xiangfeng Wang
State Key Laboratory of Plant Environmental Resilience, Department of Plant Sciences, College of
Biological Sciences, China Agricultural University, Beijing, China
Editors
Liwen Jiang Jinbo Shen
Centre for Cell and Developmental State Key Laboratory of Subtropical Silviculture
Biology Zhejiang A&F University
State Key Laboratory Hangzhou, China
of Agrobiotechnology
School of Life Sciences
The Chinese University of Hong Kong Xiangfeng Wang
Hong Kong, China State Key Laboratory of Plant Environmental
Resilience
Caiji Gao Department of Plant Sciences
School of Life Sciences College of Biological Sciences
South China Normal University China Agricultural University
Guangzhou, China Beijing, China
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-0716-4058-6 ISBN 978-1-0716-4059-3 (eBook)
https://doi.org/10.1007/978-1-0716-4059-3
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Preface
Protein secretion in eukaryotic cells involves both the conventional protein secretion (CPS)
pathway and the unconventional protein secretion (UPS) pathway. In the CPS pathway,
secretory proteins with a signal peptide are transported through the endomembrane system
consisting of multiple membrane-bound organelles with distinct functions. These include
the endoplasmic reticulum (ER), Golgi apparatus, trans-Golgi network (TGN), the endo-
some referred to as the prevacuolar compartment (PVC) or multivesicular body (MVB), and
the lysosome/vacuole. On the other hand, extracellular proteins without a signal peptide are
secreted through different types of UPS pathways.
Studying protein secretion in plant cells is of great importance due to its pivotal role in
various physiological processes and its potential applications in agriculture. Advanced exper-
imental techniques have led to a better understanding of the plant secretory system.
Researchers have discovered unique features of the secretory system in plants. For example,
unlike yeast and animal cells, the TGN and PVC/MVB in plants have been shown to
function as early and late endosomes, respectively. Additionally, in-depth studies have
been conducted in various plant cell types such as pollen tubes, pistil cells, and seed cells.
Sharing the latest and detailed experimental protocols is essential to further advance research
in this field.
Therefore, the primary goal of this book is to provide an overview and updates on the
latest developments in protein secretion research in plants, as compared to yeast and
mammalian systems. Secondly, it aims to provide researchers with a detailed and up-to-
date collection of experimental protocols for studying protein secretion in plant cells,
covering a wide range of topics, including bioinformatic analysis, proteomic studies, ultra-
structural analysis, and genetic screening methods, thus presenting a diverse and thorough
perspective.
We are grateful to the contributing authors for their expertise and dedication in
providing these valuable protocols. We hope that this book will serve as a useful resource
for researchers and students in the field of plant biology, inspiring further advancements in
our understanding of protein secretion in plant cells and beyond.
Hong Kong, China Liwen Jiang

Hangzhou, China Jinbo Shen
Guangzhou, China Caiji Gao
Beijing, China Xiangfeng Wang
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Conventional and Unconventional Protein Secretion
in Yeast and Animal Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Xiao Tang and Yusong Guo
2 An Overview of Protein Secretion in Plant Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Jing Tang, Kin Pan Chung, and Yonglun Zeng
3 Bioinformatic Analysis for Identifying Transcription Factors
Involved in Protein Secretion and Vacuole Formation . . . . . . . . . . . . . . . . . . . . . . 37
Fangfang Niu, Chudi Fan, and Liwen Jiang
4 Using AlphaFold2 and Molecular Dynamics Simulation
to Model Protein Recognition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Hiu Yan Wong and Kam-Bo Wong
5 Proteomic Analysis of Secreted Vesicle Proteins from the Plant Cells . . . . . . . . . 67
Haidi Yin, Jianying Wang, and Zhong-Ping Yao
6 Extracellular Vesicle Isolation and Mass Spectrometry-Based
Proteomic Analysis in Arabidopsis thaliana. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Jiayang Gao, Jinyu Chen, Hai Zhang, Liwen Jiang, and Yong Cui
7 Polysaccharides Analysis of Cell Wall
Using Carbohydrate Gel Electrophoresis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Zhao Wen, Jiaxi Li, Lanjun Zhang, Baocai Zhang, and Yihua Zhou
8 Acetyl Bromide Method for Total Lignin Content Determination
in Plant Biomass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Jinshan Gui
9 Utilizing Plant Protein Secretion System for Pharmaceutical
Protein Production. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Wing Man Yuen, Shijian Song, Stephen King Pong Leung,
Man Fai Leung, Sze Wan Lo, Inhawn Hwang, and Liwen Jiang
10 In Vitro Reconstitution of Plant COPII Vesicles . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Baiying Li and Liwen Jiang
11 Identification of Neighboring Proteins of Endosomal Regulators
by Using TurboID-Based Proximity Labeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Danni Lin, Jiayi Kuang, and Caiji Gao
12 Quantifying Protein Dynamics by Kymograph Analysis . . . . . . . . . . . . . . . . . . . . . 131
Xun Weng and Hao Wang
13 Polar Localization Analysis of Anionic Phospholipids
and Their Binding Proteins in Arabidopsis Pollen Tubes . . . . . . . . . . . . . . . . . . . . 145
Yang Yang, Dong Qian, Hongkai Zhang, and Yun Xiang
14 Vacuum-Based Agroinfiltration for Transient Gene Expression
in Arabidopsis Seedlings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
Linjuan Shi, Dongmei Zhu, Wenze Zhang, Jinbo Shen, and Shuai Hu
vii
viii Contents
15 Pharmaceutical Analysis in the Plant Endomembrane System . . . . . . . . . . . . . . . . 165

