Biol-K 324 Lec 14: Protein Trafficking 2

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techniques for studying the secretory pathway

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1. pulse-chase labeling

2. GFP-labeled proteins

3. sec genes

4. cell-free transport assays

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endoglycosidase D

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compartment specific oligosaccharide modifications

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sec genes

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1. found in yeast mutants

2. defective for secretion of proteins at the nonpermissive temperature

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general molecular mechanisms of vesicular traffic

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1. small vesicles form a particular parent organelle

2. fuses with another membrane of a target organelle

3. protein coats that form on these vesicles during the process control the specificity and efficiency of transport

4. v- and t- SNARE proteins bind and provide specificity for transport steps

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general vesicle budding and formation

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1. vesicle budding occurs when a protein complex forms

2. produces membrane curvature

3. eventual pinches off

4. vesicles produces can recognize appropriate target membranes via SNARE proteins

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vesicle coats

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1. COPI

2. COPII

3. Clathrin

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COPII movement

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ER to cis Golgi

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COPI movement

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cis Golgi to ER

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Clathrin movement

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trans Golgi to endosome

plasma membrane to endosome

Golgi to lysosome, melanosome, or platelet vesicles

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GTPase

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1. GTPase switch proteins control coat assembly

2. belong to Ras superfamily

3. activated when they bind to GTP

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GAP

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1. GTPase activating protein

2. inactivate Ras GTPase members

3. induces GTP hydrolysis

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GTPase Sar1

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1. activated by GEF protein Sec12

2. inserts Sar1-GTP into ER membrane

3. assembly of COPII

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COPII assembly

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1. GTPase Sar 1 is activated by GEF protein Sec12

2. inserting Sar1-GTP into ER membrane

3. induced by binding to Sar1-GTP anchors

4. after vesicle assembly, GTPase activity dissociates

5. COPII coat and releases Sar1-GDP from vesicle surface

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targeting sequences

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1. accumulate in coated vesicles

2. SNARE recognition and assembly controls target specificity

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Rab GTPases

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1. regulate vesicle docking at target

2. bound by effectors during vesicle docking

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Rabs

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1. GTPase superfamily swith proteins

2. regulate docking of vesicles with the correct target membrane

3. each Rab appears to bind a specific Rab effector associated with the target membrane

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NSF and alpha-SNAP

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control SNARE disassembly

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KDEL sequence

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1. in ER resident proteins

2. involved in retrograde pathway

3. retrieval sequence

4. recruits missorted ER proteins into retrograde COPI vesicles

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anterograde transport in the Golgi

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1. cargo from the ER arrives at the cis face of Golgi

2. cargo moves from cis to medial trans cisternae of the Golg via cisternal maturation

(rather than vesicular transport)

3. cargo processed in Golgi (N-linked glycolysatioN)

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cisternal maturation

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1. process of anterograde transport

2. depends on resident Golgi enzymes moving by COPI vesicular transport in retrograde direction

3. moves cargo from cis to medial to trans Golgi cisternae

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N-linked glycolysation

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1. produces cisternae specific carbohydrate structures

2. process cargo proteins

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later secretory pathway stages

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1. retrograde transport

2. lysosomal transport

3. late endosome

4. constitutive secretion

5. regulated secretion

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trans-Golgi network

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1. sorting compartment

2. proteins are sorted into different vesicles that are directed to different cell compartments

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what mediates transport?

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clathrin and adapter proteins

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Clathrin

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1. best characterized coat complex

2. composed of triskelion complexes

3. requires dynamin for pinching off

4. functions in vesicle traffic from the trans-Golgi network using AP1

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triskelion complexes

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1. polymerized on budding vesicles

2. composes coat complex (ex; Clathrin)

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different adapter complexes

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identify different cargo, facilitating sorting

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dynamin

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1. required for Clathrin coated vesicle pinching off

2. polymerizes the neck of the clathrin coated budding vesicle

3. mutation: neck polymer is formed

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asymmetric distributions of membrane proteins

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1. in epithelial cells

2. needed for correct ion and solute tranport

3. established by intracellular sorting to domains during biosynthesis and transport

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sorting in polarized epithelial cells

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some proteins are directed from:

1. TGN to apical plasma membrane

2. from TGN to basolateral plasma membrane

3. from TGN to baseolateral plasma membrane then back to apical plasma membrane (via transcytose)

