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Endocytosis IV April 5

Introductory Molecular and Cell Biology (McGill University)

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Endocytosis IV
Targeting of proteins from the
Golgi apparatus to the endocytic
pathway

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Review 1

Rab5 and Rab7

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Early endosomes are abnormally large in cells

overexpressing Rab5

From Bucci et al. Cell 70:715-28 (1992)

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Early endosomes are abnormally small in cells

expressing a mutant Rab5 that cannot bind GTP

From Bucci et al. Cell 70:715-28 (1992)

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Rab5 and Rab7

compared. You do
NOT need to know
any of the proteins
except those
mentioned on the
previous slide and
Rabex-5. I.e., don’t
memorize the
SNAREs except for
syntaxin13.

Rabex-5 is a GEF for

Rab5. Ypt7 is an
alternate name for
Rab7 (used primarily
by yeast people). You
do NOT need to
recognize Ypt7 on the
final.

From Kummel and Ungermann (2014) Curr. Opin. Cell Biol. 29:61-66

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Review 2

Golgi to cell surface

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Epithelial cell

Hepatocytes

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cargo

dynamin
coat
motor

mt

Tubular transport intermediate

Coated vesicle
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Secretory vesicles
In goblet cell fuse with
apical surface.

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Basolateral sorting
• Most basolateral sorting of proteins in
epithelial cells is believed to involve
clathrin vesicles and AP-1.
• This is only one possible role of clathrin
vesicles, as clathrin is a very versatile coat
thus a more detailed examination of clathrin
follows.

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General Structure of APs

• Made of four domains, specific
to type and tissue
• Can bind receptor tails via
tyrosine and dileucine motifs
• Binds clathrin through ear
• AP-1 involved in trafficking to
cell surface (one subtype) and
also to endosomes.
• AP-3 also involved in
trafficking to endosomes (later
today)
• AP-4 on Golgi, but function
unclear.

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Targeting of Enzymes to
Lysosomes

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Hydrolases, such as cathepsins, are synthesized in the ER and transferred to the

Golgi where they are tagged in two steps with M6P. The tagging involves first
the transfer of N-acetylglucosamine-1-phosphate from uridine disphosphate-N-
acetylglucosamine to selected mannose residue.

Subsequently, GlcNac phosphoglycosidase removes N-acetylglucosamine to expose

the M6Precognition signal. The two steps occur within the cis-compartment of
the Golgi.

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ICD Disease (Mutation of GlcNac Phosphotransferase)

X
Phosphotransferase

Alberts et al (1994) Molecular

Biology of the Cell

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I-Cell Disease
Protein / Gene UDP-N-acetylglucosamine N-
acetylglucosaminyl-1-
phosphotransferase (GlcNac
Phosphotransferase)
Common / Severe There are ~40 known mutations. One
Mutations causes a frame shift and another one a
premature stop codon.
Major Symptoms Severe -delayed development / short
stature / coarse facial features /
dysostosis multiplex* / cardiomyopathy
Clinical Diagnosis High lysosomal enzyme activity in
serum / Molecular genetic screening

Plante M, Claveau S, Lepage P, Lavoie EM, Brunet S, Roquis D, Morin C, Vézina H, Laprise C.
Clin Genet. 2008 Mar;73(3):236-44.
Tappino B, Regis S, Corsolini F, Filocamo M. Mol Genet Metab. 2008 Feb;93(2):129-33.

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I Cell Disease, General symptoms

Characterized by the accumulation of excessive amounts of acid

mucopolysaccharides in fibroblasts, connective tissue and
connective tissue cells, and abnormal accumulation of undigested
sphingolipids, and/or glycolipids in the lysosomes of visceral
fibroblasts and macrophages and in the lysosomes of nerve cells.
The disorder results in mental retardation and skeletal changes,
most notably dysostosis multiplex.

*Dysostosis multiplex definition: Mucopolysaccharidosis due to

deficiency of alpha-l-iduronidase, accumulation of abnormal
intracellular material and excretion of dermatan sulfate and
heparan sulfate in the urine; with severe abnormality in
development of skeletal cartilage and bone, dwarfism, deformed
limbs, limitation of joint motion, spade like hand, corneal clouding,
hepatosplenomegaly, mental retardation, and gargoyle-like faces.

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ICD: Bone Marrow Macrophages

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Adaptor (GGA)
(Sortilin)

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PROSAPOSIN

A B C D

SAPOSINS
SAPOSINS
A

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Lysosomal Storage Disorders (LSD)

There are approximately 50 hydrolases in the the lysosomes and nearly

50 known LSD due to mutations in their genes.
Almost all forms of LSD are inherited as autosomal recessive traits and
are caused by allelic mutations of the gene that encodes the hydrolase.