Baolei Li, Panpan Wang, Jing Qin, and Xiangfeng Wang
16 Observation of Vacuoles in Arabidopsis Developing Pollen
Grains with Whole-Cell Electron Tomography . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Zizhen Liang, Weiqi Wang, and Liwen Jiang
17 Whole-Cell Electron Tomography Analysis of Vacuole Biogenesis. . . . . . . . . . . . 179
Yanbin Li, Junru Zhong, Hai Zhang, Liwen Jiang, and Yong Cui
18 Microscopy Analysis of Autophagosomal Structures in Plant Cells . . . . . . . . . . . 189
Ka Kit Chung, Kai Ching Law, and Xiaohong Zhuang
19 Electron Microscopy Analysis of Membraneless Condensates in Plant Cells . . . 199
Yilin He and Ruixi Li
20 High-Pressure Freezing and Low-Temperature Processing
of Seeds for Electron Microscopy and Electron Tomography . . . . . . . . . . . . . . . . 207
Juan Li, Liangpeng Gou, Jinbo Shen, and Wenhan Cao
21 Determination of the Cryo-EM Structure of ATG9
from Arabidopsis thaliana. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Liting Zhai and Wenxin Zhang
22 Genetic Screening of Factors in the Plant Protein Secretion . . . . . . . . . . . . . . . . . 225
Haoyu Wen, Yaoyao Li, and Qiong Zhao
23 Studying Autophagy and Senescence of Arabidopsis Stigmatic Cells . . . . . . . . . . 241
Tong Zhang, Kun Wang, and Pengwei Wang
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
Contributors
WENHAN CAO • State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University,
Hangzhou, China
JINYU CHEN • State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Faculty
of Medicine and Life Sciences, Xiamen University, Xiamen, China
KA KIT CHUNG • School of Life Sciences, Centre for Cell & Developmental Biology and State
Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Sha Tin, New
Territories, Hong Kong, China
KIN PAN CHUNG • Max-Planck-Institut für Molekulare Pflanzenphysiologie, Potsdam-Golm,
Germany
YONG CUI • State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Faculty of
Medicine and Life Sciences, Xiamen University, Xiamen, China
CHUDI FAN • School of Life Sciences, Centre for Cell & Developmental Biology and State Key
Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Sha Tin, New
CAIJI GAO • Guangdong Provincial Key Laboratory of Biotechnology for Plant Development,
JIAYANG GAO • School of Life Sciences, Centre for Cell & Developmental Biology, and Key
LIANGPENG GOU • State Key Laboratory of Subtropical Silviculture, Zhejiang A&F
University, Hangzhou, China
JINSHAN GUI • State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University,
Hangzhou, China
YUSONG GUO • Division of Life Science and State Key Laboratory of Molecular Neuroscience,
The Hong Kong University of Science and Technology, Hong Kong, China
YILIN HE • Key Laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher
Education Institutes, Institute of Plant and Food Science, School of Life Sciences, Southern
University of Science and Technology, Shenzhen, China
SHUAI HU • State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University,
Hangzhou, China
INHAWN HWANG • Department of Life Sciences, Pohang University of Science and Technology,
Pohang, South Korea
LIWEN JIANG • Centre for Cell and Developmental Biology, State Key Laboratory
of Agrobiotechnology, School of Life Sciences, The Chinese University of Hong Kong,
Hong Kong, China
JIAYI KUANG • Guangdong Provincial Key Laboratory of Biotechnology for Plant
Development, School of Life Sciences, South China Normal University, Guangzhou, China
KAI CHING LAW • School of Life Sciences, Centre for Cell & Developmental Biology and State
MAN FAI LEUNG • School of Life Sciences, Centre for Cell & Developmental Biology and State
ix
x Contributors
STEPHEN KING PONG LEUNG • School of Life Sciences, Centre for Cell & Developmental
Biology and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong
Kong, Sha Tin, New Territories, Hong Kong, China
BAIYING LI • Department of Biology, Hong Kong Baptist University, Hong Kong, China
BAOLEI LI • State Key Laboratory of Plant Environmental Resilience, Department of Plant
Sciences, College of Biological Sciences, China Agricultural University, Beijing, China
JIAXI LI • Center for Genome Biology, Institute of Genetics and Developmental Biology,
Chinese Academy of Sciences, Beijing, China; University of Chinese Academy of Sciences,
Beijing, China
JUAN LI • State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University,
Hangzhou, China
RUIXI LI • Key Laboratory of Molecular Design for Plant Cell Factory of Guangdong Higher
Education Institutes, Institute of Plant and Food Science, School of Life Sciences, Southern
University of Science and Technology, Shenzhen, China
YANBIN LI • State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Faculty of
Medicine and Life Sciences, Xiamen University, Xiamen, China
YAOYAO LI • School of Life Sciences, East China Normal University, Shanghai, China
ZIZHEN LIANG • School of Life Sciences, Centre for Cell & Developmental Biology and State
DANNI LIN • Guangdong Provincial Key Laboratory of Biotechnology for Plant Development,
SZE WAN LO • School of Life Sciences, Centre for Cell & Developmental Biology and State Key
FANGFANG NIU • School of Life Sciences, Centre for Cell & Developmental Biology and State
DONG QIAN • MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life
Sciences, Lanzhou University, Lanzhou, China
JING QIN • State Key Laboratory of Plant Environmental Resilience, Department of Plant
Sciences, College of Biological Sciences, China Agricultural University, Beijing, China
JINBO SHEN • State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University,
Hangzhou, China
LINJUAN SHI • State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University,
Hangzhou, China
SHIJIAN SONG • School of Life Sciences, Centre for Cell & Developmental Biology and State
JING TANG • State Key Laboratory of Plant Diversity and Specialty Crops, South China
Botanical Garden, Chinese Academic of Sciences, Guangzhou, China; University of Chinese
Academy of Sciences, Beijing, China
XIAO TANG • Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of
Major Metabolic Diseases, College of Life Sciences, Anhui Normal University, Wuhu,
Anhui, China
HAO WANG • Department of Cell and Developmental Biology, College of Life Sciences, South
China Agricultural University, Guangzhou, China
Contributors xi
JIANYING WANG • Department of Applied Biology and Chemical Technology, The Hong Kong
Polytechnic University, Hung Hom, Kowloon, Hong Kong, China
KUN WANG • College of Life Science and Technology, Huazhong Agricultural University,
Wuhan, China
PANPAN WANG • State Key Laboratory of Plant Environmental Resilience, Department of
Plant Sciences, College of Biological Sciences, China Agricultural University, Beijing,
China
PENGWEI WANG • National Key Laboratory for Germplasm Innovation & Utilization of
Horticultural Crops, College of Horticulture and Forestry Sciences, Huazhong
Agricultural University, Wuhan, China; College of Life Science and Technology, Huazhong
Agricultural University, Wuhan, China
WEIQI WANG • School of Life Sciences, Centre for Cell & Developmental Biology and State Key
XIANGFENG WANG • State Key Laboratory of Plant Environmental Resilience, Department of
Plant Sciences, College of Biological Sciences, China Agricultural University, Beijing,
China
HAOYU WEN • School of Life Sciences, East China Normal University, Shanghai, China
ZHAO WEN • Center for Genome Biology, Institute of Genetics and Developmental Biology,
Beijing, China
XUN WENG • Department of Cell and Developmental Biology, College of Life Sciences, South
China Agricultural University, Guangzhou, China
HIU YAN WONG • School of Life Sciences, Centre for Protein Science and Crystallography,
State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong, Sha Tin,
New Territories, Hong Kong, China
KAM-BO WONG • School of Life Sciences, Centre for Protein Science and Crystallography,
YUN XIANG • MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life
YANG YANG • MOE Key Laboratory of Cell Activities and Stress Adaptations, School of Life
ZHONG-PING YAO • Department of Applied Biology and Chemical Technology, The Hong
Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China
HAIDI YIN • Multi-omics Mass Spectrometry Facility, Shenzhen Medical Academy of Research
and Translation, Shenzhen, Guangdong, China
WING MAN YUEN • School of Life Sciences, Centre for Cell & Developmental Biology and State
YONGLUN ZENG • State Key Laboratory of Plant Diversity and Specialty Crops, South China
Botanical Garden, Chinese Academic of Sciences, Guangzhou, China; University of Chinese
Academy of Sciences, Beijing, China
LITING ZHAI • School of Life Sciences, Centre for Cell & Developmental Biology and State Key
xii Contributors
BAOCAI ZHANG • Center for Genome Biology, Institute of Genetics and Developmental
Biology, Chinese Academy of Sciences, Beijing, China; University of Chinese Academy of
Sciences, Beijing, China
HAI ZHANG • State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Faculty
of Medicine and Life Sciences, Xiamen University, Xiamen, China
HONGKAI ZHANG • MOE Key Laboratory of Cell Activities and Stress Adaptations, School of
Life Sciences, Lanzhou University, Lanzhou, China
LANJUN ZHANG • Center for Genome Biology, Institute of Genetics and Developmental
Biology, Chinese Academy of Sciences, Beijing, China
TONG ZHANG • National Key Laboratory for Germplasm Innovation & Utilization of
Horticultural Crops, College of Horticulture and Forestry Sciences, Huazhong
Agricultural University, Wuhan, China; College of Horticulture Science and Engineering,
Shandong Agricultural University, Tai’an, China
WENXIN ZHANG • School of Life Sciences, Centre for Cell & Developmental Biology and State
WENZE ZHANG • State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University,
Hangzhou, China
QIONG ZHAO • School of Life Sciences, East China Normal University, Shanghai, China;
Institute of Eco-Chongming, Shanghai, China
JUNRU ZHONG • State Key Laboratory of Cellular Stress Biology, School of Life Sciences,
Faculty of Medicine and Life Sciences, Xiamen University, Xiamen, China
YIHUA ZHOU • Center for Genome Biology, Institute of Genetics and Developmental Biology,
Beijing, China
DONGMEI ZHU • State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University,
Hangzhou, China
XIAOHONG ZHUANG • School of Life Sciences, Centre for Cell & Developmental Biology and
Chapter 1
Conventional and Unconventional Protein Secretion in Yeast

and Animal Cells
Xiao Tang and Yusong Guo
Abstract
Protein secretion mediated by the secretory transport pathway is a sophisticated and highly regulated
cellular process in eukaryotic cells. In the conventional secretory transport pathway, newly synthesized
proteins pass through several endomembrane compartments to reach their destinations. This transport
occurs via small, membrane-enclosed vesicles. To ensure the fidelity of trafficking, eukaryotic cells employ
elaborate molecular machinery to accurately sort newly synthesized proteins into specific transport vesicles
and precisely deliver them to respective acceptor compartments. Leaderless cargo proteins, lacking a signal
peptide, follow an unconventional secretory pathway. This review encompasses the molecular machinery
regulating both conventional and unconventional protein secretion in yeast and animal cells.
Key words Vesicle, Cargo adaptor, Cargo receptor, Protein secretion
1 Overview of Conventional and Unconventional Protein Secretion
In any organism, each cell synthesizes diverse proteins that regulate

various cellular and physiological functions. These proteins, includ-
ing transmembrane and soluble proteins, travel to different desti-
nations via the intracellular secretory pathway. In the conventional
pathway, the ribosomes synthesize secretory proteins, which are
then guided to the endoplasmic reticulum (ER) via a signal peptide.
Here, they undergo folding and modification before being selec-
tively packaged into designated vesicles. These vesicles are sent to
the cis face of the Golgi apparatus for further modifications. Cargo-
enriched vesicles then exit the trans face of the Golgi, fusing with
specific target membranes, including the plasma membrane, endo-
somes, and lysosomes. In addition to anterograde transport, retro-
grade transport also plays an important role within the secretory
transport pathway. For instance, vesicle coat components of trans-
port vesicles need recycling to the donor membrane for the next
cycle of vesicle budding, resident proteins that have escaped need to
Liwen Jiang et al. (eds.), Plant Protein Secretion: Methods and Protocols, Methods in Molecular Biology, vol. 2841,
https://doi.org/10.1007/978-1-0716-4059-3_1,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024
1
2 Xiao Tang and Yusong Guo
be retrieved to their resident compartment, and some extracellular

signaling proteins require to be endocytosed via the plasma mem-
brane [1]. Maintaining a balance between anterograde and retro-
grade transport ensures accurate protein trafficking and stable
composition of organelles.
The secretory cargo proteins that lack the signal peptide
sequence (leaderless cargoes) utilize an unconventional secretory
transport pathway to reach their destinations. Four types of uncon-
ventional protein secretion (UPS) have been identified. Some solu-
ble cargoes translocate across the plasma membrane through pore
structures. A small fraction of cargo proteins employs ATP-binding
cassette (ABC) transporters for secretion. Certain proteins are
packaged into intracellular vesicle intermediates and released to
the extracellular environment upon fusion with the plasma mem-
brane. In response to cellular stress, some signal peptide- or trans-
membrane domain-containing proteins are directly delivered from
the ER to the plasma membrane, bypassing the Golgi. UPS plays
important roles in various physiological processes, including
immune responses, abiotic stress responses, and cell proliferation
[2, 3].
2 The Conventional Secretory Transport Pathway
2.1 Key Steps of the The key steps of the conventional secretory transport pathway
Conventional include vesicle formation, cargo sorting, vesicle transport, tether-
Secretory Transport ing, and fusion. In this section, we summarize the molecular
Pathway mechanisms of these steps.
2.1.1 Vesicle Formation In the conventional protein secretory pathway, cargo proteins are
and Cargo Sorting shuttled between different membrane-enclosed organelles by
diverse types of transport vesicles, including clathrin-coated vesicles
(CCVs) and coat protein complex I and II (COPI and COPII)
vesicles.
Clathrin-Coated Vesicles Clathrin coats are assembled at the trans-Golgi network (TGN),
endosomes, and plasma membrane, mediating the anterograde and
retrograde transport among these compartments [4]. At the donor
membrane, the small GTPases Arf or Arf-like (Arl) family proteins
play a crucial role in CCV-mediated export. Arf family proteins
exist in two states: the GTP-bound active form and the
GDP-bound inactive form. Upon association with GTP, clathrin
adaptors are recruited to the donor membrane and interact with
cargo proteins either directly or indirectly. These clathrin adaptors
include the heterotetrameric adaptor protein complex (AP), the
monomeric Golgi-localized, γ-ear-containing, Arf-binding protein
(GGA), and epsin-related protein (EpsinR) [5–7]. The AP family
Conventional and Unconventional Protein Secretion in Yeast and Animal Cells 3
comprises five members. Among them, AP-1, AP-3, and AP-4

