Lecture 12: Vesicular Trafficking I

Aims

To describe the fundamental principles of transport vesicle formation using COPII as a paradigm

Learning Objectives

● Understand the roles of GTPases, adaptors and coat proteins in transport vesicle formation

● Describe the process of cargo selection in vesicle transport.

Vesicles are targeted with high fidelity to their specific destinations, ensuring that cargo is delivered to the correct compartment.
The transport process occurs across an asymmetric lipid bilayer.
Cargo is transported in two primary forms:
- Soluble cargo: Carried within the lumen and delivered without leakage.
- Membrane-bound cargo: Maintained in a specific molecule topology within the membrane during transport.
Transport is mediated by both vesicles and tubules.
Cargo typically consists of large macromolecules, such as proteins, which are too bulky and carry a variety of charges that prevent them from passing through standard channels.
Vesicular transport maintains a balance between forward (anterograde) movement and retrograde movement (recycling), which is essential for maintaining organelle identity.

SNARE proteins are a critical part of the cargo incorporated into transport vesicles.
The process involves specific interactions between SNAREs on different membranes:
- v-SNARES: Found on the vesicle membrane.
- t-SNAREs: Located on the target compartment (Compartment B).
The interaction occurs in two distinct stages:
1. Docking: The vesicle reaches the target membrane and the SNAREs interact.
2. Fusion: The vesicle membrane merges with the target membrane (Compartment B), allowing for the delivery of soluble and membrane-bound cargo.

Transport vesicles are coated; the bilayer and the coat structure are both visible during formation.
The coat must be removed (uncoating) for membrane fusion to occur.
Transport through the secretory pathway is mediated via these coated vesicles and tubules, heading ultimately toward the Golgi apparatus.
Vesicles hook onto microtubules and move via motor proteins, which facilitates their movement through the secretory pathway.
Live-cell imaging is utilized to visualize these transport intermediates as they move within the cell.

Every instance of transport vesicle formation requires three essential components:
1. Small GTPase: Acts to recognize and select the appropriate cargo for the vesicle.
2. Adaptor proteins: Link the coat to the membrane and recognize cargo signals.
3. Coat: Provides the structural scaffold for the budding vesicle.

Small GTPases function as molecular switches that cycle between active and inactive states:
- GDP-bound form: Inactive state, usually located in the cytosol.
- GTP-bound form: Active state, typically membrane-associated.
The transition between these states is regulated by two classes of proteins:
- Guanine nucleotide exchange factors (GEFs): Promote the exchange of GDP for GTP, thereby activating the GTPase.
- GTPase Activating Proteins (GAPs): Facilitate GTP hydrolysis, converting GTP to GDP and inactivating the protein.
Ras is the founding member of this family of GTPases.
When recruited to the membrane and activated, these GTPases guide cells to proliferate.
During vesicle formation, an amphiphilic helix flips out when the GTPase is activated, allowing it to anchor into the membrane.

COPII-coated vesicles serve as the paradigm for understanding transport vesicle formation from the Endoplasmic Reticulum (ER).
The specific components of the COPII system are:
- GTPase: Sar1 (a member of the Arf family).
- Adaptor: Sec23/24 complex.
- Coat: Sec13/31 complex.
General Process of COPII Formation:
1. SAR1 Activation: Mediated by Sec12 (the Sar1-GEF) located in the ER membrane. This triggers recruitment to the membrane.
2. Adaptor Protein Recruitment: Sec23/24 are recruited. Sec23/24 have a "bowtie" crystal structure shape that allows for membrane curvature.
3. Budding: The bud becomes increasingly curved as the coat is recruited.
4. Fission: The vesicle buds off from the ER.
Chaperones in the ER ensure that newly synthesized proteins (e.g., growth factors) are properly folded before they are incorporated as cargo.

Reconstitution experiments are used to identify the minimal components required for COPII formation.
The experimental setup involves:
- Substrate/Donor compartment: ER membranes containing Ribophorin (a resident ER protein used as a control for contamination).
- Cargo: p58 (a COPII cargo protein).
- Required additives: Cytosol, ATP, and GTP.
The process involves incubating ER membranes with these components followed by sucrose gradient centrifugation to harvest the vesicles.
Analysis is performed via Western blot. If the reaction is successful, the product will contain COPII vesicles with p58, while Ribophorin remains in the donor membrane. If any essential component is left out, no vesicles form.

Sec23 functions as a GAP for Sar1.
GAP activity is enhanced following the recruitment of the Sec13/31 coat.
The activation of GAP activity promotes coat disassembly (uncoating).
Synthetic mutant GTPases are used to study these cycles:
- GDP mutant: Sequesters GEFs, preventing the activation of wild-type (WT) proteins.
- GTP mutant: Cannot hydrolyze GTP, remaining permanently active.
Expression of Sar1-GDP inhibits COPII formation, demonstrating a dominant-negative effect where the mutant overcomes the action of endogenous proteins.

Different coat systems facilitate different stages of transport:
- COPII: Uses Sar1; transports newly synthesized proteins from the ER.
- COPI: Uses Arf1; involved in retrieval transport from the vesicular tubular cluster back to the ER and within the Golgi.
- Clathrin (TGN): Uses Arf1; transports lysosomal proteins and regulated secretory proteins from the Trans-Golgi Network (TGN).
- Clathrin (Plasma Membrane): Transports endocytosed material from the plasma membrane (PM).

Adaptor proteins ensure specificity by recognizing motifs in the cytoplasmic domains of membrane proteins.
They link the coat to the membrane and select specific cargo.
Specific Adaptor Protein (AP) complexes show precise subcellular localization:
- AP1: Located at the TGN.
- AP2: Located at the Plasma Membrane (major clathrin adaptor for endocytosis).
- AP3: Located at the TGN.
Endocytic sorting signals recognized by adaptors include:
- Tyrosine-based: $Yxx\Phi$ (e.g., Transferrin receptor/TfR).
- Di-leucine: $[DE]xxxLL$ (e.g., Acetylcholine transporter).
- NPxY motif: $FXNPxY$ (e.g., LDL receptor).
- Ubiquitin: Recognized by adaptors like epsin (e.g., EGFR).
For the Transferrin receptor (TfR), the $\mu$ and $\sigma$ subunits of AP2 are critical for recognizing the $Yxx\Phi$ sorting signal.