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cell surface receptors

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1. bind ligands

2. induces assembly of clathrin-accessory protein complexes

ex: LDL receptor, transferrin receptor, mannose-6-phosphate receptor

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clathrin/AP2 complexes

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1. make up ~2% of cell surface in some cells

2. in clathrin coated vesicles

3. coat pits for internalization

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receptor-mediated endocytosis

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1. ligand bound receptor initiates formation of clathrin coated pits

2. clathrin coated vesicles pinch off

3. uncoating clathrin coated vesicles produces early endosome

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LDL endocytosis

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1. LDL particles are bound by the LDL receptor on the cell surface

2. LDL particles internalized

3. receptor recognizes teh apolipoprotein B portion of the LDL particle

4. LDL receptor/LDL complex stimulates clathrin coated pit formation

5. endosome acidification causes dissociation of LDL from the receptor

6. LDL receptor is recycled to cell surface

7. LDL is trafficked to the lysosome where the particle is degraded

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LDL receptor mutations

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were identified as the defect in hypercholesterolemia

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transferrin

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protein secreted into the blood that transports iron from the liver and intestine to tissues

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transferrin cycle

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1. transferrin binds iron

2. has high affinity for transferrin receptor

3. internalization occurs via clathrin coated pits

4. transported to late endosomes

5. low pH in endosomes dissociate iron from transferrin

6. apotransferrin recycled via the receptor (neutral pH = low affinity for receptor)

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apotransferrin

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transferrin without iron

low affinity for receptor at neutral pH

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ferrotransferrin

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transferrin binds iron

high affinity for transferrin receptor

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Sample: LDL receptor bind ____________________

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apolipoprotein B

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pulse labeling

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1. with radioactive amino acids

2. can specifically label a cohort of newly made proteins in ER

3. temp-sensitive mutant protein is retained in ER due to misfolding at nonpermissive temp

4. mutant protein will be released as a cohort for transport when cells are shifted to the permissive temp

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cell-free assays

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1. intercompartmental protein transport

2. allow for individual steps of secretory pathway to be identified

3. produce pure transport vesicles

4. test biochemical function of individual transport proteins

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coated vesicle formation

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1. formed by polymerization of cytosolic coat proteins onto a donor membrane (controlled by GTP binding proteins)

2. vesicle buds form using sorting signals

3. recruit cargo proteins into vesicle

4. eventually pinch off from membrane

5. release a complete vesicle

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what happens after vesicle release

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1. coat is shed

2. exposing proteins required for fusion with target membrane

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how are the vesicle coats disassembled?

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triggered by hydrolysis of GTP bound to ARF or Sar1

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v-SNARE

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1. found in vesicular membrane

2. binds to a complex of cognate t-SNARE proteins in target membrane

3. induces fusion of two membranes

4. after fusion, SNARE complex is disassembled in ATP-dependent reaction by other proteins

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COPII components

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1. Sar1

2. Sec23/Sec24 complex

3. Sec13/Sec31 complex

bind to membrane cargo proteins containing di-acidic or other sorting signals

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soluable cargo proteins

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targeted to COPII vesicles by binding to a membrane protein receptor

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KKXX sequence

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1. sorting signal

2. directs membrane proteins into COPI vesicles

3. bind to subunits of COPI coats

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TGN

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1. major branch point in secretory pathway

2. soluble secreted proteins, lysosomes and membrane proteins are segregated into different transport vesicles

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AP complexes and clathrin

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1. compose coats on endocytic vesicles

2. these vesicles bud from trans-Golgi network

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M6P

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1. mannose 6-phosphate

2. when soluble enzymes destined for lysosomes are modified in the cis-Golgi

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M6P receptors

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1. bind proteins bearing M6P residues

2. direct residues to late endosomes

3. receptors and ligand proteins dissociate

4. receptors recylced to Golgi or plasma membrane

5. lysosomal enzymes are delivered to lysosomes

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where are secretory proteins stored? how are they released?

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1. stored in secretory vesicles

2. released via neural or hormonal signaling

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post Golgi proteolytic cleavages

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1. yield mature, active proteins

2. occur in proteins transported through the secretory pathway

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proteolytic maturation

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1. occur in vesicles carrying proteins from the trans-Golgi network to the cell surface

2. via late endosome or lysosome

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GPI anchor

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apical to basolateral sorting signal