regulate the vesicle formation at the TGN and endosome, while
AP-2 mediates endocytosis at the plasma membrane, and AP-5
mediates endosomal trafficking [8, 9]. Clathrin adaptors recruit
clathrin to the membrane. The heterohexameric clathrin complex
is composed of three heavy chains and three light chains. The
polymerization and assembly of clathrin form a spherical polygonal
lattice coat around the cargo-containing vesicles, enabling the
delivery of cargoes from the donor membrane to the acceptor
membrane [10].
COPI Vesicles COPI vesicles regulate the intra-Golgi transport and retrograde
transport, including the movement from the Golgi or the ER–
Golgi intermediate compartment (ERGIC) back to the ER
[11]. The COPI coat is composed of a heptamer coatomer, which
includes seven core subunits: α-COP, β′-COP, ε-COP, β-COP,
δ-COP, γ-COP, and ζ-COP [12]. Arf1 plays an important role in
initiating the formation of COPI vesicles. GDP-bound Arf1 is
recognized by transmembrane protein p23 homooligomer, located
at the Golgi or ERGIC membranes. Nucleotide exchange by gua-
nine nucleotide exchange factor (GEF) on Arf1 leads to the disso-
ciation between Arf1 and p23. Consequently, the N-terminal
amphipathic helix of Arf1 is exposed and stably inserted into
Golgi or ERGIC membranes. Then, cycles of GTP hydrolysis and
GDP-to-GTP exchange result in rearrangement of p23/p24 oligo-
mers and the formation of p23/p24 heterooligomer, recruiting
coatomer [13]. COPI coat recognizes the di-lysine sorting motifs.
Cargo proteins bearing the di-lysine sorting motifs are recognized
through an interaction between sorting signals on the cargo and
the propeller domain of α- or β′-COP [14]. Several cellular factors,
such as ER vesicle (Erv)41-Erv46, Rer1, Vps74, and KDEL recep-
tor, interact with both COPI coatomer and cargo proteins, thereby
linking cargo proteins with COPI coatomer [15–17]. Finally, the
GTP hydrolysis of Arf1 triggers the disassembly of the COPI coat.
The COPI machinery is essential for protein quality control as it
retrieves cargo proteins that are erroneously packaged [17].
COPII Vesicles As one of the most extensively studied vesicles, COPII vesicles
mediate the anterograde transport from the ER to the ERGIC or
to the Golgi. The COPII coat is composed of five cytosolic pro-
teins: the small GTPase Sar1, the Sec23/24 heterodimer, and the
Sec13/31 heterotetramer [18]. The assembly of the COPII coat at
the ER is initiated by Sar1. The GEF of Sar1, Sec12, promotes the
transition of Sar1 from the GDP-bound status to the GTP-bound
status [19, 20]. The binding of GTP induces conformational
changes in the switch I/II regions of Sar1. This transformation
exposes the N-terminal amphipathic helix, enabling Sar1 to bind to
the ER membranes. Subsequently, Sar1 recruits the inner COPII
coat, Sec23/24 heterodimers, through the interaction with Sec23.

Sec24 directly or indirectly interacts with cargo proteins, forming
pre-budding complexes [21, 22]. The Sec13/31 complex is then
recruited by pre-budding complexes to form the outer coat
through the interaction between Sec23 and Sec31 [23]. Sec23
serves as the GTPase-activating protein (GAP) of Sar1 [24]. The
GAP activity of Sec23 is further activated by the Sec13/31 com-
plex. The activation of Sar1 is believed to drive membrane defor-
mation to generate COPII vesicles while GTP hydrolysis of Sar1 is
thought to drive COPII coat disassembly. The released COPII
components are then recycled for the next cycle of vesicle
budding [25].
2.1.2 Intracellular Vesicle After released from the donor compartments, vesicles need to be
Transport delivered to the correct acceptor compartment. Rab GTPases,
motor adaptor proteins, and motor proteins play critical roles in
mediating vesicle transport along the cytoskeleton
[26]. GTP-bound Rab GTPases are demonstrated to recruit
motor adaptor proteins and motor proteins to vesicle membranes.
Different factors have been shown to regulate different routes of
vesicle transport along the secretory pathway, including ER-to-
Golgi transport, the TGN export, and Golgi-to-ER retrograde
transport [27].
Rab1 has been demonstrated to regulate the trafficking of
COPII vesicle from the ER to Golgi. COPII inner coat component,
Sec23p, acts as the motor adaptor protein and interacts with the
dynein–dynactin complex [28]. The activated ATPase activity of
dynein hydrolyzes ATP, driving the movement of COPII vesicles
along the microtubule track toward the Golgi [29]. On the other
hand, Rab6a mediates the retrograde transport of COPI vesicles
from the Golgi to the ER [30]. Rab6a interacts with the COPI coat
and motor protein Rabkinesin-6 and kinesin family member 1C
(KIF1C) to facilitate the movement of COPI vesicles along the
microtubules [31]. Rab3 is reported to function to regulate the
transport of CCV from the TGN to the plasma membrane.
GTP-bound Rab3 recruits motor adaptor DENN/MADD and
motor protein KIF1A [32]. KIF1A plays a multifaceted role,
including interaction with microtubules, recruitment of vesicles
through interaction with PI(4,5)P2, and functions as a motor
protein to move vesicles on microtubules [33, 34].
2.1.3 Tethering and After budding and uncoating, the cargo-containing vesicles navi-
Fusion of the Intracellular gate through the cytoskeleton toward the target cellular compart-
Transport Vesicles ment. They tether to the target membrane by interacting with small
GTPase, such as Rab and Arls, and tethering factors. The vesicular
soluble N-ethylmaleimide-sensitive factor (NSF) attachment pro-
tein receptor (v-SNARE) on the vesicle membrane and the target
(t)-SNARE on the target membrane associate with each other to
form trans-SNARE complex. This four-helix bundle complex

enables the docking and fusion of the vesicles. Following this, the
cargo proteins within the vesicles are released to the acceptor
compartment. The SNAREs are then recycled for subsequent
fusion events [1].
Tethering Factors Tethering factors attach transport vesicles to their correct destina-
tions. They ensure that the vesicles, which carry a variety of cargo
proteins, are accurately delivered to the correct location. Tethering
factors can be categorized into two groups: long coiled-coil pro-
teins and multisubunit complexes (MTCs). Long coiled-coil teth-
ering factors are large homodimers that can tether vesicles over a
long range while MTCs comprise multiple subunit families that
tether vesicles over shorter ranges [35]. Coiled-coil tethers localize
on the Golgi membrane and endosome membrane, such as golgins
and early endosome antigen 1 (EEA1). Distinct subsets of golgins
with unique locations on the Golgi surface are responsible for
capturing vesicles from various origins [36]. EEA1 plays a crucial
role in the tethering process at the early endosomes, facilitating the
subsequent membrane fusion [37].
MTCs are grouped into three groups based on their localiza-
tion and functions. The first group of MTCs is involved in the
secretory pathway, including Dsl1p, conserved oligomeric Golgi
complex (COG), Golgi-associated retrograde protein complex
(GARP), and exocyst. The Dsl1p complex interacts with both the
vesicle coat protein COPI and the ER membrane protein Sec20p,
and effectively links the COPI vesicles with the ER, regulating the
retrograde trafficking from the ER to the Golgi [38]. COG reg-
ulates the retrograde trafficking within the Golgi and from the
Golgi back to the ER [39]. GARP is recruited to the TGN by
Arfrp1 and Arl5 to regulate the retrograde delivery from endosome
to TGN [40]. Exocyst targets vesicles to specific regions of the cell
membrane to regulate the exocytosis [41]. The second group of
MTCs participates in the endolysosomal pathway, including endo-
somal sorting complex required for transport (ESCRT) complexes,
homotypic fusion and vacuole protein sorting (HOPS), and class C
core vacuole/endosome tethering (CORVET). ESCRT sorts ubi-
quitinated proteins into intraluminal vesicles of multivesicular bod-
ies (MVBs) and regulate their degradation in the vacuole or
lysosomes [42]. HOPS complex is involved in the fusion of endo-
somes and vacuoles, as well as the fusion of autophagosomes with
lysosomes/vacuoles [43]. CORVET complex is involved in the
early stages of endosomal trafficking. It helps fuse endosomes
with each other [43]. The third group of MTCs functions in
multiple trafficking pathways, including the secretory pathway,
endolysosomal pathway, and autophagy. This group of tethers
includes the transport protein particle (TRAPP) complex
[44]. TRAPP complex has three known forms—TRAPP I, TRAPP

