For anyone having trouble understanding retrotranslocation, quality control in the ER,
and molecular chaperones (from lecture 1), here are two reviews that should help:
Spiess C, Meyer AS, Reissmann S, Frydman J. Mechanism of the eukaryotic chaperonin: protein
folding in the chamber of secrets. Trends Cell Biol. 2004 Nov;14(11):598-604. Review.
Tsai B, Ye Y, Rapoport TA. Retro-translocation of proteins from the endoplasmic reticulum into
the cytosol. Nat Rev Mol Cell Biol. 2002 Apr;3(4):246-55. Review.
March 30, 2006
Pabio552, Lecture 2
1
Lecture 2: Trafficking from ER to Golgi:
Outline:
A. Important concepts in trafficking:
1.
2.
3.
4.
5.
6.
ER as the gateway to the secretory pathway
ER translocation is co-translational
Signals direct translocation across membranes
Conservation of topology
Molecular sorting keeps membranes biochemically distinct
Vesicle targeting involves specific address tags
B. Specific events in vesicular traffic
1.
2.
3.
4.
5.
Definitions
Exocytosis
Lysosomal Sorting
Endocytosis
Vesicle fusion (covered in PM lecture)
March 30, 2006
Pabio552, Lecture 2
2
Trafficking from ER to Golgi:
Outline, cont.:
C. Machinery and Mechanisms of Trafficking
1. Overview
2. Types of coats: Clathrin, Cop I, Cop II
3. Proteins that function with coats
D. Experimental Systems
1.
2.
3.
4.
Cell-free reconstitution of Golgi transport
Biochemical analysis of synaptic vesicle membranes
Genetic dissection of yeast secretion
EM and Fluorescence microscopy
E. Examples from Pathobiology
How viruses exploit trafficking pathways
The lysosomal dogma turned on its head
March 30, 2006
Pabio552, Lecture 2
3
Trafficking : ER to Golgi to Lysosome
A. Important Concepts in Trafficking:
1. The ER as the secretory pathway gateway.
Entry into the ER allows trafficking to
specific compartments including ER, Golgi,
endosomes, lysosomes, plasma membrane
and cell exterior.
2. ER translocation is co-translational in higher
eukaryotic cells.
In contrast, import into ER of yeast (lower
eukaryotes) is post-translational.
In addition, import into nucleus,
mitochondria, and peroxisomes is posttranslational.
Note that flow in the secretory pathway
goes in multiple directions.
March 30, 2006
Pabio552, Lecture 2
The Cell: A Molecular
Approach, 3rd Ed
(Cooper). ASM Press,
2000
4
A 2. ER translocation is co-translational
March 30, 2006
Pabio552, Lecture 2
5
Please review the signal hypothesis and mechanisms of co-translational
translocation in the Alberts’ textbook!
March 30, 2006
Pabio552, Lecture 2
6
Please review the signal hypothesis and mechanisms of co-translational
translocation in the Alberts’ textbook!
March 30, 2006
Pabio552, Lecture 2
7
Trafficking : ER to Golgi to Lysosome
A. Important Concepts in Trafficking:
3. Signals direct translocation across membranes.
”N-terminal signal sequences”: the cannonical example of signals that
mediate trafficking. Entry into the secretory pathway requires a signal
sequence (SS). SS overcome unfavorable energetics in protein transfer
across membranes.
Variations in SS: stop-transfer sequences (signal anchor) & internal
signals.
At the ER: SS allow interaction of nascent chain with SRP & SRP
receptor, leading to cotranslational translocation through the translocon.
Variations in SS directed translocation occurs in bacteria, yeast,
eukaryotes, & in different compartments (i.e. co-translational vs. posttranslational, translocation into other organelles).
Other types of signals are important in trafficking besides SS - to be
covered in upcoming lectures.