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LSD Affected Soluble Enzyme

B-variant of GM2 gangliosidosis / Tay Sachs β-hexosaminidases A and S (?)
0-variant of GM2 gangliosidosis / Sandhoff β-hexosaminidases A and B
Metachromatic leukodystrophy MLD Arylsulphatase A
Gaucher’s Glucosylceramide β-glucosidase
Globoid cell leukodystrophy (Krabbe’s) Galactosylceramidase
Fabry α-galactosisidase A
Glycogen storage disease/ Pompe acid α-glucosidase
Mucopolysaccharidosis type I/ Hurler α-L-iduronidase
Mucopolysaccharidosis type II/ Hunter Iduronate-2-sulfatase
Mucopolysaccharidosis type IIIA/ Sanfilippo Heparan-N-sulfatase
Mucopolysaccharidosis type IIIB/ Sanfilippo α-N-acetylglucsaminidase
Mucopolysaccharidosis type IIID/ Sanfilippo N-acetylglucosamine-6-sulphatase
Mucopolysaccharidosis type IVA/ Morquio N-acetylgalactosamine-6-sulphatase
Mucopolysaccharidosis type IVB/ Morquio β-galactosidase
Mucopolysaccharidosis type VI/ Maroteaux-Lamy N-acetylgalactosamine 4-sulphatase
Mucopolysaccharidosis type VII/ Sly β-glucuronidase
Galactosialidosis Cathepsin A/ protective protein
Aspartylglucosaminuria Aspartyl glucosaminidase

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Gaucher’s Disease

Protein / Gene β-glucocerebrosidase / GBA

Common / Severe N370S, 84GG, L444P

Mutations
Major Symptoms Hepatosplenomegaly / anemia /
skeletal disease / dementia /
convulsions
Clinical Bone marrow aspirate / β-
Diagnosis glucocerebrosiadase activity

Chen & Wang. Arch Pathol Lab Med—Vol 132, May 2008

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Bone Marrow: Gaucher cell

Wright-Giemsa-stained bone marrow aspirate shows a Gaucher

cell containing abundant granular or fibrillary, blue-gray cytoplasm
with a wrinkled tissue paper- like appearance
Chen & Wang. Arch Pathol Lab Med, Vol 132, May 2008

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Saposins B and C assist various lysosomal

enzymes in the catabolism of sphingolipids

Glcß1-Cer
Gaucher Glucosylceramidase
Saposin C
Cer
ß-Galactosylceramidase
Saposin C
Galß1-Cer
Metachromatic Arylsulfatase A
Leukodystrophy Saposin B
SO3-Galß1-Cer

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Summary I
• Lumenal lysosomal enzymes are brought to
the late endosome by two cargo receptors.
• These are mannose-6-phosphate receptor
and sortilin
• But how do these cargo receptors traffic to
the late endosome from the Golgi
apparatus?

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Sorting Receptors for Lysosomal Proteins

Mannose 6-phosphate receptor. It is generally done by the 46 kDa

mannose 6-phosphate receptor (M6P-R). The M6P-R uses GGA
adaptor proteins to enter into clathrin coated cargo vesicles.

Sortilin is also another receptor that uses GGA adaptor proteins.

Sortilin is involved in the transport of the activator protein
prosaposin as well as a number of other activator proteins and
hydrolases.

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GGA (right) differs from the AP’s in consisting of only a single polypeptide.

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The VHS domain of GGA binds cargo targeted to lysosomes. The GAT domain binds the
Small GTPase Arf1. The GAE (ear) domain binds clathrin.

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Structure and Function of GGAs

• Made of 4 domains
• Able to bind receptor tail
motif via VHS domain
(M6PR and Sortilin)
• Binds ARF via its GAT
domain
• Binds clathrin via hinge and
terminal Ear domain

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Structure and Function of GGAs

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• COS-7 cell transfected with

Truncated GGA truncated GGA which lacks
the hinge and Ear domains to
interact with clathrin

GFP

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GGA Mutant Cells

Green Fluorescent : Cell transfected with GFP tagged GGA

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Targeting of membrane proteins

to lysosomes
• LEP, LAMPs, LIMPs etc.
• Occurs normally even if GGA is not
functioning.
• Depends on clathrin

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General Structure of APs

• Made of four domains,

specific to type and
tissue
• Can bind receptor tails
via tyrosine or
dileucine motif
• Binds clathrin to cause
vesicle formation

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Targets lysosomal
Function of AP-3 membrane proteins
TGN Membrane
such as LAMPs and
Lumen Cytosol
LIMPs.

Clathrin

LAMP-2
AP-3

Dileucine Motif

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TGN Membrane
Lumen Cytosol

Clathrin

LAMP-2

Mutant AP-3

Dileucine Motif

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AP-3 Mutant Cells

Cathepsin B has been shown to use M6P receptor

LAMP-2 uses the AP-3 transport mechanism

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Transport of most soluble hydrolases. It is generally done by the 46 kDa

mannose 6-phosphate receptor (M6P-Rc). The M6P-Rc uses GGA
adaptor proteins to enter into clathrin coated cargo vesicles.

Transport of activator proteins and some soluble hydrolases. Sortilin

is another cargo receptor that uses GGA adaptor proteins. Sortilin
is involved in the transport of the activator protein prosaposin as well
as some soluble hydrolases that we didn’t discuss.
Transport of lysosomal membrane proteins. LEP 100, LIMP 1-4
and LAMPs are membrane glycoproteins. These group of proteins
go to the lysosomes from the Golgi apparatus using adaptor
proteins. The transport of lysosomal associated protein (LAMP) is
mediated by the adaptor protein-3 (AP-3).

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Retromer
• Retromer is involved in the return of
mannose-6-phosphate receptor and sortilin
from the late endosome to the Golgi
apparatus
• Retromer is a recently discovered coat
which produces tubular buds with unusual
morphology

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Final Summary
• Sortilin and the mannose-6-phosphate receptor are
transported from the Golgi apparatus to late
endosomes via a pathway that depends on clathrin
and AP-3.
• In the acidic pH of the late endosomes, the
lysosomal enzyme is released from the receptor
• The receptor is returned to the Golgi apparatus via
retromer-coated vesicles

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