II, and TRAPP III—each with unique functions. TRAPP I is
involved in ER-to-Golgi transport, TRAPP II regulates late Golgi
trafficking, and TRAPP III mediates autophagy [45].
SNARE Complex The trans-SNARE complex is a four-helix bundle composed of

three t-SNARE and one v-SNARE [46]. The types of SNAREs
vary across different membrane, and their associations are specific,
ensuring accurate cargo delivery [47]. SNAREs can also be classi-
fied into Q-SNAREs and R-SNAREs based on the charged amino
acids in the central layer of the SNARE complex. Three glutamines
and one arginine form a central ionic layer in the SNARE complex.
The Q-SNARE and R-SNARE roughly correspond to t-SNARE
and v-SNARE, respectively [47]. After vesicle fusion, the four
subunits form a cis-SNARE complex within the same membrane
compartment. The α-soluble NSF association protein (α-SNAP)
and ATPase NSF are then recruited to the cis-SNARE complex,
leading to ATP hydrolysis and ATP-driven SNARE complex disas-
sembly. Then, the released v-SNARE is recycled to mediate the next
round of vesicular trafficking [48].
2.2 Cargo Adaptors Cargo adaptors are cytosolic proteins that directly interact with
that Regulate the sorting motifs on the cytosolic domain of transmembrane cargo
Enrichment of Cargo molecules or indirectly recognize the soluble cargo proteins by
Proteins into Transport interacting with cargo receptors to package them into transport
Vesicles vesicles.
2.2.1 Cargo Adaptor at The COPII coat subunit, Sec24, functions as a cargo adaptor at the
the ER ER [49, 50]. Multiple cargo binding sites on Sec24 have been
identified. The first site recognizes YNNSNPF motif of Sed5. The
second site binds to the di-acidic sorting motif of Sys1 [51] and the
LXXLE motif of Bet1 [52]. The third site associates with Sec22
[49, 53]. The fourth site binds to membrin and syntaxin
5 [50]. There are four isoforms of Sec24 in mammals, Sec24A-D,
each having distinct functions in cargo packaging. For instance,
Sec24A selectively packages PCSK9 into COPII vesicles to modu-
late the plasma cholesterol level [54]. Sec24B specifically sorts
Vangl2, a planar cell polarity protein, during neural tube closure
[55]. Deficiency of Sec24C or Sec24D results in an early embryonic
lethal phenotype, indicating that they regulate packaging of some
cargo proteins that are crucial in developmental processes [56, 57].
TANGO1 is proposed to act as a cargo adaptor for large cargo
proteins, such as collagen [58]. TANGO1-depleted mice show
severe defects in collagen secretion [59]. TANGO1 proteins assem-
ble into a ring at the ER exit site (ERES) to facilitate the ER export
of collagen VII [60]. It interacts with the Sec23/24 complex in the
cytoplasmic region through its proline-rich domain (PRD) and
binds to its binding partner, cTAGE5, a truncated form of

TANGO1 missing luminal domain. In the ER luminal region,
TANGO1 interacts with triple helical collagen via its SH3 domain
[61]. Then, they recruit the ERGIC/Golgi membranes to the
ERES through the NRZ (NBAS/RINT1/ZW10) tether complex
[60]. The special membrane helix organization of TANGO1 pre-
vents membrane mixing between the ER and the ERGIC/Golgi.
After collagen molecules completely enter the vesicles, TANGO1
detaches from the Sec23/24 complex and collagen so that COPII
outer coat can be recruited and bound to the inner coat. This way,
TANGO1 packages the large cargo protein into COPII
vesicles [62].
2.2.2 Cargo Adaptor at At the TGN, the adaptor protein complex-1 (AP-1) is the most
the TGN well-characterized cargo adaptor. AP-1 is composed of four sub-
units (β, γ, μ, and σ), forming a trunk domain that binds Arf
proteins, cargo proteins, and phospholipids, and two appendage
domains, which bind to some accessory proteins [5]. AP-1 directly
interacts with tyrosine-based sorting motifs and dileucine sorting
motifs on the cytosolic domain of cargo molecules [5]. AP-1 can
also recognize sorting motifs on the tertiary structure of folded
cargo proteins. For example, AP-1 recognizes a TGN sorting motif
on potassium channel Kir2.1 [63, 64]. In addition to AP-1, two
other adaptor complexes, AP-3 and AP-4, also mediate protein
sorting at the TGN [5].
Golgi-localized, γ-ear-containing, Arf-binding proteins
(GGAs) are monomeric cargo adaptors that are localized at the
TGN. There are three GGAs in mammalian cells, including
GGA1, GGA2, and GGA3, and two in yeast (Gga1, Gga2).
GGAs contain an N-terminal Vps27, Hrs, STAM (VHS) domain;
a GGA and TOM (GAT) domain; and a C-terminal γ-adaptin ear
(GAE) domain [5]. GAT and GAE are connected by a long flexible
linker. The VHS domain binds cargo proteins with acidic-cluster
dileucine motifs (DXXLL) [5]. The GAT domain interacts with Arf
and accessory proteins, and the GAE domain binds to accessory
proteins, such as p56 and γ-synergin [5].
Epsin-related proteins (epsinR) add another layer of complexity
in the cargo protein sorting process at the TGN. EpsinR in mam-
mals, along with its yeast counterparts Ent3 and Ent5, are
TGN-localized monomeric cargo adaptors. Epsins are composed
of an N-terminal Epsin N-Terminal Homology (ENTH) domain
and a long, unfolded domain at its C-terminus. The ENTH
domains play a pivotal role in binding to SNAREs and PI4P
[5]. The C-terminal unfolded regions of epsinR interact with
AP-1, GGAs, the poly-arginine TGN sorting motif on a planar
cell polarity protein, Frizzled6, and clathrin [5, 65].
In yeast, the exomer cargo adaptor is responsible for the direct

transport of specific cargo proteins such as chitin synthase III
(Chs3p) and Fus1p, from the TGN to the cell surface [66–
69]. Exomer recognizes Fus1p through a unique sorting motif
(IXTPK) located on the cytosolic domain of Fus1p [70]. It recog-
nizes the Chs3p protein through a DXE motif on the cytosolic
domain of Chs3p [71].
2.3 Cargo Receptors Transmembrane cargo receptors interact with both cargo adaptors
That Regulate the and luminal cargo molecules, thereby linking the luminal cargo
Enrichment of Cargo proteins to the cargo sorting machinery.
Proteins into Transport
Vesicles
2.3.1 Cargo Receptors at At the ER, one mammalian cargo receptor, ERGIC-53, is shown to
the ER facilitate the transport of a subset of soluble proteins, including
blood coagulation factors V and VIII (FV and FVIII), and a
cathepsin-Z-related protein [72, 73]. ERGIC-53, in conjunction
with multiple coagulation factor deficiency 2 (MCFD2), forms a
cargo receptor complex. The primary sorting signals residing in the
B domain of FVIII guide the binding of FVIII to ERGIC-53-
MCFD2 in a calcium-dependent manner [74]. The yeast homologs
of ERGIC-53, Emp46 and Emp47, recycle between the ER and the
Golgi apparatus, interacting with both COPI and COPII compo-
nents via a tyrosine-containing signal in the C-terminal region
[75]. The coiled-coil domains of Emp46 and Emp47 are responsi-
ble for their complex formation in the ER in a pH-dependent
manner [76].
The p24 family proteins in yeast such as Emp24 regulate ER
export of glycosylphosphatidylinositol (GPI)-anchored proteins
[77–79], while Erv29, another member of the p24 family, is crucial
for the efficient export of glyco-pro-α-factor, carboxypeptidase Y,
and proteinase A from the ER [80, 81]. Mammalian orthologs of
yeast Erv proteins have also been considered to function as cargo
receptors. One such ortholog, Surfeit locus protein 4 (SURF4),
that is analogous to Erv29p in yeast, regulates ER export of soluble
proteins, including lipoproteins, proprotein convertase subtilisin/
kexin type 9 (PCSK9), and Sonic Hedgehog (Shh) [82–
84]. SURF4 recognizes amino-terminal tripeptide motifs of soluble
cargo proteins and participates in ERES organization
[82, 85]. SURF4 can also induce the formation of a highly elon-
gated tubular ER–Golgi intermediate compartment (t-ERGIC)
that selectively expedites the ER-to-Golgi transport for soluble
cargoes [86]. SURF4 has been recently reported to recognize the
polybasic residues within the Cardin–Weintraub (CW) motif of Shh
to regulate the packaging of Shh into COPII vesicles at the ER [87]
(see Fig. 1A, step 1). This recognition is mediated by the direct
Fig. 1 (A) Diagram illustrating how SURF4 acts as a cargo receptor to facilitate the secretion of Shh. The
secretion process involves several steps: (1) in step 1, the triacidic motif located in the first luminal domain of
Surfeit locus protein 4 (SURF4) (depicted in A′) recognizes the basic residues on Sonic Hedgehog (Shh),
leading to the enrichment of Shh into coat protein complex II (COPII) vesicles. (2) Step 2 involves proteoglycans
competing with SURF4 to interact with Shh, resulting in the dissociation of SURF4 from Shh. (3) Step
3 demonstrates that proteoglycans play a role in mediating the transport of Shh from the trans-Golgi network
(TGN) for secretion. (4) SURF4, which is not associated with Shh, is retrieved back to the endoplasmic
reticulum (ER) to facilitate subsequent rounds of cargo sorting
electrostatic interactions between the triacidic motif in the first

luminal loop of SURF4 and the basic residues in the CW motif of
Shh (Fig. 1A, A′) [87]. Interestingly, it has been demonstrated
proteoglycans (PGs) at the Golgi compete with SURF4 to interact
with Shh, thereby promoting the dissociation of SURF4 from Shh
at the Golgi (see Fig. 1A, step 2) [87]. Subsequently, PGs play a role
in regulating TGN export of Shh (see Fig. 1A, step 3) [87]. In
addition to Shh, other secretory proteins containing the polybasic
motif, bone morphogenetic protein 8A (BMP8A) and secreted
frizzled-related protein 1 (SFRP1), are identified to interact with
the triacidic motif on the predicted first luminal domain of SURF4
to be transported from the ER to Golgi [88].
2.3.2 Cargo Receptors at At the TGN, the mannose-6-phosphate receptor (MPR) works as a
the Golgi cargo receptor, recognizing soluble lysosomal enzymes modified
with mannose-6-phosphate (M6P) and transporting them from the
TGN to endosomes [5]. The cytosolic domains of MPRs contain
dileucine motifs (DXXLL) that interact with cargo adaptor GGAs.
Another cargo receptor, sortilin, mediates trafficking of lysosomal
enzymes and nonlysosomal cargo proteins, such as brain-derived
neurotrophic factor and the TRK family of proteins [89, 90]. The
cytosolic domain of sortilin interacts with APs and GGAs. Sortlin-
related receptor with A-type repeats (SorLA), a Golgi-localized
cargo receptor, is associated with Alzheimer’s disease and regulates
trafficking and processing of APP [91, 92]. Lysosomal integral
membrane protein type 2 (LIMP-2) mediates the trafficking of
β-glucocerebrosidase (βGC) through an MPR-independent path-
way [93, 94]. Wntless is speculated as the receptor of Wnt proteins
for transport of Wnt from the Golgi to the plasma membrane [5].
Many of the soluble ER-resident proteins contain C-terminal
KDEL (HDEL in yeast) motifs that are recognized by transmem-
brane cargo receptors, the KDEL receptors (KDELRs) [95]. The
KDELRs interact with COPI through their C-terminal dilysine
motifs, thereby linking the soluble ER-resident proteins to the
COPI coat [95]. COPI then facilitates the return of these cargo
proteins back to the ER.
The variety of cargo adaptors and receptors involved in protein
sorting demonstrates the eukaryotic cells’ capability to utilize a
multitude of cellular factors to recognize unique sorting motifs
on diverse cargo proteins. This coordination guarantees the accu-
rate packaging of proteins into transport vesicles, ensuring their
delivery to their intended destinations.
3 The Unconventional Secretory Transport Pathway
In addition to conventional protein secretion, numerous cargo

