Chapter 14 - Vesicular Traffic, Secretion, and Endocytosis

Chapter 14 Overview

• Vesicular Traffic, Secretion, and Endocytosis
  • Labels track protein movement.
  • Yeast temperature-sensitive secretory (sec) mutants identify intracellular protein trafficking components.
  • Cell-free assays define secretory pathway steps.

Secretory and Endocytic Pathways

• Protein trafficking unifying principle: transport vesicles move membrane and soluble proteins between membrane-bounded compartments.
• Transport vesicles:
  • Collect cargo proteins during membrane budding from a donor compartment.
  • Deliver cargo proteins to the next compartment via fusion with the target membrane.

Secretory Pathway

• Distribution of soluble and membrane proteins from the rough ER to final destinations (cell surface, lysosomes).
• Two stages:
  • Stage 1: Rough endoplasmic reticulum
    • Step 1: Synthesis of proteins with ER signal/targeting sequence; cotranslational insertion into ER membrane or lumen.
  • Stage 2: Protein trafficking
    • Step 2: Proteins packaged into transport vesicles that bud from the ER and fuse to form cis-Golgi cisternae.
    • Step 3: ER enzymes or structural proteins are retained in the ER or retrieved by vesicles from the cis-Golgi.
    • Step 4: Each cis-Golgi cisterna and its contents move from the cis to the trans face via cisternal maturation.
    • Step 5: Retrograde transport vesicles move Golgi-resident proteins to previous Golgi compartments.
    • Step 6: Constitutive secretion (all cells): transport vesicles fuse continuously with the plasma membrane.
      • Soluble proteins are continuously secreted.
      • Membrane proteins become plasma membrane proteins.
    • Step 7: Regulated secretion (certain cell types):
      • Proteins accumulate and are stored in regulated secretory vesicles.
      • Vesicles fuse with the plasma membrane upon a neuronal or hormonal signal.
    • Step 8: Lysosome-destined proteins are transported in vesicles from the trans-Golgi to late endosomes, then to lysosomes.

Endocytic Pathway

• Step 9: Vesicles bud from the plasma membrane, take up soluble extracellular proteins, and deliver them to lysosomes via late endosomes.

Vesicle Budding and Fusion

• Three types of coated vesicles mediate protein transport.
• Small GTPase proteins direct coat protein polymerization.
• Coat shedding exposes Rab and SNARE proteins for target membrane fusion.

Vesicle Formation and Fusion

• Vesicles bud from a donor membrane and fuse with a specific target membrane.
• Protein coat assembly drives vesicle formation and cargo selection.
• (a) Vesicle budding from donor membrane:
  • Recruitment of GTP-binding G proteins.
  • Cytosolic coat protein complexes bind to the cytosolic domain of membrane cargo proteins.
  • Coat binding evaginates the membrane (vesicle diameter ~50 nm).
  • Cargo proteins act as receptors to bind soluble proteins.
  • Donor membrane-specific SNARE proteins (v-SNAREs) captured in the budding vesicle membrane.
  • Donor membrane fusion pinches off the coated vesicle.
  • Coated vesicle uncoated in cytosol, exposing v-SNAREs.
• (b) Vesicle fusion with target membrane:
  • Targeting: v-SNARE interaction with target membrane t-SNAREs.
  • Fusion.

Coated Vesicles (Table 14-1)

• COPII
  • Transport Step Mediated: ER to cis-Golgi
  • Coat Proteins: Sec23/Sec24 and Sec13/Sec31 complexes, Sec16
  • Associated GTPase: Sar1
• COPI
  • Transport Step Mediated: cis-Golgi to ER; Later to earlier Golgi cisternae
  • Coat Proteins: Coatomer containing seven different COP subunits
  • Associated GTPase: ARF
• Clathrin and adapter proteins
  • trans-Golgi to endosome: Clathrin + AP1 complexes, ARF
  • trans-Golgi to endosome: Clathrin + GGA, ARF
  • Plasma membrane to endosome: Clathrin + AP2 complexes, ARF
  • Golgi to lysosome, melanosome, or platelet vesicles: AP3 complexes, ARF
  • Note: AP complexes have four subunits; AP3 vesicle coat may or may not contain clathrin

