Unit 04 Pt2

Introduction

  • Overview of Unit 4, part 2 in BIO 331.

  • Previous discussions focused on protein coats on vesicles used for cellular trafficking and transport.

Importance of Membrane Curvature

  • Membrane bending and curvature are critical for vesicle formation (vesiculation).

  • Additional proteins called BAR domains play an essential role in membrane curvature.

BAR Domain Functions

  • BAR domains can form dimers that assist in curvature formation during vesiculation.

  • They contain alpha helices that wedge into the cytoplasmic leaflet to induce membrane curvature.

Vesicle Pinching Off

  • Vesicles must pinch off from donor membranes.

  • This process is facilitated by dynamin proteins.

Dynamin Structure and Function

  • Dynamin proteins have two key domains:

    • PIP binding domain

    • GTPase domain

  • They utilize GTP hydrolysis as energy to facilitate membrane pinching, leading to the fusion of non-cytosolic leaflets.

Historical Context

  • Early research by David Suzuki on Drosophila mutants revealed insights into endocytosis.

  • Mutant forms of dynamin hindered vesicle recycling and neurotransmitter release, causing paralysis.

  • Electron micrographs showed accumulation of vesicles at the membrane due to ineffective pinching off.

Uncoating of Vesicles

  • After pinching off, vesicles shed their coats, typically clathrin, in a rapid manner.

  • This process is aided by the PIP phosphatase co-packaged within vesicles, which weakens the association between adapter proteins and facilitates uncoating.

  • Hsp70 proteins may also function as ATPase for uncoating.

Stability of Coats

  • Different vesicle coats exhibit varying stability, with clathrin-coated vesicles being particularly notable for their stability during formation.

Role of PIP and Regulatory Mechanisms

  • PIP production is crucial for regulating clathrin assembly and coat formation alongside adapter proteins.

  • Other regulatory contexts include coat recruitment GTPases that manage coat assembly on endosomes and Golgi membranes.

GTPase Functionality

  • GDP-binding proteins act as molecular switches:

    • GTP-bound form is active.

    • GDP-bound form is inactive.

  • GTPase-activating proteins (GAPs) facilitate the deactivation of GTP into GDP.

Monomeric GTPases and Vesicle Formation

  • Monomeric GTPases such as SAR1 play a crucial role in vesicular transport and have distinct activating proteins for COPI and COPII vesicles.

  • The transition between GDP and GTP states is essential for their function.

SAR1 Activation Process

  • SAR1 transitions from an inactive GDP-bound state to an active GTP-bound state, leading to a conformational change that allows insertion into the membrane.

  • Active SAR1 recruits additional proteins (Sec23 and Sec24) that further aid in coat assembly and vesicle formation.

COPII Coat Assembly

  • COPII coat assembly involves additional proteins (Sec13 and Sec31) forming an outer shell, analogous to clathrin structures.

  • The transition between active and inactive states (GTP to GDP) is regulated through GTPase activity.

Assembly vs. Disassembly

  • The assembly of vesicle coats must outrun disassembly to ensure efficient trafficking of vesicles.

  • COPII coats are generally more stable compared to clathrin and COPI coats, with slower shedding post-pinching.

Tubular Vesicle Formation

  • Vesicular structures can vary in shape, with examples like tubular vesicles forming around linear proteins such as collagen.

  • Live cell imaging studies exhibit the formation of these tubular structures from the trans Golgi network.

RAB Proteins and SNAREs

  • RAB proteins act as markers for specific vesicular transport and are key players in identifying destination compartments.

  • SNARE proteins mediate the membrane fusion process at target sites.

RAB Protein Functionality

  • RAB proteins have distinct roles in facilitating docking and fusion of vesicles, utilizing GEFs for activation.

  • RAB proteins stay bound to GDP when inactive and are tethered by GDI for solubility.

Interactions and Complexity of RAB Proteins

  • The interplay of RAB proteins with effectors and SNAREs highlights the complexity of vesicular transport.

  • Specific examples show RAB proteins binding to multiple effector proteins, showcasing functional diversity.

Conclusion

  • Summary of vesicular transport mechanisms and the specificity involved in trafficking to various cellular compartments.