proteins are translocated to their destinations through unconven-
tional secretory transport pathway. There are four types of UPS.
Some of these cargo proteins do not contain any signal peptide or
transmembrane domain. They are secreted into the extracellular
environment through pore-mediated translocation (type I),
ABC-transporter secretion (type II), or organelle-based secretion
(type III). The signal peptide- or transmembrane domain-
containing cargoes can also be delivered directly from the ER to
the plasma membrane, bypassing the Golgi apparatus (type
IV) [96].
3.1 Type I Pore- Fibroblast growth factor 2 (FGF2) and human immunodeficiency
Mediated virus type 1 transactivator of transcription (HIV-Tat) utilize type I
Translocation UPS to be secreted. Different from most of the UPS, which is
stimulated by stress, the secretion of FGF2 and HIV-Tat happens
constitutively [97, 98]. First, mature FGF2 and HIV-Tat are
recruited to the inner leaflet of the plasma membrane by phospha-
tidylinositol 4,5-bisphosphate (PI(4,5)P2) and α1-subunit of the
Na/K-ATPase (ATP1A1) [99], where FGF2 and HIV-Tat undergo
phosphorylation by Tec kinase and self-oligomerization
[100, 101]. The oligomerization of cargoes induces membrane
insertion and pore formation. Finally, the oligomers are disas-

sembled on the outer leaflet of the plasma membrane by heparin
sulfate PGs to be released [102]. FGF2 avoids the undesired modi-
fication and maintains the biological activity through UPS [102].
Type I UPS is also utilized by a quick release of cytokines under
inflammation, such as interleukin-1β (IL-1β) [103]. Caspase-1, a
member of aspartate-specific cysteine-dependent proteases, is acti-
vated by inflammasomes under inflammation situations. Pro-IL-1β
is processed by Caspase-1 to form the mature form. Caspases can
also cleavage gasdermin-D (GSDMD) to form large β-barrel pores
on the plasma membrane, enabling the release of mature IL-1β
through GSDMD pores [104]. Pore formation UPS is also found
in the bacteria [105].
3.2 Type II ABC- Only a few cargo proteins utilize type II UPS, which is ABC-
Transporter Secretion transporter-based secretion. Those cargoes include acetylated
apurinic endonuclease-1/redox factor-1 (AcAPE1/Ref-1), macro-
phage migration–inhibitory factor (MIF), and heat shock 70-kDa
protein (HSP70) [106–108]. ABC-transporter mediates package
of HSP70 into endolysosomal vesicles on lysosomal membranes.
Then, these vesicles are secreted from the plasma membrane [108].
3.3 Type III Some leaderless cargo proteins utilize type III UPS, organelle-
Organelle-Based based translocation, under stress conditions. These proteins are
Secretion captured by organelles such as ERGIC, MVBs, autophagosomes,
endosomes, and lysosomes [109]. It has been reported that the
transmembrane emp24 domain-containing protein 10 (TMED10)
promotes the translocation of UPS cargo proteins into ERGIC
[110]. During this process, UPS cargo proteins triggers the oligo-
merization of TMED10, causing TMED10 to form a channel on
ERGIC membranes to mediate the translocation process
[110]. Some misfolded proteins are also shown to be ubiquitinated
and delivered to late endosomes. Then, late endosomes are fused
with the plasma membrane and misfolded proteins are released
[111]. In yeast, acyl-CoA-binding protein 1 (Acb1) is secreted
through type III UPS and functions in many physiological activ-
ities. The cytosolic Acb1 is engulfed by autophagosomes and then
fused with early endosomes. Acb1-containing endosomes go
through direct fusion with yeast cell surface or MVB sorting path-
way to be delivered outside the cell. In the MVB pathway, Acb1-
containing endosomes enter MVBs using ESCRT machinery and
then fuse with the plasma membrane. Both endosome-plasma
membrane fusion and MVB-plasma membrane fusion require the
assistance of the plasma membrane t-SNARE Sso1 [112].
3.4 Type IV Golgi- Under stress conditions, some signal peptide- and transmembrane
Bypassed Secretion domain-containing cargoes can be transported from the ER to the
plasma membrane, bypassing the Golgi (type IV UPS). Some cargo
proteins bearing mutations cannot be fully folded in the ER, such as
H723R-mutated pendrin (pendrin*) and Phe508-deleted cystic
fibrosis transmembrane conductance regulator (CFTR*)
[113, 114]. Accumulated pendrin* and CFTR* in the ER induce
ER stress, leading to the phosphorylation of Golgi reassembly
stacking protein 55 (GRASP55). Then, the phosphorylated
GRASP55 monomerizes and translocated from the Golgi to the
ER, where it recognizes and interacts with CFTR* to regulate UPS
[115]. Under ER stress, DNAJC14, the co-chaperone of HSP70, is
upregulated and interacts with pendrin* [113]. Subsequently,
CFTR* and pendrin* are packaged into carriers and delivered to
the plasma membrane, bypassing the Golgi.
4 Conclusion
The mechanisms of protein secretion are complex, dynamic, and

meticulously regulated. Both conventional and unconventional
pathways have been found to contribute significantly to this pro-
cess. In the conventional secretory pathway, yeast and animal cells
utilize a highly conserved trafficking machinery to deliver newly
synthesized secretory proteins along a similar path, from the ER to
the Golgi and from the Golgi to the plasma membrane, ultimately
resulting in protein secretion. Transport between different com-
partments occurs in several steps, and each transport step is regu-
lated by specific components in the trafficking machinery: cargo
adaptors regulate the sorting of secretory proteins into specific
transport vesicles; motor proteins move vesicles along the cytoskel-
eton; tethering factors and SNAREs regulate tethering and fusion
of transport vesicles with the acceptor compartments. The coordi-
nated actions of these transport steps ensure that the secretory
proteins are accurately delivered to their specific destinations.
Meanwhile, UPS pathway bypasses the traditional ER–Golgi
route, allowing for the secretion of leaderless cargo proteins
through pore-mediated translocation, ABC-transporter secretion,
or organelle-based secretion. Under certain stress conditions, some
cargoes with signal peptides or transmembrane domains can bypass
the Golgi for secretion. Different types of stimuli induce different
UPS pathways and determine cell destiny. The balance and inter-
play between conventional and unconventional protein secretion
pathways constitute a vital aspect of cellular biology, with significant
implications for both physiological functions and pathological
conditions.
Acknowledgments
X.T. acknowledges the support from the National Natural Science

Foundation of China (NSFC32300589), Anhui Province Higher
Education Research Project (2022AH050174), the Outstanding
Innovative Research Team for Molecular Enzymology and Detec-
tion in Anhui Provincial Universities (2022AH010012), the Uni-
versity Synergy Innovation Program of Anhui Province (GXXT-
2022-067), the Key Laboratory of Biomedicine in Gene Diseases
and Health of Anhui Higher Education Institutes, and the Auhui
Provincial Engineering Research Centre for Molecular Detection
and Diagnostics. Y.G. acknowledges the support from the Hong
Kong Research Grants Council Grants 16102921, 16104423,
16103622, C4002-20W and T13-602/21-N and the Innovation
and Technology Commission (ITCPD/17-9).
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Chapter 2
An Overview of Protein Secretion in Plant Cells

Jing Tang, Kin Pan Chung, and Yonglun Zeng
Abstract
Newly synthesized proteins are delivered to the apoplast via conventional or unconventional protein
secretion in eukaryotes. In plants, proteins are secreted to perform various biological functions. Conserved
from yeast to mammals, both conventional and unconventional protein secretion pathways have been
revealed in plants. In the conventional protein secretion pathway, secretory proteins with a signal peptide
are translocated into the endoplasmic reticulum and transported to the extracellular region via the endo-
membrane system. On the contrary, unconventional protein secretion pathways have been demonstrated to
mediate the secretion of the leaderless secretory proteins. In this chapter, we summarize the updated
findings and provide a comprehensive overview of protein secretion pathways in plants.
Key words Conventional protein secretion, Unconventional protein secretion, Coat protein complex
I, Coat protein complex II, Exosome
1 Conventional Protein Secretion in Plants
1.1 Endoplasmic In higher eukaryotes, nascent secretory proteins synthesize on

