Study Notes on Vesicular Trafficking and GTPases

Vesicular Trafficking and the Roles of GTPases

Introduction

  • Overview of vesicular trafficking and GTPases as key regulators.

Protein Trafficking from the Endoplasmic Reticulum (ER)

  • Once proteins are folded in the ER, they can go to multiple destinations:

    • Nuclear Envelope

    • Lysosome

    • Endoplasmic Reticulum

    • Late Endosome

    • Early Endosome

    • Cytosol

    • Cisternae Secretory Vesicle

    • Golgi Apparatus

    • Plasma Membrane

Basics of Vesicular Trafficking

  • Most trafficking steps necessitate a vesicle for transport:

    • Exocytosis: The process of trafficking lipids and proteins to the cell surface.

    • Endocytosis: The uptake of lipids and proteins from the cell surface.

    • Importance of balance between exocytosis and endocytosis. (Further explanation required for 'why')

Specifics of Exocytosis

  • Example:

    • Exocytosis of a GFP-marked protein from the Golgi complex to the cell surface.

Steps in Vesicular Transport

  • Overview of the sequence of events involved in vesicular transport:

    1. Cargo Recruitment

    2. Vesicle Formation:

    • a) Budding

    • b) Scission

    1. Transport and Targeting

    2. Tethering

    3. Fusion

1. Cargo Selection and Recruitment

  • Cargo selected based on:

    • Lumenal proteins are bound by specialized cargo receptor proteins.

    • Transmembrane proteins are recruited to sites of vesicle formation (ER lumen and cytoplasm).

2. Vesicle Formation

  • Process involved in vesicle budding:

    • Clue found in appearance of “coated” vesicles.

Clathrin Proteins

  • Function as coat proteins:

    • Form triskelion structures composed of 3 heavy chains and 3 light chains.

    • Triskelions serve as units for higher-order clathrin assembly leading to basket formation.

3. Cargo and Coat Protein Interaction

  • How cargo associates with coat proteins:

    • Adaptor Proteins bind to cargo receptors.

    • Adaptor proteins help recruit coat proteins.

    • Sequence of events in cargo recruitment:

    1. Cargo receptor proteins bind to specific proteins for trafficking.

    2. Adaptor proteins bind to the cargo receptor proteins.

    3. Adaptor protein binding concentrates cargo receptors, thus recruiting cargo to budding vesicles.

    4. Adaptor proteins recruit clathrin for basket formation.

4. Scission Process

  • Separation of vesicle from membrane is distinct from basket formation:

    • Clathrin baskets cannot sever the lipid bilayer.

    • Dynamin is recruited to the bud neck and cuts the neck to free the vesicle.

GTPases Overview

  • GTPase proteins exist in two states:

    • Active (GTP-bound)

    • Inactive (GDP-bound)

  • Guanine Exchange Factors (GEFs) promote activation of GTPases.

  • GTPase-Activating Proteins (GAPs) promote inactivation.

Regulation of Coat Formation by GTPases

  • GTPases such as Arf and Sar1 play crucial roles:

    • Arf proteins regulate clathrin and COPI assembly.

    • Sar1 proteins regulate COPII assembly.

    • Cargo receptor proteins function as GEF or GAP for Sar1 and Arf proteins.

    • Sar1-GTP extends an amphipathic helix, aiding in the recruitment of adaptor and coat proteins.

Timers of Coated Vesicles

  • GTPases function as timers:

    • Eventually, Sar1 and Arf will hydrolyze GTP, return to their inactive states, retract the amphipathic helix, leading to disassembly of the coat and adaptor proteins from the vesicle.

5. Vesicle Transport and Targeting

  • Mechanism of guidance to target organelles via Rab proteins:

    • Act as address labels for vesicles.

    • GTP binding makes amphipathic helix accessible (similar to Sar1).

    • Over 60 types of Rab proteins exist, each targeting specific organelles.

    • Rab proteins facilitate interaction with:

      • Motor proteins for intracellular movement.

      • Tethering proteins for localization near target compartment.

Different Rab Proteins

  • Guide movement of vesicles to various organelles, such as:

    • RAB8: Cilium

    • RAB3: Secretory vesicles

    • RAB5: Early endosome

    • RAB6: Golgi

    • RAB7: Late endosome

    • RAB1: TGN

    • RAB14 and others for various specific cellular functions.

6. Vesicle Tethering

  • Tethering proteins help recognize vesicles by their target compartments:

    • Tethering proteins attach to target compartments and form long multi-protein complexes that extend into the cytoplasm.

    • Tethering proteins specifically bind to Rab proteins, effectively localizing vesicles near the target membrane.

7. Vesicle Fusion

  • Fusion of lipid bilayers facilitated by SNAREs:

    • Comprises of v-SNAREs (vesicle) and t-SNAREs (target).

    • One v-SNARE interacts with three t-SNAREs to form a 4-helix bundle.

    • Formation of the bundle drives bilayer apposition and water displacement between membranes, enabling fusion.

Conclusion: Influenza Virus Mechanism

  • Influenza employs both endocytic and exocytic mechanisms to enter host cells:

    • Key components include envelope glycoproteins and viral RNA.

    • Viral proteins enable escape from vesicles into the host cytoplasm, ensuring successful infection.