Lecture 18 - Vesicular Transport of Secretory Proteins

mechanism through which proteins synthesized by ribosomes on the ER is delivered to the plasma membrane, lysosome or secreted out of the cells with the help of vesicles

secretory pathway

delivers proteins from ER to the cell membrane and to organelles that lie along the secretory pathway (ER, Golgi, endosome, lysosome)

secretory pathway

Series of interconnected, closed, membrane-bound organelles and vesicles

Endomembrane System

nuclear envelope

endoplasmic reticulum

Golgi apparatus

Lysosome

Vesicles

Endosomes

plasma membrane

Endomembrane System

Works together to modify, package and transport Lipids and proteins

Important in trafficking cellular contents from one location to another

Endomembrane System

Transport of proteins happens from one membrane bound organelle to another via coated membrane bound vesicles

Transport vesicles

As transport vesicles bud from one membrane and fuse with the next, the same face of the membrane remains oriented towards what

the cytosol

Once a protein has been inserted into the membrane or the lumen of the ER, that protein can be carried along what without being translocated across another membrane or altering its orientation within the membrane

secretory pathway

How do you determine the Fate of secretory proteins

Pulse chase experiment

Established the order in which proteins move along the secretory pathway

Showed that proteins are transported in membrane bound vesicles

Pulse chase experiment

Add radioactive amino acid (e.g., radioactive leucine)

Pulse

The squiggles show radioactive proteins

3 minutes pulse

Majority of radioactive proteins have moved to the periphery of the Golgi complex

7 minutes pulse

Radioactive proteins are in vesicles moving towards the plasma membrane

37 minutes pulse

Radioactive proteins are in the extracellular space

117 minutes pulse

A way to label a group of proteins in an early compartment so that their transfer to later compartments can be followed over time

A way to identify the compartment where the labelled protein resides

Requirements for an assay to study inter-compartmental transport

Viral protein that mediates the fusion between the virus envelope and the host cell membrane

Vesicular stomatitis virus glycoprotein

When VSV-G is expressed in mammalian cells it inserts into the

ER membrane

At what degree is mutant VSV-G is unable to fold

40C

Shift to 32°C mutant VSV-G-GFP folds properly (still in the ER)

0 minutes

most mutant VSV-G–GFP concentrated in the Golgi

40 minutes

most mutant VSV-G–GFP is at the cell surface (plasma membrane)

180 minutes

Studies in which cells helped identify proteins that are important in vesicle trafficking

yeast cells

Several temperature sensitive yeast mutants were identified that were unable to secrete proteins at non-permissive (higher) temperatures

sec mutants

Accumulation in the cytosol

Transport into ER

Class A

Accumulation in rough ER

Budding of vesicles from the rough ER

Class B

Accumulation in ER to Golgi transport vesicles

Fusion of transport vesicles with Golgi

Class C

Accumulation in Golgi

Transport from Golgi to secretory vesicles

Class D

Accumulation in secretory vesicles

Transport from secretory vesicles to cell surface

Class E

Characterization of which mutants helped identify components and molecular mechanisms of vesicle trafficking

sec mutants

How will you identify the order of genes involved in the secretory pathway using yeast mutants?

Make double sec mutants

Accumulation in rough ER

Budding of vesicles from the rough ER

Gene X

Accumulation in secretory vesicles

Transport from secretory vesicles to cell surface

Gene Y

Accumulation in rough ER

Budding of vesicles from the rough ER

Masking occuring

Gene X & Y

Gene X is involved in earlier or later stages compared to Gene Y

earlier

Gene ? -- > ER-Golgi-transport vesicles

Y

ER-Golgi-transport vesicles ----> Gene ?