Sorting Signals (Table 14-2)

• LUMINAL SORTING SIGNALS
  • Lys-Asp-Glu-Leu (KDEL): ER-resident soluble proteins, KDEL receptor in cis-Golgi membrane, COPI
    KDEL receptor in cis-Golgi membrane
  • Mannose 6-phosphate (M6P): Soluble lysosomal enzymes after cis-Golgi processing, M6P receptor in trans-Golgi membrane, Clathrin/AP1
  • Secreted lysosomal enzymes: M6P receptor in plasma membrane, Clathrin/AP2
• CYTOPLASMIC SORTING SIGNALS
  • Lys-Lys-X-X (KKXX): ER-resident membrane proteins, COPI α and β subunits, COPI
  • Di-arginine (X-Arg-Arg-X): ER-resident membrane proteins, COPI δ and β subunits, COPI
  • Di-acidic (e.g., Asp-X-Glu): Cargo membrane proteins in ER, COPII Sec24 subunit, COPII
    Asp-X-Glu: Cargo membrane proteins in ER
  • Asn-Pro-X-Tyr (NPXY): LDL receptor in plasma membrane, AP2 complex, Clathrin/AP2
  • Tyr-X-X-: Membrane proteins in trans-Golgi, AP1 (μ1 subunit), Clathrin/AP1
  • Plasma membrane proteins: AP2 (μ2 subunit), Clathrin/AP2, AP2 complexes
  • Leu-Leu (LL): Plasma membrane proteins, Clathrin/AP2
  • X = any amino acid

Rab GTPases

• Rab GTPases control docking of vesicles on target membranes.
  • (a) Fusion of secretory vesicles with the plasma membrane:
    • Step 1: Transport vesicle docking: Rab protein (tethered via lipid anchor) binds to an effector protein complex on the target membrane.
    • Step 2: v-SNARE (VAMP) forms a stable coiled-coil interaction with cognate t-SNAREs (syntaxin and SNAP-25).
      • SNARE complexes hold the vesicle close to the target membrane.
    • Step 3: Fusion of the two membranes drives dissociation of the SNARE complexes.
      • Rab-GTP is hydrolyzed to Rab-GDP, dissociating Rab from the Rab effector.
  • (b) v-SNARE–t-SNARE complex:
    • Four α helices (two from SNAP-25, one each from syntaxin and VAMP) form a four-helix coiled-coil via noncovalent interactions.

Early Stages of Secretory Pathway

• COPII-coated vesicles transport proteins with Golgi-targeting sequences from the rough ER to the cis-Golgi (anterograde).
• COPI-coated vesicles transport ER/Golgi-resident proteins in the retrograde direction, supporting Golgi cisternal maturation.

ER and cis-Golgi Trafficking

• Vesicle transport between ER and cis-Golgi: initial stage of the secretory pathway.
• Forward (anterograde) transport: COPII vesicle-mediated ER to cis-Golgi transport.
  • Cargo: newly synthesized proteins.
  • Step 1:
    • COPII coat assembles on the ER membrane, forming vesicles with:
      • v-SNAREs
      • Membrane cargo proteins
      • Soluble cargo proteins (bound to receptors)
    • COPII vesicle pinches off the ER membrane.
  • Step 2: COPII coat disassembly exposes v-SNAREs for fusion targeting.
  • Step 3:
    • Rab-effector interaction promotes v-SNARE association with t-SNAREs on the cis-Golgi membrane.
    • Membrane fusion releases vesicle contents into the cis-Golgi.
• Reverse (retrograde) transport: COPI vesicle-mediated cis-Golgi to ER transport.
  • Cargo: recycles the membrane bilayer, v-SNAREs, and missorted ER-resident proteins.
  • Step 4: ARF-GTP recruits COPI coat.
  • Step 5: COPI-coated vesicles pinch off the cis-Golgi membrane.
  • Step 6: cis-Golgi v-SNARE interaction with ER t-SNARE mediates vesicle fusion with the ER membrane.