March 30, 2006
Pabio552, Lecture 2
8
J. R. Lingappa,
Pabio
Trafficking : ER to Golgi to Lysosome
A. Important Concepts in Trafficking:
552, lecture 2-7
Pro
3. Signals direct translocation across membranes.
How was the critical role of signal sequences demonstrated?
cytosol
Translation of a secreted glycosylated protein in a cell-free system +/tunicamycin (tun), +/- microsomal membranes (mb), +/- protease (pro)
added at various times (t):
tun _
mRNA transcript
Pro _
_
Mb
cytosol
_
t
(min)
AAA
_
_
+
0
_
_
+
30
+
_
+
0
_
+
+
0
microsomes
+aa,
35-S met,
GTP,
cell extract,
(containing
ribosomes,
translation
factors,
other factors)
March 30, 2006
_
_
+
_
_
+
+
30
microsomal
lumen
We’ll fill these lanes in during class
Pabio552, Lecture 2
9
Trafficking : ER to Golgi to Lysosome
A. Important Concepts in Trafficking:
4. Conservation of topology:
i. Membrane sidedness (lumenal vs. cytoplasmic) is maintained throughout
the secretory pathway:
Why? Because there is lateral mobility of lipid & protein in lipid bilayers, but
typically no spontaneous flip-flop across bilayers (this is energetically
unfavorable).
cytoplasm
PM
lumen
TGN
lumen
TV
ER
March 30, 2006
extracellular
Pabio552, Lecture 2
10
Trafficking : ER to Golgi to Lysosome
A. Important Concepts in Trafficking:
4. Conservation of topology:
ii. Lumen = lumen = extracellular space
Why is this? It follows from how the ER evolved in primitive eukaryotes.
Evolution of the Eukaryotic ER
March 30, 2006
Pabio552, Lecture 2
11
Trafficking : ER to Golgi to Lysosome
A. Important Concepts in Trafficking:
4. Conservation of topology:
ii. Lumen = lumen = extracellular space
cytoplasm
PM
lumen
TGN
lumen
TV
ER
March 30, 2006
extracellular
Pabio552, Lecture 2
12
Trafficking : ER to Golgi to Lysosome
A. Important Concepts in Trafficking:
4. Conservation of topology:
cytoplasm
PM
lumen
TGN
lumen
TV
ER
March 30, 2006
extracellular
Pabio552, Lecture 2
13
Trafficking : ER to Golgi to Lysosome
A. Important Concepts in Trafficking:
4. Conservation of topology:
March 30, 2006
Pabio552, Lecture 2
14
Trafficking : ER to Golgi to Lysosome
A. Important Concepts in Trafficking:
5. Molecular sorting keeps membranes in each compartment "biochemically
distinct" despite continuous vesicular traffic between compartments.
Involves balance of forward and backwards transport.
Types of sorting:
A. Selection of specific components during formation of TV
B. Segregation of vesicular container from cargo after fusion.
C. Retrieval of specific components for retrograde transport.
i.e. proteins bearing KDEL and KKXX sequences bind to specific recycling
receptors in the Golgi and are selectively transported back to the ER.
= PM TM protein
ER
TV
Golgi
= Golgi TM protein
= KDEL receptor
= soluble ER protein
= secretory protein
TV = transport vesicle
RTV = retrograde TV
March 30, 2006
RT V
TV
Pabio552, Lecture 2
15
Trafficking : ER to Golgi to Lysosome
A. Important Concepts in Trafficking:
6. Vesicle targeting involves specific address tags that identify donor vesicle
and target organelle.
examples: V-SNARES vs. T-SNARES,
Rab GTPases that are specific for different organelles or vesicles.
The Cell: A Molecular Approach, 3rd Ed (Cooper). ASM Press, 2000
March 30, 2006
Pabio552, Lecture 2
16
Trafficking : ER to Golgi to Lysosome
B. Specific Events in Vesicular Trafficking:
1. Definitions:
Exocytosis - fusion of vesicles derived from TGN with the PM resulting in insertion of
transmembrane proteins into PM or secretion of soluble proteins into extracellular space.
cytoplasm
cytoplasm
PM
TGN
PM
TGN
V
V
extracellular
extracellular
Endocytosis - process by which particles, solutes, membrane proteins (including
receptor-ligand complexes) and lipids are taken up by vesicles from the PM. Also
used by parasites and bacteria to get into the host cell (to be discussed in Nancy
Freitag's upcoming lecture).
Phagocytosis - uptake of pathogens as a defense, clearance of cell debris.
Pinocytosis - uptake of extracellular fluid through endocytosis.
