20250203_Trafficking Lecture 3_v2

Intracellular Trafficking Overview

• Title: Intracellular Trafficking

• Course: ANAT212/BIOC212

• Instructor: Katie Cockburn, PhD

• Date: February 3, 2025

• Reference: Molecular Biology of the Cell (7th Edition), Chapter 13

Rab GTPases

• Definition: Large family of compartment-specific GTPases, pivotal in cellular trafficking.

• Key Components:

  • Rab motors

  • Tethers

  • SNARE proteins

  • Effector proteins

• Functions:

  • Vesicle targeting and fusion

  • Involved in various intracellular transport mechanisms.

Tethering Proteins

• Role of Tethers:

  • Rab effectors located at target membranes required for vesicle tethering.

• Components:

  • Cargo receptors

  • v-SNARE and t-SNARE proteins

  • Rab-GTP complexes

• Structure of Tethers:

  • Coiled-coil tethers

  • Multisubunit tethers (e.g., TRAPPI)

  • Interaction with Golgi and other organelles.

Multisubunit Tethers

• Importance:

  • Essential for numerous secretory and endocytic pathways.

• Families:

  • TRAPPI for ER to Golgi and within Golgi

  • CATCHR family for Golgi to plasma membrane

  • CORVET/HOPS for endosomal trafficking.

Exocyst Complex

• Formation:

  • After vesicle uncoating, Rab-GTP binds to a 6-protein complex (exocyst).

  • Completes to an 8-mer when binding additional plasma membrane proteins.

• Function:

  • Assists in SNARE recruitment for membrane fusion; anchored by PI(4,5)P2.

Endocytosis Pathway

• Process:

  • Vesicles traffic between plasma membrane, early endosomes, and Golgi.

  • Early endosome (Rab5) matures into multivesicular body and late endosome (Rab7).

  • Late endosomes can mature into lysosomes.

Endosome Tethers (CORVET/HOPS)

• Fusion Mechanisms:

  • Early endosome fusion with vesicles (Rab5-GTP).

  • Late endosome fusion with lysosomes (Rab7-GTP).

  • Core subunits are conserved while end subunits bind different Rabs.

Clustering of Tethers

• Mechanism:

  • Rab5 effectors have GEF or PI kinase activity, boosting local Rab5-GTP concentrations.

  • PI-phosphates increase binding sites for tethers, forming a microdomain for vesicle landing.

Rab Cascades

• Activation:

  • Rab5-GTP effector (CORVET) activates Rab7.

  • As Rab5 vesicles fuse with early endosomes, Rab7 activation increases while Rab5 decreases.

  • Critical process in endosome maturation.

Rabs

• Example Rabs:

  • Sar1 (COP-II)

  • Arf1 (COP-I)

• Questions to Consider:

  • Where is the GEF located?

  • What are the effectors?

  • When/where does the GAP function?

  • Compare and contrast different Rabs.

Protein Cargo Transport

• Key Questions:

  • How are vesicles formed?

  • How are vesicles accurately transported?

  • How do vesicles fuse with their target compartments?

SNAREs: Targeting and Fusion

• Definition: SNARE proteins facilitate vesicle fusion.

• Recruitment: Rabs and tethers assist in recruiting SNAREs to the fusion site.

• Interaction: v-SNAREs on vesicles interact with t-SNAREs on target membranes to initiate fusion.

Characteristics of SNAREs

• v-SNAREs:

  • Composed of monomers with a single transmembrane domain.

• t-SNAREs:

  • Trimers of transmembrane and peripheral subunits.

• Fusion Mechanics: Multiple SNARE complexes induce vesicle fusion at target sites.

SNARE Folding

• Initial State: v-SNAREs exist as unstable monomers.

• Stable Formation: Folding into a stable 4-helix bundle occurs with t-SNAREs, enabling closer membranes.

• Physical Dynamics: Strain from folding pulls membranes together without using ATP/GTP.

Membrane Fusion

• Process:

  • SNARE complexes encircle the fusion site leading to membrane layers fusing in sequence.

  • SNARE transmembrane domains bend and strain, facilitating membrane interaction.

SNARE Dissociation

• Post-Fusion State:

  • The SNARE complex is inactive and stable post-fusion.

• Recycling: v-SNAREs are recycled back by vesicles, while t-SNAREs are reactivated.

• Role of NSF:

  • NSF dissociates SNAREs, essential for continuous vesicle trafficking.

NSF Mechanism

• Binding: NSF interacts with SNARE complexes via adaptor protein (α-SNAP).

• Function: ATPase activity unwinds SNARE helices through mechanical pulling effects during hydrolysis.

Homotypic Fusion

• Definition: Fusion events where donor and target membranes are identical.

• Examples:

  • COP-II vesicles forming the cis-Golgi

  • Early endosomes fusing

  • Reformation of ER and Golgi post-cell division.

• Importance: Identical SNAREs present in both membranes necessitate NSF for separation post-fusion.

Synaptic Vesicles

• Function: Neurons secrete neurotransmitters using specialized secretory vesicles called synaptic vesicles.

• Conditions for Fusion:

  • Requires specific signaling (e.g., [Ca2+])

  • Must occur rapidly (up to ~1000 times per second).

Synaptic Vesicle Fusion (Part 1)

• Initial Steps: Synaptic vesicles dock and begin forming a SNARE tetramer.

• Complexin Role: Complexin stabilizes SNARE complexes in a metastable state, preventing spontaneous fusion.

Synaptic Vesicle Fusion (Part 2)

• Completion of Fusion:

  • Transition to stable tetramer allows for rapid vesicle fusion upon Ca2+ influx.

  • Displacement of complexin by synaptogamin further initiates fusion.