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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 ... – PowerPoint PPT presentation

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Title: Lecture 1: Trafficking from ER to Golgi:


1
  • 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)

2
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
3
Please review protein translation (see the
assigned Alberts textbook chapter).
4
Please review the signal hypothesis and
mechanisms of co-translational translocation in
the Alberts textbook and in the assigned review.
5
Please review the signal hypothesis and
mechanisms of co-translational translocation in
the Alberts textbook.
6
  • 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
7
  • 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.

8
  • 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.

10
  • 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.

11
(No Transcript)
12
Cell-free systems for studying trafficking
Molecular Biology of the Cell, 4th edition
13
  • 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).

14
  • Trafficking ER to Golgi to Lysosome
  • B. Important Concepts in Trafficking
  • 5. Conservation of topology
  • ii. Lumen lumen extracellular space

15
  • Trafficking ER to Golgi to Lysosome
  • B. Important Concepts in Trafficking
  • 4. Conservation of topology

16
  • 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
17
  • Trafficking ER to Golgi to Lysosome
  • B. Important Concepts in Trafficking
  • 5. Conservation of topology

18
  • 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.

19
  • 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
20
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.
21
  • 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.

22
  • 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
24
  • 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
25
  • 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
27
Overview of Trafficking to the Lysosome
The Cell A Molecular Approach, 3rd Ed (Cooper).
ASM Press, 2000
28
(No Transcript)
29
  • 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
30
Different Coat Proteins Act at Specific Points in
the Secretory Pathway
31
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).

32
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
33
  • 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
34
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
35
Properties of membrane lipids and detergents
36
Nonionic detergent solubilizes membranes and
proteins but doesnt denature proteins
Ionic detergent denatures proteins
37
Nonionic detergent solubilizes membranes and
proteins but doesnt denature proteins
Isolated, solubilized membrane proteins Still
functional, still in complexes, still in their
native state.
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