March 30, 2006
Pabio552, Lecture 2
17
Trafficking : ER to Golgi to Lysosome
B. Specific Events in Vesicular Trafficking
2. Exocytosis
a. Events that occur in the ER (review):
Translocation, signal cleavage, N-linked core glycosylation
Proper folding by ER resident chaperones
Post-translational modifications: trimming core sugars, adding GPI anchors
Retention of ER proteins via KDEL sequences
b. Trafficking from ER to Golgi complex:
Transport vesicles (TV): bud from mb of one organelle & fuse with mb of next organelle
Golgi complex: a series of stacked membranes
where proteins from ER are further processed (i.e. glycosylation, trimming, and CHO
addition), & sorted for transport to final destinations: outside cell, PM, or lysosomes
Consists of cis Golgi, Golgi stack (medial and trans), and trans Golgi network (TGN)
Distinct polarity of Golgi: entry via the cis face; exit from the trans face.
TGN
cytoplasm
PM
TV
TV
extracellular
ER
March 30, 2006
Pabio552, Lecture 2
18
Lingappa, Pabio 552, lecture 2-12
Trafficking :
ER to Golgi to Lysosome
B. Specific Events in
Vesicular Trafficking
2. Exocytosis
Direction of traffic:
ER to ERGIC
to cis Golgi
to medial Golgi
to trans Golgi
to trans Golgi network (TGN)
to lysosomes, PM or exterior.
March 30, 2006
The Cell: A Molecular Approach,
3rd Ed (Cooper). ASM Press, 2000
Pabio552, Lecture 2
19
Trafficking : ER to Golgi to Lysosome
B. Specific Event, Vesicular Trafficking
3. Lysosomal sorting
J. R. Lingappa, Pabio 552, Lecture 2-14
The Lysosome
A. Lysosomes:
v Organelle containing enzymes that degrade
proteins, nucleic acids, CHO and lipids.
v Delivery from exterior (via receptor-mediated
endocytosis or phagocytosis) or from ER via
Golgi.
Lysosomal enzymes:
v Acid hydrolases (i.e. cathepsin D),
v Active at pH 5 (pH maintained within lysosomes)
v Inactive at neutral pH. Protects cell in case of
release into the neutral cytoplasm.
v Acid pH maintained by ATP-dependent H+ pump
in membrane.
B. Trafficking of lysosomal enzymes:
1. 14-sugar N oligo-saccharide core added in the
ER to lysosomal enzymes.
2. One mannose and 3 glucoses are removed while
protein is still in the ER.
March 30, 2006
Pabio552, Lecture 2
20
Trafficking : ER to Golgi to Lysosome
3. Lysosomal sorting
B. Trafficking of lysosomal enzymes, cont.:
3. Mannose residues on lysosomal enzymes are phosphorylated creating M-6-P
residues.
enzyme: N-acetyl-glucosamine (GlcNAc) phosph-transferase, which
recognizes a specific conformation (signal patch) present only on lys. enzymes.
site: cis Golgi.
4. Another enzyme removes GlcNac leaving M-6-P residue on lysosomal enzyme.
March 30, 2006
The Cell: A Molecular Approach, 3rd Ed (Cooper). ASM Press, 2000
Pabio552, Lecture 2
21
Trafficking : ER to Golgi to
Lysosome
4. Lysosomal sorting, cont.
B. Trafficking of lysosomal
enzymes, cont.:
5. The M-6-P residue binds to
M-6-P receptors located in
trans Golgi. Binding occurs
at pH = 6.5 - 7 (pH of Golgi),
but not at pH < 6.
6. Clathrin-coated vesicles
bud from trans Golgi, become
uncoated, & fuse with late
endosome.
March 30, 2006
Pabio552, Lecture 2 The Cell: A Molecular Approach, 3
rd
Ed (Cooper). ASM Press, 2000
22
Trafficking : ER to Golgi to Lysosome
4. Lysosomal sorting, cont.
B. Trafficking of lysosomal enzymes,
cont.:
7.
Late endosome (LE) pH = 5.5 so
lyso enzyme is released from M-6-P
receptor. Phosphatase in LE
removes phosphate to prevent
rebinding.
8.
Transport vesicles (TV) transport
enzymes to lysosomes
9.
Some lysosomal enzymes need to
undergo proteolytic cleavage (in
lysosome) to become active
10. A different TV recycles M-6-P
receptor back to trans-Golgi
The Cell: A Molecular Approach, 3rd Ed (Cooper). ASM Press, 2000
March 30, 2006
Pabio552, Lecture 2
23
Trafficking : ER to Golgi to Lysosome
4. Lysosomal sorting
C.