Reticulum: The Entry cytoplasmic ribosomes before or concomitant translocation across
Point of Secretory the endoplasmic reticulum (ER) membrane via a translocon formed
Pathway primarily by the Sec61 complex, which is a heterotrimer composed
of Sec61α, Sec61β, and Sec61γ and resides in the ER membrane
[1]. Although encoding Sec61 paralogs in the genomes, the func-
tion of Sec61 remained uncharacterized in plants. The first isolated
plant Sec61 homolog was from the algae [2]. Preserving the con-
served function in protein translocation as yeast and mammalian
Sec61, the plant Sec61 is essential for wheat storage protein depo-
sition and the barely susceptibility to powdery mildew
[3, 4]. Intriguingly, recent studies have shown that the plant
Sec62, a component of Sec62/63 complex, which is the Sec61
channel partner and essential for the post-translational transloca-
tion, is required for plant growth and ER-phagy [5, 6]. After trans-
location into ER, secretory and membrane proteins are subjected to
protein folding processes through interactions with distinct
Liwen Jiang et al. (eds.), Plant Protein Secretion: Methods and Protocols, Methods in Molecular Biology, vol. 2841,
https://doi.org/10.1007/978-1-0716-4059-3_2,
© The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024
19
20 Jing Tang et al.
molecular chaperones, lectins, as well as folding enzymes. In plants,

some major ER chaperones, including BiPs (binding immunoglob-
ulin protein), HSP90 (heat shock protein 90), and the BiP
co-chaperones ERdj proteins (ER-localized DNAJ domain-
containing proteins), have been demonstrated to play important
roles in protein folding and plant development [7]. Incompletely
folded or unassembled proteins are recognized by a constitutively
active ER-mediated protein quality control (ERQC) system that
differentiates aberrant proteins and targets them for destruction in
the cytosol via an evolutionary-conserved degradation process
known as ER-associated degradation (ERAD) that involves multi-
ple steps, including ubiquitination, retro translocation, and cyto-
solic proteasome [8]. Conserved and unique functions (involved in
stress pathway and pathogen defense) of plant ERQC/ERAD
mechanisms have been revealed by recent studies combining bio-
chemical approaches and genetic analysis [9, 10].
1.2 Plant ER-to-Golgi Upon properly folding in the ER, the secretory proteins exit the ER
Transport to the Golgi apparatus for further protein modifications. The
molecular basis for ER protein export has been built on the isola-
tion and characterization of sec mutants that accumulate ER mem-
branes at the nonpermissive temperature in yeast by Schekman
laboratory [11, 12]. Based on the genetic screening, Schekman
and colleagues established an in vitro vesicle reconstitution assay
to unveil and demonstrate that the vesicle coat proteins, collectively
termed the coat protein complex II (COPII), are responsible for
the ER–Golgi transport in yeast, which are later further verified in
animal by Rothman laboratory [13, 14]. The COPII vesiculating
machinery mainly consists of five cytosolic components: Sar1,
Sec23, Sec24, Sec13, and Sec31. The small GTPase Sar1 [15–18]
is first activated and recruited onto the ER membrane by the
guanosine nucleotide exchange factor (GEF) Sec12, which is an
ER-localized integral membrane protein [19]. Subsequently, the
GTPase-activating protein (GAP) Sec23 that stimulates the enzy-
matic activity of Sar1 [20] and the adaptor protein Sec24 [21] are
recruited to the ER membrane as a heterodimer by Sar1-GTP to
form the pre-budding complex [22]. This complex in turn recruits
a Sec13/Sec31 heterotetramer, forming the outer layer of the
COPII coat and completing the vesicle formation. Eventually, the
secretory proteins recognized and recruited by the Sec24 or the
cargo receptors into the nascent COPII vesicles accumulate at ER
export sites (ERESs) and are further transported to the Golgi
apparatus for modifications (Fig. 1). Despite increasing under-
standing of COPII function in early secretory pathway and
human diseases in yeast and mammals, studies on the COPII
function in plant are still in their infancy. In higher plants, Sar1
and Sec12 are the first COPII components being characterized for
their roles in ER-to-Golgi trafficking [23, 24]. Until recently, other
An Overview of Protein Secretion in Plant Cells 21
Fig. 1 Overview of protein secretion pathways in plant cells. In the conventional

protein secretion pathway (indicated by black arrows), newly synthesized pro-
teins with signal peptides are translocated into the endoplasmic reticulum (ER)
before being anterogradely transported to the Golgi and subsequently to the
trans-Golgi network (TGN). In the TGN, some proteins are delivered to the plasma
membrane while others are transported to the multivesicular body (MVB), which
are subsequently targeted to the vacuole. In the unconventional protein secretion
pathway (indicated by red arrows), leaderless secreted proteins are delivered to
the extracellular space via different routes: (1) Golgi-bypass pathway; (2) secre-
tion through MVB–plasma membrane fusion; (3) secretion through vacuole–
plasma membrane fusion; and (4) exocyst-positive organelle (EXPO)-mediated
secretion pathway
22 Jing Tang et al.
COPII proteins have also been studied for their functions in early
secretory pathway as well as plant development and stress response
[25–33]. In particular, the existence of greater Arabidopsis COPII
isoforms in number than other eukaryotes remains a mystery. In
Arabidopsis, there are five Sar1, two Sec13, two Sec31, seven Sec23,
and three Sec24 isoforms encoding in the genome [33, 34]. How-
ever, the significance of this diversification remains poorly under-
stood, raising the question as to whether tissue specificity or stress-
related functional diversity exists for plant COPII isoforms. Several
studies have pointed toward the functional diversity for COPII
paralogs in ER protein export and stress pathway in Arabidopsis
recently. Genetic screening has identified a recessive missense point
mutation (R693K) in Sec24A, which induces the formation of ER
and Golgi membrane clusters and leads to the Golgi and secretory
protein redistribution in the clusters [27, 28]. Interestingly, expres-
sion of Sec24B and Sec24C is incapable of complementing the
missense mutation phenotype, indicating the existence of func-
tional diversity among the Arabidopsis Sec24 paralogs. Recently,
Sec24A has been further shown to be essential for male fertility
[29] while Sec24B and Sec24C have been proved to exhibit redun-
dant function in male and female gametogenesis in Arabidopsis
[31]. Strikingly, Arabidopsis Sar1 homolog AtSar1A was reported
to exhibit distinct inhibitory effects in ER protein export in both
tobacco protoplasts and Arabidopsis plants [26, 32]. Cell biology,
biochemistry, and structural approaches revealed the function het-
erogeneity of AtSar1A was caused by an evolutionary amino acid
substitution, which is crucial for the recognition of unique Sar1-
GAP AtSec23a [32]. Further microarray analysis has indicated the
specific pairing of AtSar1A and AtSec23A, especially their potential
role in plant stress pathway [33]. Indeed, using a newly established
plant in vitro vesicle reconstitution system [35], a recent study has
further elucidated the bona fide role of AtSar1A in plant stress
responses, in particular drought and ABA-related abiotic stresses
[36]. AtSar1A is promptly upregulated upon ABA-related abiotic
stresses and modulated the biogenesis of a novel population of
giant COPII vesicles in plants [36]. Coincidentally, a distinct
Sec23 homolog in Physcomitrens patens has also been shown to be
responsible for the formation of giant COPII/ERES with differen-
tial effects during cell-polarized growth [37]. In Arabidopsis,
although no evidence postulates that the Sec23 homologs regulate
the giant COPII vesicle formation, AtSec23A and AtSec23D have
been demonstrated to regulate pollen wall development and exine
patterning [38]. Lastly, Sec31A and Sec31B, a component of
COPII outer coat protein, are essential for gametogenesis and
interchangeable in pollen development in Arabidopsis [39].
Although accumulating evidence demonstrated the bona fide
role of COPII components in ER-to-Golgi transport, the molecu-
lar details of ER-to-Golgi transport have been the subject of
intensive debate. Besides COPII-mediated protein ER export,

nascent secretory cargoes have been found to exit the ER in a
COPII-independent manner by forming the ER-derived carriers
in mammals [40]. Using state-of-the-art imaging techniques,
recent studies have found that the ER–Golgi transport of secretory
cargo is achieved by a tubule which COPII components are con-
fined to the neck of the tubular carrier [41, 42]. In plants, a pioneer
work a decade ago has also shown the direct ER–Golgi tubular
connections in tobacco leaves by osmium impregnation and trans-
mission electron microscope (TEM), suggesting the potential role
of tubular structures in mediating the ER-to-Golgi protein traffic
[43] (Fig. 1). Although the presence of tubules in electron micro-
graphs does not prove they are directly involved in protein trans-
port in plants, increasing application of advanced cell imaging
techniques in plant cell biology research may further elaborate the
molecular function of the tubular structures in ER-to-Golgi trans-
port in future [44].
1.3 Intra-Golgi In mammals, newly synthesized secretory proteins are delivered to

