Golgi complex,

secretion and
protein transport
Biology I – Faculty of Pharmacy

Dr. Eszter Lajkó

Department of Genetics, Cell- and Immunobiology

03.10.2016
 Protein targeting and sorting
Transport of proteins from the synthesis site to their destinations
“Roadmap” of protein traffic
Key components of the protein transport
1. Sorting signal

2. Receptors: recognize sorting signal and guide proteins to

their appropriate destination
3. Way of protein transfer
• Gated transport through the nuclear pore
• Translocation across the membrane (transmembrane
protein traslocator = translocon)
• Vesicular transport
4. Energy
 Protein sorting

Co-translational
Protein synthesis transmembrane Endoplasmic reticulum
transport
in cytoplasm (free ribosome) (membrane-bounded ribosome)
(hydrophobic aa at N-term)
Gated
transport
Post-translational Vesicular transport
Cytoplasm
(no signal) transmembrane transport
Golgi
Nucleus
(NLS)
Late endosome Secretory
Mitochondria
vesicle
(N-term. Lysosome
positively
Early endosome
charged aa) Peroxisomes
(3 aa at C-term)
Plasma membrane
 Vesicular transport
• transport between membrane-enclosed compartments
• transport of macromolecules (soluble and membrane-bound) from
the donor compartment to the target compartment

Further information about the vesicular transport in 6th week lecture

Main vesicular transport pathways
Inward transport Outward transport
Endocytotic pathway Secretory pathway
Golgi – „Traffic manager” of the cell
 Golgi apparatus
"internal reticular apparatus"

Camillo Golgi
(1843-1926)

Nobel prize 1906

Metal impregnation
 Structure of Golgi apparatus

• consist of saccules (cisternae), tubules and vesicles

• structural-functional unit: dictyosome (4-6 saccules)
• structure is polarized into sub-compartments

Endoplasmic reticulum

entry
Cis Golgi network
(CGN)

Cis Golgi (CG)

Medial Golgi (MG)
Trans Golgi (TG)

Trans Golgi network

(TGN)

exit
Plasma membrane Lysosome
Position

Nucleus
Microtubule
Golgi
 Role of microtubules in
maintance of Golgi structure

Intact microtubules Disintegration of microtubules

Golgi- green
Microtubules - red

2002 by Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, and Peter Walter.
Visualisation of Golgi with different techniques
Silver impregnation for LM TEM

1. Golgi dyctiosomes

Multiphoton fluorescence image

 Main functions
Sructural and functional polarization

Cis face: arrival side

1. post-translational modifications of
proteins (and lipids)
2. sorting
3. formation of transport vesicles
4. transport

trans face:
departure side
Vesicular tubular cluster

• continually generated from ER-derived vesicles

• separate compartment from ER and Golgi
• transport container
• moving along the microtubules to the Golgi
 Cis Golgi Network – Entry side
• collection of fused vesicular tubular clusters
• the proteins and lipids arrive from the ER in vesicles

ER-resident protein move back to the ER

proteins of Golgi
targeting to the cell surface move to the cis Golgi
or another compartments cisterna

they are N-glycosylated

no sorting in the ER
 Glycosylation
N-glycosylation
• Adding oligosaccharide chain to
O-glycosylation
protein (lipids)

• Made by glycosyl transferases

soluble glycoportein

• N-glycosylation (to Asn) is made in

ER and modified in ER and Golgi O-glycosylation
(longer chains)
Glycolipid
N-glycosylation

• O-glycosylation (to Ser, Thr) is

made in Golgi
(shorter chains)

Membrane
glycoportein
 N-glycosylation
• Preformed oligosaccharide chain is transfered to asparagine side
chains of a polypeptide in ER
• Processing of oligosaccharide chain starts in ER and continues in Golgi

cytoplasm

Asparagine
side chain

ER lumen
Golgi:
further processing of
N-linked
olgosaccharide chain
Modification of N-oligosaccharide chains of proteins
1. phosphorylation of the mannoses of the lysosomal proteins
­ cis Golgi network
2. no change (high mannose)
­ cis Golgi
3. change of mannoses to other monosaccharides
1. medial and trans Golgi

• each cisternae containing a characteristic mixture of enzymes involved

in processing of N-linked oligosaccharides
• enzymes are all membrane bound
• processing depends on the position of oligosaccharides in the protein
Sorting and modification of lysosomal
enzymes

Mannose-6-phosphate (M-6-P) signal

•based on the recognition of
lysosomal hydrolases
•recognition of the “signal patches” is required
•main working enzyme:
GlcNAc-phosphotransferase
Lysosomal enzymes get a M-6-P signal in CGN
 Significance of M-6-P labelling

Lysosomal
enzyme

Lysosome
Mannose-6-
phosphate
M-6-P
Lysosomal enzymes with M-6-P signal are
­not modified further in Golgi
­recognised by receptors in trans Golgi
network
­transported to the lysosome