Trafficking PM to lysosome:
1. Receptors take up cargo via receptor-mediated endocytosis (next
lecture), into clathrin-coated endocytic vesicles which bud into cell from
PM and fuse with early endosomes.
2. Early endosomes mature into late endosomes, with recycling of
membrane components back to PM.
3. Late endosomes mature into lysosomes, resulting in lowering pH to
5.5, allowing the lysosomal enzyme activation.
Note: M-6-P receptor is also transported to the PM where it binds
extracellular phosphorylated lysosomal enzymes that are occasionally
secreted. Thus proteins that are accidentally sent to one compartment
can be rescued and brought back to another compartment.
March 30, 2006
Pabio552, Lecture 2
24
Overview of Trafficking to the Lysosome
The Cell: A Molecular Approach, 3rd Ed (Cooper). ASM Press, 2000
March 30, 2006
Pabio552, Lecture 2
25
Trafficking : ER to Golgi to Lysosome
C. Machinery and Mechanisms of Trafficking:
1. Overview of Coats and Adaptors:
a. Two functions of coats:
1. Drive budding: deforms planar into curved.
2. Select cargo destined for forward transport.
b. Three types of coats:
i. clathrin
ii. COP I
iii. COP II
c. Principles of how coats act:
1. Oligomerization of coat proteins into lattices.
2. Connection to specific adaptors.
3. Additional cellular proteins regulate bud formation
and pinching off.
4. Uncoating required for vesicle docking and fusion.
5. Coat components are recycled.
March 30, 2006
Pabio552, Lecture 2
26
Trafficking: ER to Golgi to Lysosome
J. R. Lingappa, Pabio 552, Lecture 2-21
C. Machinery and Mechanisms of Trafficking
2.
Types of coats:
a. Clathrin
Acts during uptake of extracellular molecules at PM in endoctyosis
Acts during lysosomal sorting in the TGN
Structure: three-legged trimer of 3 HC and 3 LC
Oligomerizes to form polyhedral lattice in coated pit
Undergoes rearrangement to form curvature that results in budding
Assembly/disassembly regulated by Hsp70 using ATP hydrolysis
Clathrin Structure
March 30, 2006
Clathrin-Coated Vesicles:
Pabio552, Lecture 2
27
Different Coat Proteins Act at Specific Points in the Secretory Pathway
March 30, 2006
Pabio552, Lecture 2
28
J. R. Lingappa, Pabio 552, Lecture 2-22
Trafficking: ER to Golgi to Lysosome
C. Machinery and Mechanisms of Trafficking
2. Types of Coats :
b. CopI: Made of coatamer subunits.
Mediates retrieval of proteins from Golgi to ER (retrograde transport).
COPI vesicles transport ER resident proteins with KKXX or RRXX signals.
Uses GTP binding protein ARF (as does clathrin).
Note: The drug Brefeldin A inhibits activation of the ARF protein by
inhibiting nucleotide exchange, and thereby inhibits budding of COPI vesicles.
c. CopII: Mediates forward movement of vesicles from ER to Golgi
(anterograde transport).
Regulated by a GTP binding protein Sar1.
Budding of COPII is not inhibited by Brefeldin A (which is specific for Arf).
March 30, 2006
Pabio552, Lecture 2
29
Trafficking : ER to Golgi to Lysosome
C. Cellular Machinery, Coats and Adaptors:
3. Proteins that function with clathrin:
i. Adaptor proteins promote clathrin assembly, linking clathrin to the membrane and
interacting with membrane proteins encoding signals for sorting into CCVs.
ii. GTP-binding proteins (include ARF and Sar1) regulate coat protein binding:
Sar1 or ARF bound to GTP recruits coat proteins to vesicle.
Coat proteins promote bud formation.
After budding occurs, GTP is hydrolyzed to GDP resulting in dissociation of
coat proteins from vesicle.
Guanine Exchange Factors (GEFs) exchange GDP and replace with GTP
March 30, 2006
Pabio552, Lecture 2
30
Trafficking: ER to Golgi to Lysosome
C. Machinery and Mechanisms of Trafficking
3. Proteins that function with coats:
c. Dynamin, a GTPase protein:
Localizes to membrane-bud junction to cause vesicle closure (coated pit
becomes vesicle).
Purified dynamin can constrict vesicles to form long tubelike structures.
Dynamin activity is probably regulated by a kinase-phosphatase cycle.
