Lecture 8 – Intracellular Trafficking & Vesicular Transport

Intracellular Trafficking Machineries

  • Three major coat systems assemble vesicles and sculpt membranes
    • Clathrin – endocytosis, TGN→endosome traffic
    • COPI – retrograde Golgi→ER & intra-Golgi transport
    • COPII – anterograde ER→Golgi export
  • Characteristic cage diameter ≈ 100 nm

Vesicular Transport – Historical Context

  • 2013 Nobel Prize in Physiology & Medicine (J. Rothman, R. Schekman, T. Südhof)
    • Rothman elucidated how vesicles fuse with specific membranes ensuring cargo is delivered to the correct organelle/cell surface
    • Clinical relevance: neurotransmission, hormone release, immune responses

Two Core Questions in Protein Targeting

  • Directionality – Rab GTPases
    • Select the correct destination membrane
  • Fusion – SNAREs
    • Drive membrane merger once the vesicle arrives

The Rab GTPase Family – Molecular Postal Codes

  • >70 human Rab isoforms act as organelle‐specific molecular markers
  • Representative localization table
    • Rab1 – ER/Golgi
    • Rab2 – cis-Golgi network
    • Rab3A – synaptic/secretory vesicles
    • Rab4 / Rab11 – recycling endosome
    • Rab5 – early endosome & plasma membrane
    • Rab6 – medial & trans-Golgi
    • Rab7 – late endosome
    • Rab8 – cilia + clathrin-coated vesicles
    • Rab9 – late endosome ↔ TGN
    • Rab27A – melanosomes & cytotoxic granules

Molecular Cycle

  • GDP-bound form = inactive, soluble, shielded by GDI (GDP Dissociation Inhibitor)
  • Membrane recruitment
    • \text{Rab–GDP} + \text{GEF} \rightarrow \text{Rab–GTP} + \text{GDP}
    • GTP binding exposes a geranylgeranyl lipid anchor → tight membrane association
  • Active Rab–GTP interacts with effector proteins (motors, tethering factors, fusion regulators)
  • Inactivation
    • GAP-stimulated hydrolysis: \text{Rab–GTP} \xrightarrow{\text{GAP}} \text{Rab–GDP} + P_i
    • GDI extracts Rab–GDP back to cytosol for reuse

SNAREs – The Universal Fusion Machine

  • v-SNAREs (vesicle) pair with t-SNAREs (target) forming a trans-SNARE complex
  • Zippering pulls bilayers together → docking → hemifusion → full fusion
  • Post-fusion disassembly
    • NSF + accessory factors + \text{ATP} \rightarrow \text{ADP} + P_i

Clinical Case Study – RAB27A Mutation

  • 6-month-old boy: partial albinism + recurrent infections
    • Genetic screening → RAB27A loss-of-function
  • Guiding questions
    • Are Rab proteins cytosolic or membrane-anchored?
      ▸ Both – cycle between cytosol (GDP-bound) & membrane (GTP-bound)
    • Healthy RAB27A function?
      ▸ Governs exocytosis of lysosome-related organelles (LROs) in immune cells & melanocytes
    • Pathogenesis link
      ▸ Defective melanosome transport ⇒ hypopigmentation (albinism)
      ▸ Failed cytotoxic-granule release by CTLs/NK cells ⇒ immunodeficiency / infections
  • Diseases: Griscelli syndrome type 2 (pigment dilution + HLH) mirrors this molecular defect

Rab27A in Immune Cells (CTLs & NK Cells)

  • Step 1 – Organelle convergence
    • Effector MUNC13-4 tethers Rab11-positive recycling endosomes to Rab7-positive late endosomes / LROs
  • Step 2 – Vesicle fusion after T-cell-receptor (TCR) triggering → perforin & granzymes loaded into exocytic granules
  • Step 3 – Plasma-membrane tethering via SLP1 / SLP2 (Rab27 effectors) → targeted release at immunological synapse

Rab27A in Melanocytes

  • Regulates two distinct stages using different effectors
    1. Actin-based transport of melanosomes to cell periphery
    2. Docking of mature melanosomes to plasma membrane for transfer to keratinocytes
  • Silencing (shRNA) or mutation of RAB27A causes pigment retention in melanocytes and transfer failure

Melanosome Biogenesis – Four Morphological Stages

  • Stage I – endosomal precursor
  • Stage II – appearance of internal fibrils (gp100/PMEL)
  • Stage III – melanin polymer deposition begins
  • Stage IV – fully melanized, ready for exocytosis & transfer to keratinocytes

Sorting Decisions After the Golgi

  • Proteins exit the trans-Golgi network (TGN) via three routes
    1. Signal-mediated diversion to lysosomes via Mannose-6-Phosphate (M6P) receptor
    2. Signal-mediated diversion to secretory vesicles for regulated secretion
    3. Constitutive secretory pathway to plasma membrane (default)

Mannose-6-Phosphate (M6P) Tagging Pathway

  • Two-step reaction in the cis-Golgi catalyzed by GlcNAc-1-phosphotransferase
    1. Transfer of GlcNAc-P onto a terminal mannose residue
    2. Removal of GlcNAc leaving M6P exposed
  • M6P-tagged hydrolases bind the M6P receptor → packaged into clathrin-coated vesicles → early/late endosome
  • Acidic endosomal pH triggers dissociation; receptors recycle via retromer coat

I-Cell (Inclusion-Cell) Disease (ML II/III)

  • Loss-of-function mutations in phosphotransferase
    • Lysosomal enzymes remain unphosphorylated ⇒ missorting to extracellular space
  • Cellular phenotype: densely packed inclusion bodies, multi-systemic pathology
  • Highlights importance of post-translational modifications in protein targeting

Constitutive vs Regulated Secretion

  • Constitutive pathway
    • No sorting signal needed
    • Continuous vesicle fusion replenishes membrane lipids & secretes ECM proteins, antibodies, etc.
  • Regulated pathway
    • Cargo sorted into dense-core secretory vesicles in TGN
    • Fusion blocked until intracellular Ca²⁺ rise or hormonal/neurotransmitter signal
  • Example triggers: acetylcholine for synaptic vesicles, glucose for insulin granules

Vesicle Maturation & Cargo Concentration

  • Immature secretory vesicles bud from TGN containing excess membrane/fluid
  • Clathrin-coated retrieval vesicles recycle surplus components to Golgi/endosome
  • Progressive condensation yields 200 nm dense-core mature granules ready for stimulus-dependent exocytosis

Proteolytic Processing During Maturation – Renin Example

  • Preprorenin mRNA → preprorenin polypeptide synthesized on rough ER
  • Signal peptide cleavage → Prorenin enters ER lumen
  • Glycosylation in Golgi; packaging into constitutive vs regulated carriers
  • In dense-core vesicles, proteases activate renin (angiotensin cascade regulator)
    • Ensures enzyme activity only after exocytosis, preventing intracellular damage

Broader Implications & Connections

  • Trafficking defects underlie diverse diseases: Parkinson’s (Rab35), Charcot-Marie-Tooth (Rab7), Hermansky-Pudlak (LRO biogenesis)
  • Therapeutic prospects
    • Small-molecule Rab modulators to correct trafficking in neurodegeneration
    • Gene therapy for Rab27A-related immunodeficiency/albinism
  • Ethical note: Precision treatments demand equitable access; early genetic testing can prevent life-threatening HLH in Rab27A deficiency