Transport in Plant ER–Golgi intermediate compartment (ERGIC) before arriving at
cis-Golgi and progressing through the Golgi complex [45]. How-
ever, the presence of ERGIC in higher plants remains doubtful as
Golgi stacks are closely associated with the ERES in plant cells as in
yeast cells. Nevertheless, a recent study suggests that most cis-Golgi
cisternae are biosynthetically inactive and may function as the
mammalian ERGIC, which is the site of membrane assembly and
cargo sorting [46]. Further, BFA treatments in tobacco BY-2 cells
show that punctate structures containing some cis-Golgi compo-
nents near the ERES act as the scaffold for Golgi stack regenera-
tion, suggesting their ERGIC-like properties in plants
[47, 48]. Thus, distinct from mammals, cis-Golgi may function as
the bona fide ERGIC in plants. Once arriving at cis-Golgi, secretory
proteins may undergo carbohydrate modifications and proteolytic
processing in a sequential manner as cargo passing through distinct
Golgi compartments. In mammals and yeasts, a long-lasting debate
about how secretory proteins are transported through the stack of
Golgi cisternae raises two major models: one is the COPI-
dependent vesicular transport (stable compartments) model, and
the other is the cisternal maturation model [49–51]. The stable
compartments model, which described the Golgi as discrete uncon-
nected sub-compartments and retains distinct sets of matrix pro-
teins that establish Golgi compartmental identity and maintain
Golgi architecture in each stack, was first postulated by Rothman
and colleagues. Such a scenario is supported by the observation of
COPI transport vesicles at the cisternal rims [52, 53] as well as cell-
free reconstitution assay using a biochemical approach [54]. The
model was further modified in which COPI vesicles move between
intra-Golgi cisternae bidirectionally, with anterograde vesicles
24 Jing Tang et al.
carrying secretory proteins and retrograde vesicles recycling traf-

ficking components [55]. Nevertheless, the basic concept of the
cisternal maturation model appears to be widely accepted as
(1) cargoes much larger than conventional COPI vesicles can travel
across the Golgi stacks [49]. For instance, procollagen aggregates
in mammalian fibroblasts are shown to progress across the Golgi
cisternae without getting into small vesicles [56, 57]. (2) Retrograde
COPI-dependent cargo concentration and transport are favored,
while the existence of anterograde vesicles remains to be confirmed
[58]. In higher plants, the existence of COPI vesicles was demon-
strated using a cell-free reconstitution assay [59]. Later, distinct
population of COPI vesicles, COPIa and COPIb, which bud exclu-
sively from cis-cisternae and exclusively from medial- and trans-
Golgi cisternae, respectively, were observed using a combination of
electron tomography and immunolabeling techniques in plants
[60]. These studies support the cisternal maturation model in
plants [60]. Recently, observations of Golgi regeneration after
BFA treatment and removal in plant cells have revealed that the
Golgi stacks regenerate in a cis-to-trans manner, which is consistent
with the cisternal maturation model [47]. Therefore, the basic
concept of the cisternal maturation model appears to be more
applicable in higher plants than in other eukaryotes.
While the secretory proteins pass through distinct Golgi com-
partments and undergo carbohydrate modifications and proteolytic
processing, the immature cargoes, escaped ER-resident proteins, as
well as Golgi-resident proteins need to be retrieved by COPI
vesicles to proper loci. Plants’ COPI vesicles, particularly the dis-
tinct paralogs of COPI coat proteins, have been shown to play a
differential role in female and male gametophyte development
[61], cell plate formation [62], plant growth [63, 64], stress toler-
ance [65, 66], and acceptance of compatible pollen [67]. COPI
vesicles recognize and transport cargoes or cargoes receptors with
specific signal. The COPI-interacting signal, canonical dilysine
motif (KKXX and KXKXX), was first identified on the cytoplasmic
C terminus of adenoviral E3 19 kDa (E19) protein in mammals
[68, 69]. In plants, dilysine motifs can also be found in many type I
integral membrane proteins, such as Cf-9 in Lycopersicon esculen-
tum and p24 family proteins in Arabidopsis thaliana [70–
72]. Although the molecular function of p24 family proteins has
been widely explored in yeast and mammals, their specific function
in plants remains largely elusive. Recently, many studies have shown
the trafficking and localization of plant p24 [70–72] and revealed
its function as putative cargo receptors and regulators of protein
trafficking along the early secretory pathway [73–75]. Interestingly,
a novel COPI-interacting signal, the KXD/E motif, is responsible
for the Golgi retention of polytopic integral membrane protein
EMP70 (endomembrane protein 70) in Arabidopsis [76]. Sequence
alignment analysis and further studies suggested the conservative of
the KXD/E motif function in COPI-dependent Golgi retention in

all eukaryotes [77, 78]. Nonetheless, the bona fide function of
EMP70 in early secretory protein trafficking remains unknown.
For tethering of intra-Golgi COPI vesicles to designated mem-
branes, distinct proteins and complex such as coiled-coil tethers
p115, golgin-84, CASP, as well as SNAREs (soluble N-ethylmalei-
mide-sensitive fusion protein attachment protein receptors) and
multi-subunit complexes TRAPPII and COG that are suggested
to be involved have been characterized in plants. For instance,
Arabidopsis p115 homolog, which is isolated as GOLGIN CAN-
DIDATE 6 (GC6) and MAIGO4, was shown to localize to the
restricted domain of the cis-Golgi cisternae as well as COPII vesicles
in Arabidopsis root cells and tobacco BY-2 cells by confocal and
immunoelectron microscopy [79, 80]. Mutant analysis showed that
the maigo4 mutant (Arabidopsis p115) accumulates seed storage
proteins in the ER, indicating its essential role in the secretory
pathway. Similarly, golgin-84 (identified as GC1 and GC2) and
AtCASP have been shown to localize at the cis-Golgi cisternal
rims [79]. However, the physiological roles and the tether func-
tions of p115, golgin-84, and AtCASP remain largely uncharacter-
ized in plants. Meanwhile, the distinct components of COG
tethering complex, namely COG7, COG3, and COG8, have been
shown to be responsible for Golgi morphology maintenance, plant
embryo development, as well as pollen tube growth [81, 82]. Addi-
tionally, SNARE proteins such as Qc-SNARE BET12 have been
shown to interact with COPI component and may be essential for
COPI vesicle fusion with the target membrane [83]. Nevertheless,
the TRAPII complex components and their functions in COPI
vesicle tethering are still elusive. Future studies need to elucidate
how the tether proteins mediate the COPI vesicles targeting the
designated membrane and their physiological impacts on the plant
development.
1.4 Post-Golgi After modification in the Golgi apparatus, the secretory proteins
Trafficking of will continually be processed by resident enzymes and eventually
Secretory Proteins to sorted at trans-Golgi network (TGN), which was defined as a
the Plasma Membrane specialized compartment on the trans-most cisterna of the Golgi
in Plants composed of tubular-like membrane structures [84]. In plant cells,
the ultrastructure of TGN was observed as a branched and tubular
membrane structure with clathrin-coated buds by electronic
microscopy [85, 86]. Intriguingly, besides acting as a sorting sta-
tion for the post-Golgi pathways, the TGN in plant cells may also
function as an early endosome (EE), which is distinct from yeast
and mammals [85–87]. Live-cell analysis using spinning disk con-
focal and super-resolution confocal shows the existence of two
types of TGN in plant cells [86, 88]: (i) the Golgi-associated
TGN, which is located on the trans-side of the Golgi apparatus,
and (ii) the Golgi-released TGN, which is a mobile and
26 Jing Tang et al.
independent organelle locating away from the Golgi apparatus.

Function diversity of these distinct populations of TGN remains
uncharacterized in plants. Recently, cargo sorting zones in TGN
have been further identified using multicolor high-speed and high-
resolution spinning disk confocal microscopy [89]. Furthermore,
unlike in mammals, the secretory protein trafficking en route to the
plasma membrane in plant cells are largely underinvestigated. A
recent study has elucidated the mechanism underlying the TGN
subdomain in secretory cargo trafficking such as PIN2 polarized
transport [90]. Notably, chemical genomic approach has identified
a chemical compound ES16 (endosidin 16) perturbed apically
localized PM proteins without affecting basal polarity, further sup-
porting the existence of TGN subdomain or subpopulation in
mediating secretory cargo trafficking to PM [91]. LRR-receptor-
kinases IRK and KOIN, which control root cell division, are recog-
nized, sorted, and secreted differentially through distinct pathways
from TGN to PM [92]. Using fluorescent-tagged SCAMP2, the
secretory vesicle cluster (SVC) generated from TGN that moves in
the cell and eventually fuses with the plasma membrane was identi-
fied. The SVC was found in Arabidopsis thaliana as well as rice
(Oryza sativa) cells and moved to the cell plate in dividing tobacco
cells, indicating that the SVC is a dynamic structure involved in
secretory protein transport from the Golgi apparatus or TGN to
the plasma membrane in plant cells [93, 94]. In addition, a recent
study in Arabidopsis adaptor protein complexes 1 (AP1), the con-
served protein complex participating in TGN to PM protein traf-
ficking in mammals, has shown that loss of function of the large
subunit γ-adaptin in AP-1 complex leads to the defective secretory
protein trafficking to the vacuole, plasma membrane, and cell plate
[95, 96]. SNARE proteins and Rab GTPase have been shown to
regulate protein trafficking from TGN to PM in mammals. In
plants, the SNARE and Rab GTPase functions in TGN-PM traf-
ficking remain largely unclear. Intriguingly, the R-SNAREs
VAMP721 and VAMP722 have been shown to mediate the post-
Golgi trafficking of auxin transporters to the PM from the TGN
subdomains, substantially contributing to plant growth [97]. In
addition, PM-localized Q-SNARE SYP121 interacts with
Q-SNARE VAMP721 and the exocyst component EXO70 to
mediate the protein secretion from TGN during the vegetative
growth [98]. Meanwhile, SYP121 binds to the potassium channels
and triggers the assembly of the secretory machinery for exocytosis
[99]. Interestingly, a small GTPase homolog RABA2a has been
shown to recruit and interact with the VAMP721/722-SYP121-
SNAP33 SNARE ternary complex for membrane fusion, probably
playing an essential role in TGN to PM exocytosis [100]. These
studies indicate the existence of the conserved TGN to PM traffick-
ing pathway in higher plants (Fig. 1). However, the vesicles med-
iating this trafficking process and the underlying mechanism remain
elusive. Future studies on the TGN to PM trafficking pathway in

