For anyone having trouble understanding retrotranslocation, quality control in the ER, and molecular chaperones (from lecture 1), here are two reviews that should help:

1
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 Nov14(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
Apr3(4)246-55. Review.
2

• Lecture 2 Trafficking from ER to Golgi
• Outline
• Important concepts in trafficking
• 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
• Specific events in vesicular traffic
• Definitions
• Exocytosis
• Lysosomal Sorting
• Endocytosis
• Vesicle fusion (covered in PM lecture)

3
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. Cell-free reconstitution of Golgi
transport 2. Biochemical analysis of synaptic
vesicle membranes 3. Genetic dissection of
yeast secretion 4. EM and Fluorescence
microscopy E. Examples from Pathobiology How
viruses exploit trafficking pathways The
lysosomal dogma turned on its head
4

• Trafficking ER to Golgi to Lysosome
• 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 post-translational.
• Note that flow in the secretory pathway goes in
  multiple directions.

The Cell A Molecular Approach, 3rd Ed (Cooper).
ASM Press, 2000
5
A 2. ER translocation is co-translational
6
Please review the signal hypothesis and
mechanisms of co-translational translocation in
the Alberts textbook!
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Please review the signal hypothesis and
mechanisms of co-translational translocation in
the Alberts textbook!
8

• 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.
  post-translational, translocation into other
  organelles).
• Other types of signals are important in
  trafficking besides SS - to be covered in
  upcoming lectures.

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Well fill these lanes in during class
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• 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).

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• Trafficking ER to Golgi to Lysosome
• 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
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• Trafficking ER to Golgi to Lysosome
• Important Concepts in Trafficking
• 4. Conservation of topology
• ii. Lumen lumen extracellular space

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• Trafficking ER to Golgi to Lysosome
• Important Concepts in Trafficking
• 4. Conservation of topology

14

• Trafficking ER to Golgi to Lysosome
• Important Concepts in Trafficking
• 4. Conservation of topology

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• Trafficking ER to Golgi to Lysosome
• 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.

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• Trafficking ER to Golgi to Lysosome
• 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
17
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.
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.
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• Trafficking ER to Golgi to Lysosome
• 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.

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The Cell A Molecular Approach, 3rd Ed (Cooper).
ASM Press, 2000
20

• Trafficking ER to Golgi to Lysosome
• Specific Event, Vesicular Trafficking
• 3. Lysosomal sorting
• 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.

J. R. Lingappa, Pabio 552, Lecture 2-14
The Lysosome
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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.
The Cell A Molecular Approach, 3rd Ed (Cooper).
ASM Press, 2000
22

• Trafficking ER to Golgi to Lysosome
• 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 lt 6.
• 6. Clathrin-coated vesicles bud from trans
  Golgi, become uncoated, fuse with late
  endosome.

The Cell A Molecular Approach, 3rd Ed (Cooper).
ASM Press, 2000
23

• Trafficking ER to Golgi to Lysosome
• Lysosomal sorting, cont.
• B. Trafficking of lysosomal enzymes, cont.
• Late endosome (LE) pH 5.5 so lyso enzyme is
  released from M-6-P receptor. Phosphatase in LE
  removes phosphate to prevent rebinding.
• Transport vesicles (TV) transport enzymes to
  lysosomes
• Some lysosomal enzymes need to undergo
  proteolytic cleavage (in lysosome) to become
  active
• A different TV recycles M-6-P receptor back to
  trans-Golgi

The Cell A Molecular Approach, 3rd Ed (Cooper).
ASM Press, 2000
24

• Trafficking ER to Golgi to Lysosome
• 4. Lysosomal sorting
• Trafficking PM to lysosome
• 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.
• Early endosomes mature into late endosomes, with
  recycling of membrane components back to PM.
• 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.

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Overview of Trafficking to the Lysosome
The Cell A Molecular Approach, 3rd Ed (Cooper).
ASM Press, 2000
26
(No Transcript)
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• Trafficking ER to Golgi to Lysosome
• Machinery and Mechanisms of Trafficking
• 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

J. R. Lingappa, Pabio 552, Lecture 2-21
Clathrin Structure
Clathrin-Coated Vesicles
28
Different Coat Proteins Act at Specific Points in
the Secretory Pathway
29
J. R. Lingappa, Pabio 552, Lecture 2-22

• Trafficking ER to Golgi to Lysosome
• 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).

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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
31

• Trafficking ER to Golgi to Lysosome
• 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
32

• Trafficking ER to Golgi to Lysosome
• Machinery
• 4. Summary

The Cell A Molecular Approach, 3rd Ed (Cooper).
ASM Press, 2000
33
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 of radiolabeled proteins
off mRNA transcripts to study protein
trafficking. Particularly useful for identifying
novel cellular machinery. 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
Molecular Biology of the Cell, 4th edition
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Cell-free systems for studying trafficking
Molecular Biology of the Cell, 4th edition
35
Trafficking ER to Golgi to Lysosome E.
Examples from Pathobiology
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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) Reprogr
amming 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 1598
(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 158409-414 (2002)
(minireview) Roy, C. R. and L. G. Tilney. The
road less traveled transport of Legionella to
the endoplasmic reticulum. J. Cell Bio
158415-419 (2002) (minireview) Russell, D. G.,
H. C. Mwandumba, and E. E. Rhoades.
Mycobacterium and the coat of many lipids. J.
Cell Bio 158421-426 (2002) (minireview) Andrews
, N. W. Lysosomes and the plasma membrane
trypanosomes reveal a secret relationship. J.
Cell Bio 158389-394 (2002) (minireview)