20250205_Trafficking Lecture 4

Intracellular Trafficking Overview

• Lecture by Dr. Katie Cockburn, focusing on cellular mechanisms for material transport within cells.

• Fundamentals outlined in "Molecular Biology of the Cell (7th Edition): Chapter 13."

Rabs and Tethers

• CORVET and HOPS:

  • Types of endosome tethers involved in membrane dynamics.

  • Facilitate positive and negative feedback loops for Rab5/7 activation.

SNAREs and Vesicle Fusion

• Function of SNAREs:

  • Mediate vesicle fusion between membranes.

  • Key components include:

    • v-SNARE: Synaptobrevin

    • t-SNARE: Syntaxin-1, SNAP25

• Vesicle Fusion Mechanism:

  • NSF (N-ethylmaleimide sensitive factor) separates SNARE complexes using ATP.

Rabs and COP Proteins

• Key Rabs: Sar1 (COP-II) and Arf1 (COP-I).

• Location of GEF:

  • Donor membrane or target membrane (e.g., TRAPPI complex).

• Effectors include tethers, motor proteins, GEFs, GAPs, and kinases.

Endocytosis Pathway

• Path composed of:

  • Plasma membrane → early endosome → multivesicular body → late endosome → lysosome.

• Processes facilitated by microtubule-mediated transport and can include recycling elements.

Receptor Mediated Endocytosis

• Mechanism:

  • Enriches specific macromolecules via CCVs (clathrin-coated vesicles).

  • Endosome membrane invaginates, forming multivesicular bodies.

  • Receptors may recycle back to plasma membrane or further degrade in lysosomes.

Cholesterol Uptake Process

• Steps:

  1. Cholesterol packaged in LDL and recognized by receptors for endocytosis.

  2. Early endosome lowers pH, causing dissociation of LDL and receptors.

  3. LDL directed to lysosome for degradation, while receptors recycled.

Retrograde Traffic

• Involves movement from:

  • Endosomes back to the plasma membrane

  • Endosomes to the trans-Golgi network.

• Utilizes Retromer protein complex to form tubular structures.

Retromer Formation

• Initiation by Rab5-GTP or Rab7-GTP.

• Steps in formation:

  1. Adaptor proteins bind Rab-GTP and cargo.

  2. SNX complex promotes membrane curvature, leading to tube formation.

  3. Fission and Uncoating:

     • Dynamin pinches off tube structures.

     • GAP converts Rab-GTP to Rab-GDP, facilitating uncoating.

Endosome Maturation

• Proteins modified by mono-ubiquitin are marked for endocytosis.

• If ubiquitin is cleaved, proteins can recycle to the plasma membrane; if not, they proceed to lysosome.

Multivesicular Body Formation

• Involves ESCRT complexes:

  • ESCRT-0 captures mono-Ub proteins.

  • ESCRT-I and II push membrane inward, forming vesicle necks.

  • ESCRT-III facilitates membrane bending and vesicle detachment.

Lysosomes

• Formed from MVB fusion with protease-containing vesicles.

• Characteristics:

  • Acidic lumen (pH 5) where proteins unfold, degrading quickly.

  • Maintained by ATP-dependent proton pumps.

Pathways for Lysosomal Degradation

• Include:

  • Endocytosis.

  • Macropinocytosis (fluid intake).

  • Phagocytosis (large particle engulfment).

  • Autophagy (cellular self-digestion).

Autophagy Process

• Large-scale degradation of cellular components.

• Steps:

  1. Initiation: Formation of phagophore from membrane vesicles.

  2. Phagophore grows, eventually enclosing material to form autophagosome.

  3. Fusion with lysosome to digest contents.

Selective Autophagy

• Cargo-specific receptors ensure proper material degradation, utilizing ubiquitination to signal degradation.

• Variability in autophagy types exists based on cargo, cell type, and environmental conditions.

Comparison of Trafficking Mechanisms

• COP-II, COP-I, CCV, Retromer, ESCRT:

  • Differences in initiation, cargo signals, coat dynamics, and fission/uncoating mechanisms.