KDEL Receptor

• Role of the KDEL receptor in retrieval of ER-resident luminal proteins from the Golgi.

Later Stages of Secretory Pathway

• The trans-Golgi network sorts proteins into vesicles for different destinations.
• Lysosomal enzymes have M6P residues, recognized by M6P-receptors, delivered by clathrin-coated vesicles to lysosomes.
• Regulated secretory proteins are concentrated and stored; constitutively secreted proteins are continuously delivered to the plasma membrane.
• Some proteins are processed after leaving the trans-Golgi network.

trans-Golgi Network

• Distal sorting compartment
• Sorts proteins into five vesicle types:
  • (1) COPI vesicles: retrograde transport of Golgi enzymes (cisternal progression).
  • (2) AP complex vesicles (± clathrin): transport lysosomal enzymes directly to lysosomes.
  • (3) Clathrin-coated (+AP2) vesicles: transport lysosomal enzymes to late endosomes.
  • (4) Constitutive secretory vesicles (unknown coat):
    • Transport constitutively secreted proteins and plasma membrane proteins.
    • Cargo: ECM proteins, blood proteins, immunoglobulins.
  • (5) Regulated secretory vesicles (unknown coat):
    • Store and process secreted proteins until signaled to fuse with the plasma membrane.
    • Cargo: digestive enzymes, peptide hormones.

Clathrin Coats

• Bud from the trans-Golgi and plasma membranes
• Clathrin coat interaction with a membrane is mediated by AP (adapter protein) complexes.
  • Clathrin subunit triskelion structure:
    • Three long, bent heavy chains form a triskelion
    • Three light chains are associated with HCs near the vertex
    • Intrinsic curvature due to the bend in the heavy chains
• Vesicle uncoating: clathrin coat depolymerization
  • ARF GTP hydrolysis regulates timing.
  • Mediated by cytosolic Hsp70 (chaperone, mechanism unknown), activated by auxilin (co-chaperone) which stimulates Hsp70 ATP hydrolysis
  • Releases triskelions for reuse
  • Exposes v-SNAREs for fusion

Dynamin

• Required for pinching off of clathrin-coated vesicles
• Polymerizes around the vesicle neck
• Hydrolyzes GTP, causing a conformational change coupled to membrane fusion and vesicle release

Receptor-Mediated Endocytosis

• Extracellular ligands bound to cell-surface receptors (with cytoplasmic domain AP2-targeting sequences) are internalized by clathrin-coated vesicles.
• The endocytic pathway delivers some ligands (e.g., LDL particles) to lysosomes for degradation.
• The late endosome acidic environment dissociates receptor-ligand complexes, receptors are recycled to the plasma membrane, and ligands are degraded in lysosomes.
• The iron endocytosis pathway releases Fe^{3+} in the late endosome but recycles the transferrin carrier proteins with the receptor to the plasma membrane.

LDL Internalization

• Endocytic pathway for internalizing low-density lipoprotein (LDL).

Iron Endocytosis

• The iron endocytosis pathway releases Fe^{3+} in the late endosome but recycles the transferrin carrier proteins with the receptor to the plasma membrane.

Lysosome Targeting

• Endocytosed membrane proteins for degradation in the lysosome are incorporated into vesicles that bud into the interior of the endosome.
• Cellular components (e.g., ESCRT) mediate endosome membrane budding and pinch off enveloped viruses (e.g., HIV) from the plasma membrane of infected cells.
• Autophagy envelopes a region of cytoplasm or an organelle into a double-membrane autophagosome for delivery to a lysosome.

Plasma Membrane Protein Degradation

• Delivery of plasma-membrane proteins to the lysosome interior for degradation.