Lecture 1: Trafficking from ER to Golgi:

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• Lecture 1 Trafficking from ER to Golgi
• Outline
• A. Review translation and translocation on your
  own
• B. Important concepts in trafficking
• Four types of proteins are made using the
  secretory pathway
• The ER is 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
• C. Specific events in vesicular traffic
• Definitions
• Exocytosis
• Lysosomal Sorting
• Endocytosis
• (Vesicle fusion covered in PM lecture)

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Trafficking from ER to Golgi Outline,
cont. D. Machinery and Mechanisms of
Trafficking 1. Overview 2. Types of coats
Clathrin, Cop I, Cop II 3. Proteins that
function with coats E. 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 F. Additional concepts you need for
Thursdays paper
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Please review protein translation (see the
assigned Alberts textbook chapter).
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Please review the signal hypothesis and
mechanisms of co-translational translocation in
the Alberts textbook and in the assigned review.
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Please review the signal hypothesis and
mechanisms of co-translational translocation in
the Alberts textbook.
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• Trafficking ER to Golgi to Lysosome
• B. Important Concepts in Trafficking
• 1. Translocation into the ER and trafficking
  through the secretory pathway is required for
  four important categories of proteins
• i. Secretory proteins
• ii. Transmembrane proteins at the PM
• iii. Soluble proteins that reside in organelles
  of the secretory pathway (ER, Golgi, vesicles,
  endosomes, lysomes)
• iv. Membrane proteins that reside in organelles
  of the secretory pathway (ER, Golgi, vesicles,
  endosomes, lysomes)

The Cell A Molecular Approach, 3rd Ed (Cooper).
ASM Press, 2000
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• Trafficking ER to Golgi to Lysosome
• B. Important Concepts in Trafficking
• 2. The ER is the gateway to the secretory
  pathway.
• Entry into the ER allows trafficking to specific
  compartments including ER, Golgi, endosomes,
  lysosomes, plasma membrane and cell exterior.

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• Trafficking ER to Golgi to Lysosome
• B. Important Concepts in Trafficking
• 3. 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 is
  anterograde and retrograde.

The Cell A Molecular Approach, 3rd Ed (Cooper).
ASM Press, 2000
9

• Trafficking ER to Golgi to Lysosome
• B. Important Concepts in Trafficking
• 3. ER translocation is co-translational in
  higher eukaryotic cells, cont.

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• Trafficking ER to Golgi to Lysosome
• B. Important Concepts in Trafficking
• 4. Signals direct translocation across
  membranes.
• N-terminal signal sequences (SS) the
  cannonical example of signals that mediate
  trafficking. Entry of proteins into the
  secretory pathway requires a SS to overcome the
  unfavorable energetics in protein transfer across
  membranes.
• Variations in SS during translocation
  stop-transfer sequences (signal anchor)
  internal SS or start transfer sequences.
• At the mammalian ER SS allow interaction of
  nascent chain with SRP SRP receptor, leading to
  co-translational translocation through the
  translocon.
• Variations in translocation directed by SS occur
  in bacteria, yeast, eukaryotes, in different
  compartments (i.e. co-translational vs.
  post-translational, translocation into other
  organelles).
• Other types of signals besides SS are important
  in trafficking - to be covered in upcoming
  lectures.

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Cell-free systems for studying trafficking
Molecular Biology of the Cell, 4th edition
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• Trafficking ER to Golgi to Lysosome
• B. Important Concepts in Trafficking
• 5. Conservation of topology
• i. Membrane sidedness (lumenal or extracellular
  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
• B. Important Concepts in Trafficking
• 5. Conservation of topology
• ii. Lumen lumen extracellular space

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

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• Trafficking ER to Golgi to Lysosome
• B. Important Concepts in Trafficking
• 5. 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
• B. Important Concepts in Trafficking
• 5. Conservation of topology

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• Trafficking ER to Golgi to Lysosome
• B. Important Concepts in Trafficking
• 6. Vesicles are used for transport from one
  compartment to the next in the secretory pathway.
  Molecular sorting keeps membranes in each
  compartment "biochemically distinct" despite
  continuous vesicular traffic between
  compartments.
• Forward transport is balanced by retrograde
  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
• B. Important Concepts in Trafficking
• 7. Vesicle targeting involves specific address
  tags that identify donor vesicle and target
  organelle.
• examples of targeting factors
• Rab GTPases - targeting factors that direct
  vesicles to the right targets.
• Tethering factors Cognate v-SNARES and t-SNARES

The Cell A Molecular Approach, 3rd Ed (Cooper).
ASM Press, 2000
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Trafficking ER to Golgi to Lysosome C.
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. 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
• C. Specific Events in Vesicular Trafficking
• 2. Exocytosis
• a. Events that occur in the ER (next lecture)
• ?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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• Trafficking ER to Golgi to Lysosome
• C. Specific Events in Vesicular Trafficking
• Exocytosis
• Direction of traffic
• ER to ERGIC
• To cis Golgi
• To medial Golgi
• To trans Golgi network (TGN)
• To lysosomes, PM, or exterior

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

• Trafficking ER to Golgi to Lysosome
• C. Specific Events in Vesicular Trafficking
• 3. Lysosomal sorting
• 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.

J. R. Lingappa, Pabio 552, Lecture 2-14
The Lysosome
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• Trafficking ER to Golgi to Lysosome
• Specific Events in Vesicular Trafficking
• 3. Lysosomal sorting
• b. Trafficking of lysosomal enzymes, cont.
• 3. Mannose residues on lysosomal enzymes are
  phosphorylated creating M-6-P.
• 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
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• Trafficking ER to Golgi to Lysosome
• C. Vesicular Trafficking
• 3. Lysosomal sorting, cont.
• c. 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
26

• Trafficking ER to Golgi to Lysosome
• C. Vesicular Trafficking
• 3. Lysosomal sorting, cont.
• c. 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
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Overview of Trafficking to the Lysosome
The Cell A Molecular Approach, 3rd Ed (Cooper).
ASM Press, 2000
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(No Transcript)
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• Trafficking ER to Golgi to Lysosome
• D. 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
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Different Coat Proteins Act at Specific Points in
the Secretory Pathway
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J. R. Lingappa, Pabio 552, Lecture 2-22

• Trafficking ER to Golgi to Lysosome
• D. 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 D.
Cellular Machinery, Coats and Adaptors 3.
Proteins that function with coat proteins
A. 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
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• Trafficking ER to Golgi to Lysosome
• D. Machinery and Mechanisms of Trafficking
• 3. Proteins that function with coats
• B. 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
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Trafficking ER to Golgi to Lysosome E.
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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Properties of membrane lipids and detergents
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Nonionic detergent solubilizes membranes and
proteins but doesnt denature proteins
Ionic detergent denatures proteins
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Nonionic detergent solubilizes membranes and
proteins but doesnt denature proteins
Isolated, solubilized membrane proteins Still
functional, still in complexes, still in their
native state.