Unit 04 Pt2

Unit Overview

  • Title: Intracellular Trafficking & Coats

  • Course: BIOL 331 – Advanced Cell Biology

  • Source: Molecular Biology of the Cell, 6th edition. Alberts B, Johnson A, Lewis J, et al. New York: Garland Science; 2022.

  • Unit Range: Chapter 13, pages 749 - 807

Part 2 Outline of Topics

  • Membrane-bending Proteins: Help deform the membrane during vesicle formation.

  • Cytoplasmic Proteins: Regulate the pinching off and uncoating of coated vesicles.

  • Monomeric GTPases: Control coat assembly.

  • Coat-Recruitment GTPases: Participate in coat disassembly.

  • Diversity of Transport Vesicles: The shape and size are diverse.

  • Rab Proteins: Guide transport vesicles to their target membrane and alter organelle identity.

Membrane-Bending Proteins

  • Clathrin Coat Formation:

    • The force generated by clathrin coat alone is insufficient for vesicle formation.

    • BAR Domains: Membrane-bending proteins with crescent-shaped domains that wedge into the cytoplasmic leaflet to induce curvature.

    • Clathrin assembly nucleates actin filaments to introduce tension for vesicle budding.

Vesicles: Pinching Off and Uncoating

  • Invagination Process:

    • As a clathrin-coated pit forms a bud, proteins including dynamin are recruited to the bud's neck.

    • Dynamin: Facilitates the “pinching off” process by bringing the non-cytosolic leaflets of the membrane together to fuse.

Mutant Analysis: Drosophila

  • Dynamin Deficiency:

    • Studied in the temperature-sensitive shibire mutant causing reversible paralysis due to disrupted clathrin-mediated endocytosis.

    • Neurotransmitter release is inhibited by blocking synaptic vesicle membrane recycling at restrictive temperatures.

Shedding the Clathrin Coat

  • Mechanism:

    • After pinching off, a vesicle sheds its clathrin coat. A co-packaged PIP phosphatase depletes PI(4,5)P2, weakening adaptor protein association.

    • Hsp70: Acts as an uncoating ATPase possibly activated by auxillin.

    • Early stage stabilization of the clathrin coat must occur to enable vesicle formation.

GTPase Regulation in Coat Assembly

  • Role of GTPases:

    • Coat recruitment GTPases regulate assembly of clathrin, COPI, and COPII coats.

    • Key proteins: GTP-bound forms are active, while GDP-bound forms are inactive, regulated by GEFs (guanine nucleotide exchange factors) and GAPs (GTPase activating proteins).

Coat Recruitment GTPases

  • Monomeric GTPases:

    • Arf proteins and Sar1 proteins associated with COPI and COPII coats respectively.

    • Activation: Sar1-GEF embedded in ER membrane activates SAR1 by exchanging GDP for GTP, leading to conformational change that inserts Sar1-GTP into ER membrane, initiating COPII assembly.

Formation of COPII-Coated Vesicle

  • First Layer Assembly:

    • GTP-bound Sar1 recruits two COPII proteins, Sec23 and Sec24, which binds the cytoplasmic tails of cargo receptors.

  • Second Layer Formation:

    • Additional COPII proteins, Sec13 and Sec31, form an outer shell, structurally similar to clathrin.

COPII Coat Disassembly

  • Mechanistic Understanding:

    • GTP hydrolysis mechanism for Sar1-GTP remains unknown, possibly involves Sar1 itself.

    • COPII coats are more stable than clathrin, and their disassembly requires successful vesicle formation that ‘outruns’ the disassembly process.

Vesicle Shape Diversity

  • Vesicle Variability:

    • Not all vesicles are spherical; for instance, collagen constructs long rod-like structures that cannot fit into standard vesicles.

Tubular Transport

  • Live Imaging Findings:

    • Endosomes and trans-Golgi network observed sending out long tubules, important for transporting membrane proteins due to a higher surface area-to-volume ratio.

Vesicle Targeting Mechanisms

  • Transport Pathways: All proteins begin in the ER and are selectively packaged into vesicles via adaptor proteins.

  • Vesicle Identification: Markers on transport vesicles allow them to recognize target membranes via complementary receptors, involving Rab and SNARE proteins for specificity and fusion.

Rab Protein Role

  • Functionality: Rabs are monomeric GTPases crucial for vesicular transport specificity, acting as molecular markers to identify vesicles and target organelles.

  • Rab-GTP Activation: Cycles between inactive Rab-GDP and active Rab-GTP forms, playing vital roles in membrane tethering and vesicle transport.

Tethering, Docking, and Fusion

  • Mechanism: Active Rab proteins initiate contacts between membranes through Rab effectors, with SNARE proteins facilitating final docking and fusion.

Binding at Endosomal Membrane

  • Rab5 Activation: Rab5-GEF activates Rab5, which in turn activates PI3-kinase, leading to the recruitment of effectors and the creation of specialized membrane patches for vesicles.

Summary of Key Questions

  • What roles do dynamin and coat recruitment proteins play?

  • Explain the COPII vesicle assembly and the role of small GTPases.

  • Do transport vesicles always retain a spherical shape?

  • How do vesicles find their targets post-coating? Define Rab proteins.