X

Gene ? --- > secretory vesicles ----> outside the cells

X

Membrane bound vesicles are the fundamental functional elements in which pathways

secretory and endocytic

to the membrane of the target organelle

Fuse

from the membrane of the parent or donor organelle

Bud

At each step they employ a which type of vesicle

different

transport proteins from the ER to the Golgi

COP II vesicles

transport proteins from the plasma membrane (cell surface) and the trans-Golgi network to late endosomes

Clathrin-coated vesicles

mainly transport proteins in the retrograde

direction between Golgi cisternae and from the cis-Golgi back to the ER

Retrograde between Golgi Cisternae and from Golgi to ER

COP I vesicles

Assembly of the different coat proteins to form the vesicle

Pinching off of completed vesicle from parent membrane

GTPases

GTPase: ARF protein

COP I and Clarithan

GTPase: Sar1 protein

COP II

only GTPase protein activated at the ER membrane

Sar1

Only which vesicles bud from ER membrane

COPII-coated

ARF-GDP weakly tethers to the membrane with the help of a myristate anchor on the

N-terminus

What will happen if a mutation in Sar1 or ARF prevents GTP hydrolysis

No disassembly of coat protein

Vesicles cannot fuse at target membrane (as v-SNAREs are not accessible)

What is needed for pinching of buds

GTPase

How else can you cause vesicles to not dissociate

Non-hydrolyzable GTP

COP II

Anterograde

COP I

Retrograde

Vesicle bud needs to distinguish between

Membrane and soluble cargo proteins

Cytoplasmic Sorting Signals

Signal-Bearing Protein: ER-resident membrane proteins

Proteins with Signal: COPI α and β subunits

Signal Receptor: COPI

Lys-Lys-X-X (KKXX)

Cytoplasmic Sorting Signals

Signal-Bearing Protein: ER-resident membrane proteins

Proteins with Signal: COPI α and β subunits

Signal Receptor: COPI

Di-arginine (X-Arg-Arg-X)

Cytoplasmic Sorting Signals

Signal-Bearing Protein: Cargo membrane proteins in ER

Proteins with Signal: COPII Sec24 subunit

Signal Receptor: COPII

Di-acidic (e.g., Asp-X-Glu)

Cytoplasmic Sorting Signals

Signal-Bearing Protein: LDL receptor in plasma membrane

Proteins with Signal: AP2 complex

Signal Receptor: Clathrin/AP2

Asn-Pro-X-Tyr (NPXY)

Cytoplasmic Sorting Signals

Signal-Bearing Protein: Membrane proteins in trans-Golgi

Proteins with Signal: AP1 (μ1 subunit)

Signal Receptor: Clathrin/AP1

Tyr-X-X-Φ (YXX Φ)

Cytoplasmic Sorting Signals

Signal-Bearing Protein: Plasma membrane proteins

Proteins with Signal: AP2 complexes

Signal Receptor: Clathrin/AP2

Leu-Leu (LL)

Luminal Sorting Signals

Signal-Bearing Protein: ER-resident soluble proteins

Proteins with Signal: KDEL receptor in cis-Golgi membrane

Signal Receptor: COPI

Lys-Asp-Glu-Leu (KDEL)

Luminal Sorting Signals

Signal-Bearing Protein: Soluble lysosomal enzymes after processing in cis-Golgi

Proteins with Signal: M6P receptor in trans-Golgi membrane

Signal Receptor: Clathrin/AP1

Mannose 6-phosphate (M6P)

small GTP-binding proteins

Rab GTPases

Key regulators of fusion of transport vesicles to the target membrane

Rab GTPases

isoprenoid anchor that allows them to become tethered to the transport vesicle membrane

Rab proteins

Activated Rab binds to different proteins called what that leads to docking of the vesicle to the appropriate target membrane

Rab effectors

Every type of transport vesicle is labeled with one or more specific

Rab proteins

SNAP RECEPTOR

SNARE

How many members in yeast SNARE proteins

24

more than ? members in mammals SNARE proteins

60

Responsible for fusion of the vesicle with the target membrane

SNARE proteins

Present on the transport vesicle. Incorporated during vesicle formation

v-SNAREs

Present on the target membrane to which the vesicle is docked

t-SNAREs