Golgi
Main types of N-oligosaccharide chains

-Asn
-Asn

-Asn
Steps of processing and subsequent sugar addition is rigidly ordered, but the
complex oligosaccharides can be heterogeneous

cis Golgi medial Golgi medial Golgi

trans Golgi

negatively charged

trans Golgi
N- and O-linked glycosylation

Threonine,
Serine
 O-linked glycosylation

-Ser/Thr

-Ser/Thr

• takes place mainly in the medial- and trans Golgi

• shorter oligosaccharide chains
• adding monosaccharides one by one to Ser/Thr
• each step requires different enzymes
Significance of glycosylation
• folding
• sorting
• protection against proteolytic enzymes
• makes proteins hydrophylic
• cell adhesion (leukocytes and endothel –
cell adhesion molecules)
• antigenity (A,B,O blood groups)
• glycocalyx (external coat)
 Proteoglycan synthesis
Basic structure of proteoglycans

Serine

linker: tetrasaccharide
Glycosaminoglycan
(GAG)
core protein

• Proteins with polisacharide side chains (long, unbranched)

• Found in plasmambrane and extracellular matrix
• Major components: glycosaminoglycan (GAG)
­ composed of repated disaccharides
­ containing sulphate group negatively charged
­ e.g. hyaluronic acid, chondroitin sulphate,
dermatan sulphate, heparan sulphate
 Proteoglycans

N and O oligosaccharides are

not shown
Modifications of proteins and lipids in
Golgi
1. Glycosylation
• modification of oligosaccharide chains of proteins,
• binding of newly synthesized oligo- or polysaccharide chains
- M-6-P group (lysosomal proteins)
- no change (high mannose)
- change of mannoses to other monosaccharides
- O-glycosylation
- proteoglycans synthesis

• adding of -SO4 group

­ carbohydrates: GAG, proteoglycans
­ amino acid: Tyr residue of proteins
chondroithine
sulphate
• synthesis of lipids
1.glycolipids
2.sphingomyelin

• proteolysis
 Synthesis of membrane lipids
Golgi

Sphingomyelin

ER

Monosaccharide Oligosaccharide

Clycolipid Cerebroside Gangliosode

 A,B,0 blood group antigens
(glycolipids = ganglioside)

ceramide

sugar
residues
 Golgi is a major protein sorting

• back to ER
­ ER resident proteins (soluble) – KDEL signal
­ membrane proteins – KKXX signal cis Golgi network
cargo receptors and from all cisternae
proteins needed for vesicule formation and fusion

• retaining Golgi proteins

­ aggregation of Golgi proteins in all cisternae

• to lysosome
­ M-6-P signal
trans Golgi network
• to plasmamembrane and ECM
• to cell exterior (for secretion)
Main pathways going in and out Golgi
 Bidirectional transport between ER and Golgi

Vesicular tubular cluster

Secretory
protein

ER-resident
protein receptor
(KDEL receptor)

ER-resident
protein with KDEL
signal sequence
 Transport through the Golgi apparatus

Cisternal maturation model Vesicle transport model

• Cisternae mature from early to late by • Cisternae remain at the same place with
acquiring and then losing specific Golgi- characteristic set of Golgi proteins
resident proteins • Cargo protein are moved forward by
• Cisternae moves through the dictyosome with transport vesicle
cargo in its lumen • Retrograd pathway of vesicles return the
• Retrograde transport of Golgi enzymes escaped proteins to the previous cisternae
 Golgi resident proteins
- enzyme-content of the different compartments
-
cis Golgi medial Golgi trans Golgi
Unstained mannosidase I mannosidase II nucleoside diphosphatase

cis face trans face Osmium

impregnation
Bidirectional transport of proteins
 Forward – anterograd transport
• lysosomal enzymes
• secretory proteins
• extracellular matrix and plasma membrane components
• Golgi-resident proteins

 Backward – retrograd transport

• proteins of the ER recycled
­ membrane-bound or soluble
­ retention signal is required
• protein of each cisterna
• M-6-P receptor from the lysosomes
• membrane components of transport vesicles from the
plasma membrane
 Main transport pathways from TGN
Proteins are seggregated into different transport packages and dispatched

exocytosis
transport from the TGN to
secretory vesicles the cell exterior
(fusion of transport vesicles
secretion with plasma membrane)
synthesis, modification lysosome
and release (exocytosis) of
endosomal-lysosomal
different compounds
compartment
(e.g. proteins, lipids)

•central organelles – ER and

Golgi
•types:
consitutive secretion
regulated secretion
Constitutive secretion (exocytosis)

• default pathway
• presents in all cells
• continuously
• non-selective
• no accumulation of vesicles
• ECM proteins, membrane
lipids and proteins
signal
Regulated secretion (exocytosis)

• typical for glandular cells

signal and neurons
• signal is needed for the
exocytosis
• accumulation of vesicles
• hormones, eurotransmitters,
digestive enzymes
Pathways of protein sorting in the TGN
Modifications of secretory vesicles

• selective aggregation - TGN

• further modifications and sorting

­ inactive precursor - active enzyme or hormone
(e.g. preproinsulin - proinsulin - insulin)

• concentration - loss of water

• hydratation - e.g. proteoglygans

• uptake some cytoplasmatic substances e.g. histamine

Immature secretory
vesicle contains
missorted proteins

Removal of
missorted proteins

Acidification and
condensation
Proteolysis of proteins in secretory vesicles

proteolysis
pro-
hormone
inactive
active
hormone
hormone
active
Secretory
Synthesis of
vesicle with
pre-pro-hormone Glycolistaion
pro-hormone
inactive of pro-hormone
inactive
inactive
Network of membran flow in eukaryotic
cells