plant cells will shed light on the study of plant cell polarity and
development.
2 Unconventional Protein Secretion in Plants
2.1 Plant Although increasing evidence has demonstrated that the plant
Unconventional conventional protein secretion pathway is highly conserved
Protein Secretion among higher eukaryotes with some unique characteristics, the
unconventional protein secretion pathway remains largely elusive
in plants. In mammals, two types of extracellular vesicles (EVs)
expressed by all types of cells, namely the exosomes (ILVs from
MVBs) and ectosomes (assembled from the initial microdomain of
the plasma membrane and secreted out), have been shown to
mediate unconventional protein secretion. Interestingly, more
than 50% of the secreted proteins lack signal peptide as revealed
by the analysis of the plant secretome [101]. These leaderless
secretory proteins (LSPs) are not translocated into the ER to join
the conventional protein secretion pathway, thus raising an alterna-
tive route termed unconventional protein secretion (UPS). In gen-
eral, LSP bypassing the ER–Golgi protein transport pathway, that
is, (i) trafficking unaffected by BFA and (ii) without post-
translational modification, probably make use of the UPS pathway
for their extracellular delivery [102]. Proteomic studies revealed
the majority of the LSP are related to stress or pathogen infection
[103], implicating the essence of the UPS pathway in dealing with
various environmental cues in plants. However, the vesicles and
their underlying mechanism in regulating the protein unconven-
tional secretion are elusive.
2.1.1 Direct Different types of UPS pathway have been described in plants over
Translocation of LSP the last decade. For instance, certain LSP may be secreted directly
Across the Plasma from cytosol without the involvement of other organelles; while in
Membrane some cases, the secretion of LSP is mediated by vacuole, MVB, or
exocyst-positive organelle (EXPO), respectively (Fig. 1). Although
the direct translocation of LSP across the plasma membrane has not
been proved in plants yet, previous studies suggested that the
leaderless cytosolic enzyme mannitol dehydrogenase (MTD) is
being secreted into apoplast in response to salicylic acid, a plant
defense hormone, in tobacco [104]. It was shown that MTD
secretion is Golgi-independent since the treatment with BFA did
not interfere with its extracellular trafficking [105]. Similarly,
another cytoplasmic enzyme hygromycin phosphotransferase
(HYGR) that is commonly used for the selection of hygromycin B
resistance was found to be secreted into extracellular space in a
BFA-insensitive manner in Arabidopsis [106]. In yeast, it has been
shown that farnesylated peptides such as α-factor and M-factor are
28 Jing Tang et al.
transported through the ABC-transporter-based secretion

[107]. However, it is not clear whether any protein or channel
present on the plasma membrane would assist the translocation
and secretion of the cytoplasmic MTD and HYGR. Nevertheless,
MTD and HYGR represent LSP that utilize a Golgi-independent
pathway and therefore belong to the UPS category.
2.2 UPS via Vacuole In addition to the plasma membrane direct translocation approach,
and Multivesicular LSP could be secreted with the help of other organelles as carriers.
Bodies Upon pathogen infection, fusion of the vacuole with the plasma
membrane at the site of attack has been reported in Arabidopsis
[108]. The UPS mediated by the vacuole–plasma membrane fusion
allows the release of vacuolar enzyme in response to the pathogen
attacks, thus representing a strategy for plant survival. During
fungal infection, the accumulation of MVB at the site of the inva-
sion papillae has been reported [109]. It is plausible that the fusion
of MVB with the plasma membrane would release the intraluminal
vesicles as exosomes in the fungal haustorium [110]. Indeed, pre-
vious studies implicated the presence of exosome-like structures in
the papillae matrix. In barley leaves infected by powdery mildew
fungus, vesicle-like bodies were frequently observed while some of
them were identified as MVB and paramural bodies (PVBs). It was
suggested that antimicrobial compounds were contained in the
MVB and PVB and their subsequent discharge could be used
against fungal penetration. However, how the LSP such as papilla
building block and antimicrobial compounds are being sequestered
into the MVB and undergo UPS is still unclear. Interestingly, a
recent study has shown that plant cells secrete MVB-derived EVs to
deliver small RNAs upon infection by the fungal pathogen Botrytis
cinereal [111]. Many antimicrobial peptides are known to be
secreted out of the cells to regulate the plant immunity
[112]. Future studies would be of great interest to investigate
whether these structures are also indispensable for the secretion
of LSPs upon pathogen infection in plants.
2.3 Other Plant UPS UPS is not only involved in pathogen response but may also be
Pathway responsible for plant growth and development.
S-adenosylmethionine synthetase 2 (SAMS2), an enzyme involved
in lignin biosynthesis that could contribute to the cell wall architec-
ture, is proposed to be secreted to the extracellular space via EXPO.
EXPO is a double-membrane compartment marked by the exocyst
subunit AtExo70E2 [113]. EXPO showed no response to BFA,
wortmannin, nor concanamycin A, suggesting its distinctive
nature, and is independent of the conventional protein trafficking
pathway. Fluorescence signals of AtExo70E2-GFP did not overlap
with other fluorescence-tagged organelle-specific markers, includ-
ing the Golgi, TGN, and MVB. Transmission electron microscopy
and immunogold labeling revealed the ultrastructure of EXPO and
confirmed its presence in the extracellular space, indicating that the

EXPO, together with its LSP cargoes, is ultimately secreted
[113]. In the EXPO-mediated UPS pathway, it was proposed that
the cytosolic LSP cargoes are sequestered into the forming EXPO,
which the completed EXPO would then eventually fuse with the
plasma membrane and release a single-membrane bound vesicle to
the extracellular space [103]. Nonetheless, the nature of EXPO and
the relationship between EXPO and secreted autophagosomes is
yet to be investigated.
2.4 Techniques and Although the underlying mechanisms for the UPS pathway in
Approaches for Future plants remain elusive, enormous efforts have been put into advanc-
UPS Study ing our knowledge in this field. Among various approaches, pro-
teomic study of the plant secretome represents a major technique in
studying plant UPS. However, it should be noted that the purity of
the fractions obtained for analysis is a matter of utmost importance.
Contamination with cytosolic proteins caused by the breakage of
cells during fraction preparation would cause deviation and discrep-
ancy of the secretome data, which could hinder the further explo-
ration of the UPS in plants. A recent study reported a successful
purification of the EV fraction from the leaf apoplast [114]. How-
ever, the purity of the EVs purified from this method was
challenged by a recently developed protocol [115]. Further devel-
opment of the protocol on EV isolation and discovery of markers
for different EV populations is important to understand the uncon-
ventional protein secretion pathway in plants. Additionally, proteo-
mic data suggested that proteins involved in biotic and abiotic stress
are enriched in the EVs purified, while the amount of secreted EV is
increased upon pathogen and salicylic acid treatment. It is reason-
able to assume that plant secretome and the secretion activity are
altered in response to different conditions. Thus, exposing plants
toward various environmental cues and comparing the
corresponding protein secretome data may aid in the identification
of specific LSP in plants. On the other hand, in-depth studies of the
proteins involved in UPS are required to elucidate the molecular
mechanisms of EV biogenesis. Super-resolution in vivo real-time
imaging can be used to monitor the LSP trafficking and their
dynamic behavior with the UPS machinery. For instance, the
sequestration of cargoes into EVs may be able to be tracked using
advanced imaging techniques. From the ultrastructural perspective,
cryo-electron microscopy is a promising tool to study the architec-
ture of the EVs as well as the machinery regulating the EV forma-
tion, which could provide insight into the formation of EVs in
plants.
In summary, more and more evidence points to the existence
and importance of UPS in plants. UPS is not only reported in
model plant Arabidopsis thaliana but also in many plant species,
indicating that it is an efficient protein trafficking pathway that is
30 Jing Tang et al.
widely adopted. Due to the complexity and lack of genetic mutant

information, UPS study in plants remains challenging and is under-
investigated. Nevertheless, combining the proteomic data and the
latest cell biological technique would certainly advance our knowl-
edge of the UPS in plants.
Acknowledgments
This work was supported by grants from the National Natural

Science Foundation of China (311540011), Strategic Priority
Research Program of the Chinese Academy of Sciences
(XDA0450000), Hundred Talents Program of the Chinese Acad-
emy of Sciences (361030011), Guangzhou Basic and Applied Basic
Research Scheme (SL2023A04J01893), and Science and Technol-
ogy Projects in Guangzhou to YZ.
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Plant Protein Secretion 1st Edition Liwen Jiang

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Plant Epigenetics Methods and Protocols 2nd Edition Igor

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Methods and Protocols

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Hong Kong, China Liwen Jiang

15 Pharmaceutical Analysis in the Plant Endomembrane System . . . . . . . . . . . . . . . . 165

Conventional and Unconventional Protein Secretion in Yeast

Key words Vesicle, Cargo adaptor, Cargo receptor, Protein secretion

1 Overview of Conventional and Unconventional Protein Secretion

In any organism, each cell synthesizes diverse proteins that regulate

be retrieved to their resident compartment, and some extracellular

2 The Conventional Secretory Transport Pathway

comprises five members. Among them, AP-1, AP-3, and AP-4

coat, Sec23/24 heterodimers, through the interaction with Sec23.

form trans-SNARE complex. This four-helix bundle complex

[44]. TRAPP complex has three known forms—TRAPP I, TRAPP

SNARE Complex The trans-SNARE complex is a four-helix bundle composed of

binds to its binding partner, cTAGE5, a truncated form of

In yeast, the exomer cargo adaptor is responsible for the direct

electrostatic interactions between the triacidic motif in the first

3 The Unconventional Secretory Transport Pathway

In addition to conventional protein secretion, numerous cargo

insertion and pore formation. Finally, the oligomers are disas-

The mechanisms of protein secretion are complex, dynamic, and

X.T. acknowledges the support from the National Natural Science

An Overview of Protein Secretion in Plant Cells

1 Conventional Protein Secretion in Plants

1.1 Endoplasmic In higher eukaryotes, nascent secretory proteins synthesize on

molecular chaperones, lectins, as well as folding enzymes. In plants,

Fig. 1 Overview of protein secretion pathways in plant cells. In the conventional

intensive debate. Besides COPII-mediated protein ER export,

1.3 Intra-Golgi In mammals, newly synthesized secretory proteins are delivered to

carrying secretory proteins and retrograde vesicles recycling traf-

the KXD/E motif function in COPI-dependent Golgi retention in

independent organelle locating away from the Golgi apparatus.

elusive. Future studies on the TGN to PM trafficking pathway in

2 Unconventional Protein Secretion in Plants

transported through the ABC-transporter-based secretion

confirmed its presence in the extracellular space, indicating that the

widely adopted. Due to the complexity and lack of genetic mutant

This work was supported by grants from the National Natural

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