Vesicle Budding and Fusion: The Secretory and Endocytic Pathways

Conceptual Framework: Intracellular transport is mediated by the movement of vesicles between membrane-bound organelles. These pathways are categorized into three primary flows: * Secretory Pathway (Anterograde): Transports materials from the Endoplasmic Reticulum (ER), through the Golgi apparatus (cisternae), to the plasma membrane or lysosomes. This includes the use of secretory vesicles for extracellular release. * Endocytic Pathway: Moves materials from the plasma membrane into the cell via early endosomes, late endosomes, and eventually to lysosomes for degradation. * Retrieval Pathway (Retrograde): Returns proteins and membranes to their original compartments (e.g., from Golgi back to ER).
Organelle Components: The system involves the nuclear envelope, ER, Golgi apparatus, various endosomes (early, late, recycling), and lysosomes.

Nobel Prize (2013): Awarded to Rothman, Schekman, and Südhof for discovering the machinery regulating vesicle traffic.
Biochemical Approaches (Rothman): * Utilized cell-free transport assays to isolate individual steps of vesicular transport. * Enabled the fractionation of cytosol, allowing for the precise characterization of specific protein complexes.
Genetic Approaches (Schekman): * Utilized budding yeast (Saccharomyces cerevisiae) to generate mutants with defects in secretion, known as 'sec' mutants. * Temperature-sensitive mutants: A solution to legal defects in cells. Mutants function normally at permissive temperatures (approx. $23^{\circ}C$ ) but exhibit defects at restrictive temperatures (approx. $36^{\circ}C$ ), where the mutant protein becomes dysfunctional. * Classification: For example, Class A 'sec' mutants (e.g., Sec61) have a dysfunctional protein conduction channel.

Experimental Mechanism: Monitoring the processing of N-linked oligosaccharides in the Golgi. * Typical Golgi Processing Steps: 1. Golgi mannosidase I 2. N-acetylglucosamine (NAG) transferase I 3. Golgi mannosidase II 4. N-acetylglucosamine transferase II 6. Galactosyl transferase 7. N-acetylneuraminic acid (sialic acid) transferase
Rothman's VSV G-Protein Assay: * Used Vesicular stomatitis virus (VSV) G-protein as cargo. * System composed of two types of membranes: 1. Donor: Wild-type (WT) membranes containing NAG transferase but no VSV G-protein. 2. Acceptor: Mutant membranes (lacking NAG transferase) expressing VSV G-protein. * Process: Vesicles bud from WT donor membranes and fuse with mutant acceptor membranes. In the presence of ATP, Cytosol, and $^{3}H$ -NAG, NAG is attached to the mannose sugars of the VSV G-protein.
NSF Identification: The first protein identified from the cytosol using this assay was NSF (N-ethylmaleimide Sensitive Factor). Activity was tracked via radioactivity (counts per minute, $cpm \times 10^{-3}$ ) across different protein fractions and potassium chloride ( $KCl$ ) concentrations.

The Three Main Protein Coats: * Clathrin: Mediates transport from the plasma membrane to early endosomes and from the Golgi to late endosomes. * COPII: Mediates anterograde transport from the ER to the cis-Golgi. * COPI: Mediates retrieval/retrograde transport from the Golgi back to the ER.
Common Features of Coats: * Double Layer Construction: The inner layer selects cargo proteins; the outer layer provides the structural curvature required to form a sphere. * Accessory Proteins: Different locations using the same coat type utilize distinct accessory/adaptor proteins. * Coat Removal: The protein coat must be shed before the vesicle can fuse with its target membrane.
Clathrin-Coated Vesicle Structure: * Composed of triskelions consisting of three heavy chains and three light chains. * Pinching Off: Requires dynamin, a large GTP-binding protein, and GTP hydrolysis to sever the membrane neck. * Uncoating: Involves PI(4,5)P2 phosphatase, HSP70 chaperone, and ATP hydrolysis.

Organelle Specificity: Different phosphoinositides (PIs) define membrane identity: * $PI(3)P$ : Found in early endosomes. * $PI(4,5)P2$ : Concentrated at the plasma membrane. * $PI(4)P$ : Present in the Golgi.
The AP2 Adaptor: At the plasma membrane, $PI(4,5)P2$ binding to the AP2 adaptor induces a conformational change ("opening"), exposing binding sites for cargo receptors containing endocytosis signals (e.g., $\mu 2$ and $\sigma 2$ subunits).

Mannose 6-Phosphate (M6P) Tagging: Occurs in the cis-Golgi network. * Enzymes: GlcNAc phosphotransferase recognizes a signal patch on lysosomal hydrolases and transfers $P-GlcNAc$ from $UDP-GlcNAc$ . A phosphodiesterase then removes the GlcNAc, leaving a M6P residue. * Mechanism: The M6P receptor in the trans-Golgi network (TGN) binds the tagged enzyme, packaging it into clathrin-coated vesicles for delivery to lysosomes.
ER Resident Retrieval: * KDEL Signal: Soluble ER proteins (like Protein Disulphide Isomerase, PDI) have a $Lys-Asp-Glu-Leu$ C-terminal sequence. They bind to the KDEL receptor in the Golgi (favored by decreasing pH) and are returned via COPI vesicles. * KKXX Signal: Membrane-bound ER proteins have a $Lys-Lys-X-X$ C-terminal sequence which binds directly to COPI coats. * Experiment (Munro and Pelham, 1987): Fusing a KDEL sequence to lysozyme-myc redirected the protein from secretory vesicles/Golgi to the ER.

Initiation Factors: * Sar1: Regulates COPII assembly at the ER membrane. * Arf Proteins: Mediate COPI and clathrin coat assembly at the Golgi.
The Rab Protein Family: * Over $60$ members providing specificity in membrane docking. * Cycle: Inactive Rab is bound to Rab-GDI (GDP dissociation inhibitor) in the cytosol. Rab-GEF (GTP Exchange Factor) on the membrane activates Rab by exchanging GDP for GTP. Rab-GTP then binds to specific Rab effectors on target membranes.
Localization Examples: * Rab1: ER and Golgi. * Rab5: Early endosomes, plasma membrane. * Rab7: Late endosomes. * Rab11: Recycling endosomes.

Fusion Machinery: Mediated by SNAREs (Soluble NSF Attachment Receptors). * v-SNAREs: Located on the vesicle. * t-SNAREs: Located on the target membrane. * Mechanism: Four long $\alpha$ -helices (two from t-SNAREs like SNAP-25, one from v-SNARE like VAMP/synaptobrevin, and one from the t-SNARE syntaxin) form a stable coiled-coil structure, pulling membranes together.
SNARE Disassembly: After fusion, the complex must be recycled. This requires NSF, accessory proteins ( $Sec17$ in yeast), and ATP hydrolysis.
Conservation: Machinery is conserved from yeast to humans (e.g., Yeast Sec18 is the homolog of mammalian NSF; Sec22 is a v-SNARE).

COPII: Step: ER to cis-Golgi; Proteins: Sec23/Sec24, Sec13/Sec31, Sec16; GTPase: Sar1.
COPI: Step: cis-Golgi to ER (retrograde); Proteins: Coatomers ( $7$ subunits); GTPase: ARF.
Clathrin + AP1: Step: trans-Golgi to endosome; GTPase: ARF.
Clathrin + AP2: Step: Plasma membrane to endosome; GTPase: ARF.
Clathrin + AP3: Step: Golgi to lysosome/melanosome; GTPase: ARF.