Other proteins (i.e. amphiphysin) implicated dynamin recruitment from cytosol.
Temperature-sensitive dynamin mutants in drosophila (shibire) undergo
paralysis due to accumulation of long-neck coated pits and failure to
generate coated vesicles in neurosecretory cells.
Model for Dynamin Action
Vesicles in Shibire mutant
March 30, 2006
Pabio552, Lecture 2
31
Trafficking: ER to Golgi to Lysosome
C.
Machinery
4. Summary
March 30, 2006
Pabio552, Lecture 2
The Cell: A Molecular Approach, 3rd Ed (Cooper). ASM Press, 2000
32
Trafficking : ER to Golgi to Lysosome
D. Experimental systems used to study trafficking:
1. Cell-free reconstitution of Golgi transport (i.e. Rothman & colleagues)
Uses cytoplasmic extracts, ER plus Golgi membranes, and de novo synthesis
radiolabeled proteins off mRNA transcripts to study protein trafficking.
Particularly useful for identifying novel cellular machinery.
of
2. Biochemical analysis of different membranes.
3. Genetic dissection of yeast secretion (i.e. Schekman & colleagues).
Sec mutants = yeast mutants defective in various stages of vesicular transport, i.e.
protein secretion, vacuolar transport, or retrieval of ER resident proteins.
Isolation of mutants led to molecular cloning of genes.
4. Electron microscopy; Fluorescence microscopy
Example of
Genetics Used
For Studying
Trafficking
March 30, 2006
Pabio552, Lecture 2
Molecular Biology of the Cell, 4th edition
33
Cell-free systems for studying trafficking
in vitro transcript
+ unlabeled aa,
35-S met,
GTP, ATP
cell extract
- ribosomes
- factors for translation,
translocation, trafficking
microsomes
Molecular Biology of the Cell, 4th edition
March 30, 2006
Pabio552, Lecture 2
34
Trafficking: ER to Golgi to Lysosome E. Examples from Pathobiology:
How Viruses Exploit the Trafficking Pathways of the Cell during Virion Formation
GOLGI
ER
PM
HIV Env
HBV capsid
HIV - Env glycoprotein cotranslationally translocated
into ER as a
transmembrane protein,
trafficks via secretory pathway to Golgi, where it is
cleaved by a furin protease, then moves to PM.
Capsid assembles at PM
HIV virion where Env is present.
Herpesvirus - Capsid
assembly in the nucleus,
two models proposed for
subsequent trafficking: deenvelopment at outer nucl.
mb. vs. exit within a
vesicle.
NUCLEUS
Herpes
HBV - capsid assembly in
the cytoplasm, buds into
ER, exits via secretory
pathway.
Coronavirus
March 30, 2006
ERGIC
Pabio552, Lecture 2
Coronavirus - assembles
by glycoprotein budding
into the ERGIC. Helical
capsid not needed for bud ding to take place.
35
March 30, 2006
Pabio552, Lecture 2
36
Additional Reading on Pathogens and Intracellular Trafficking Pathways
Overviews:
Knodler, L. A., J. Celli, and B. B. Finlay. Pathogenic Trickery: deception of host cell processes. Nature Rev.
Mol. Cell. Bio. 2: 578-588 (2001)(review)
Reprogramming the phagocytic pathway - intracellular pathogens and their vacuoles. Mol. Memb. Biol. 15:
103-121 (1998)
Specific Organisms:
Stegmann, T. Membrane fusion mechanisms: The influenza hemagglytinin paradigm and its implications for
intracellular fusion. Traffic 1:598 (2000).
Portnoy, D. A., V. Auerbuch, and I. J. Glomski. The cell biology of Listeria monocytogenes infection: the
intersection of bacterial pathogenesis and cell-mediated immunity. J. Cell Bio 158:409-414 (2002) (minireview)
Roy, C. R. and L. G. Tilney. The road less traveled: transport of Legionella to the endoplasmic reticulum. J.
Cell Bio 158:415-419 (2002) (minireview)
Russell, D. G., H. C. Mwandumba, and E. E. Rhoades. Mycobacterium and the coat of many lipids. J. Cell Bio
158:421-426 (2002) (minireview)
Andrews, N. W. Lysosomes and the plasma membrane: trypanosomes reveal a secret relationship. J. Cell Bio
158:389-394 (2002) (minireview)
March 30, 2006
Pabio552, Lecture 2
37