In-Depth Notes on Protein Sorting and Transport Mechanisms

Protein Sorting Mechanisms

  • Quality Control in ER:
    • Ensures proteins are properly folded and assembled.
    • Misfolded proteins are retained by ER-localized chaperones.
  • Active Cargo Selection:
    • Specific cargo is collected in ER regions for vesicle formation.
    • Soluble Cargo: Recognized by integral membrane proteins.
    • Membrane Cargo: Identified by cytoplasmic proteins facilitating vesicle assembly.

Protein Retrieval Signals

  • KDEL Signal:
    • Present in soluble ER proteins, attracts KDEL receptors for retrieval.
    • KDEL receptors cycle between the Golgi and ER.
  • KKXX Signal:
    • Found in resident ER membrane proteins, recognized by COP I coat proteins for retrieval.

Vesicle-Mediated Transport

  • Conservation Across Eukaryotes:
    • Yeast mutants help identify essential proteins for secretory mechanisms.
  • Vesicle Formation:
    • Driven by coat protein complexes.
    • Classifications:
    1. Clathrin: Involved in transport from Golgi and plasma membrane.
    2. COP I: Mediates transport from cis-Golgi to ER.
    3. COP II: Facilitates transport from ER to cis-Golgi.

Roles of Protein Coats

  • Functions of Vesicle Coats:
    1. Shape the donor membrane into a bud.
    2. Capture cargo proteins into vesicles.
  • Role of Rab Proteins:
    • Small GTP-binding proteins that regulate coat formation and vesicle transportation.
    • Cycle between active (GTP) and inactive (GDP) forms, controlled by GEFs and GAPs.

COP II Assembly Process

  1. Activation of Sar1 (Rab protein) by GEF.
  2. Insertion of Sar1 into the membrane, bending it.
  3. Recruitment of Sec23 and Sec24 which continue to deform the membrane and act as cargo receptors.
  4. Final assembly involves Sec13 and Sec31 for the outer COP II coat layer.

Vesicle Fusion Process

  • Common features during fusion:
    1. Removal of vesicle coat.
    2. Specific recognition of vesicle by target membrane.
    3. Fusion and mixing of vesicle content with target organelle.

Tethering and Docking

  • Tethering: First contact between vesicle and target membrane, involving various classes of tethers (e.g., multiprotein complexes).
  • Docking: Heavier interaction mediated by SNARE proteins:
    • v-SNARE (on vesicle) and t-SNARE (on target).
    • The formation of a 4-helix bundle from paired SNAREs drives the fusion.

Membrane Fusion Mechanism

  • SNARE Pairing:
    • Zippering of SNAREs releases energy to bring the vesicle and target membranes close.
    • Fusion occurs in three stages:
    1. Hemifusion intermediate formation.
    2. Expansion allows inner leaflets to fuse.
    3. Mixing of soluble materials occurs after inner leaflet fusion.

Golgi Transport Models

  • Vesicle Transport Model:
    • Golgi cisternae are static, while cargo is transported in vesicles.
  • Cisternal Maturation Model:
    • Enzymes migrate retrograde, converting cis Golgi to trans Golgi while cargo remains stationary.

Glycosylation in the Golgi

  • Functions as a glycosylation factory, modifying proteins:
    • Enzymatic modifications happen at different Golgi regions - cis, medial, and trans.
    • Unique modification for lysosomal enzymes, leading to mannose-6-phosphate targeting.

Endocytic Pathways

  • Types of Endocytosis:
    1. Bulk-phase (non-specific).
    2. Receptor-mediated (specific and clathrin-dependent).
  • Clathrin-coated Vesicles:
    • Formed from triskelion structures; adaptor proteins engage receptors.

Endosome Maturation

  • Early endosomes transform into late endosomes which exhibit:
    • Lower pH for ligand dissociation.
    • Association with different Rab proteins (Rab5 to Rab7).

Fate of Receptor/Ligand Complexes

  1. Ligands dissociate from receptors; receptors recycled to the membrane.
  2. Ligands and receptors remain together, recycled to cell surface.
  3. Both complex sent to lysosome for degradation (e.g., EGF receptors).