Vesicle Transport and Cargo Delivery

BIOL2056: Cell Biology - Vesicle Transport and Cargo Delivery

Course Information

  • Instructor: Dr. David Tumbarello

  • Email: D.A.Tumbarello@soton.ac.uk

Reading List

  • Chapters to Review:

    • Chapter 13 and 16

    • Chapter 14: Introduction, Sections 14.2 - 14.3 (first half), 14.4 (limited), 14.5, 14.6

    • Chapter 17: Introduction, Sections 17.1 - 17.3, 17.5

    • Chapter 18: Introduction, Sections 18.1, 18.2, 18.4

  • Additional Reading:

    • 2 review articles on vesicle coat proteins and Rab GTPases (available on Blackboard)

Learning Outcomes

By the end of this session, students should be able to:

  • Explain the requirements for vesicle transport.

  • Describe the different types of vesicle coat proteins and their functions.

  • Describe the molecular composition of clathrin-coated vesicles.

  • Explain the mechanism of clathrin coat formation.

  • Describe the function of dynamin.

The Endomembrane System and Vesicle Transport

  • The endomembrane system divides the cell into different membrane-bound compartments, regulating various cellular functions such as:

    • Translation, modification, and trafficking of proteins.

    • Activation and inactivation of signal transduction pathways.

    • Components include:

    • Nuclear envelope

    • Endoplasmic reticulum (ER)

    • Golgi apparatus

    • Secretory vesicles

    • Endosomes

    • Lysosomes

    • Autophagosomes

    • Plasma membrane

Vesicle Transport

  • Vesicles bud off and fuse with various compartments, carrying 'cargo' which includes both membrane-associated and soluble molecules.

  • Each vesicle is selective for certain cargo and must fuse with an appropriate target membrane.

Pathways of Vesicle Transport:

  1. Secretory Pathway:

    • Flow of membrane-bound and soluble proteins destined for specific organelles or extracellular space.

    • Sequence: ER  Golgi  Plasma Membrane via secretory vesicles.

  2. Endocytic Pathway:

    • Captures extracellular components via the plasma membrane and internalizes membrane proteins into vesicles.

    • Results in recycling of receptors or degradation of contents via lysosomes.

  • This process maintains the integrity of organelles, ensuring proper morphology and lipid/protein composition.

Visualization of Protein Transport

  • A viral membrane glycoprotein (VSV-G) labeled with GFP serves as a temperature-sensitive reporter for studying synchronous transport from the ER to Golgi and onwards to the plasma membrane.

Requirements for Vesicle Transport

  • Key components involved in vesicle transport include:

    • Identification of specific cargo

    • Sorting of vesicles and associated cargo

    • Role of cytoskeletal motor proteins

    • Mechanisms for:

    • Fission

    • Tethering

    • Fusion

Vesicle Coat Proteins

  • Transport vesicles typically exhibit coat proteins that:

    • Provide shape to membranes facilitating curvature and budding.

    • Determine the size and shape of vesicles.

    • Concentrate proteins within vesicles.

    • Provide selectivity for cargo.

    • Influence the destination of the vesicle.

  • Types of Coated Vesicles:

    • Clathrin-Coated Vesicles: Trans-Golgi Network (TGN) to endosome/plasma membrane (via endocytosis).

    • COPI-Coated Vesicles: From Golgi complex back to ER (retrieval).

    • COPII-Coated Vesicles: From ER to Golgi.

Clathrin-Coated Vesicles

  • Composed of clathrin subunits made of 3 large (heavy) and 3 small (light) polypeptides forming triskelions on the TGN or plasma membrane.

  • These vesicles transport materials from the plasma membrane and between endosomes and the Golgi apparatus, forming an outer protein lattice.

Endocytosis Mechanisms

  • Types include:

    1. Receptor-Mediated Endocytosis:

    • Clathrin-dependent

    • Caveolin-dependent (in lipid rafts, sphingolipids, GPI-anchored proteins)

    • Clathrin- and caveolin-independent

    1. Phagocytosis: Engulfment of large particles.

    2. Pinocytosis: Uptake of fluids and small molecules.

Clathrin Coat Formation

  • During endocytosis at the plasma membrane, recruitment of the AP2 adaptor protein complex is necessary for clathrin recruitment and coat assembly.

  • Binding of AP2 adaptor to specific phospholipids triggers conformational changes allowing attachment to cargo receptors, instigating membrane curvature.

  • The AP-2 complex is characterized as heterotetrameric with subunits (α, β2, σ2, µ2).

Dynamin's Role in Vesicle Fission

  • Dynamin oligomerizes into a helical ring around the neck of the bud, recruitment of other proteins occurs, and it tethers itself to the membrane via lipid-binding domains.

  • Constriction occurs in the presence of GTP, and hydrolysis of GTP facilitates membrane fission.

  • Temperature-sensitive mutants (e.g., Shibire in Drosophila) aid in understanding dynamin function, halting vesicle fission and allowing visualization of arrested buds.

COP I and COP II Coated Vesicles

  • COP II Vesicle Formation:

    • The COP II coat consists of 5 protein subunits plus an associated GTPase (Sar1).

    • Sar1-GEF (Sec12) embedded in donor membrane recruits and activates Sar1 by loading it with GTP.

    • Sar1-GTP combines with Sec23/24 (inner coat) and Sec13/31 (outer coat) to facilitate vesicle formation.

  • COP I Vesicular Transport:

    • Proteins returning to the ER from the Golgi possess the KDEL sequence, recognized by KDEL receptors for retrieval via COP I vesicles.

  • The COP I vesicle coat features multiple subunits organized as a coatomer, recruited en bloc to membrane sites, with ARF1 GTPase activating the coatomer.

Rab GTPases and Vesicle Transport

  • Rab GTPases are crucial for regulating intracellular transport and organelle identity with 61 known members, facilitating formation, budding, transport, and fusion of vesicles through effector protein interaction.

Rab GTPase Activation

  • The activation pathway involves GDI (keeping Rab inactive in cytosol), GDF (GDI displacement factor) that allows Rab membrane association, and GEF-mediated GDP to GTP exchange triggering conformational changes for interaction with effectors.

Role of SNARE Proteins

  • SNAREs (v-SNAREs on vesicles and t-SNAREs on target membranes) mediate vesicle fusion, with interactions initiated by Rab proteins binding to tethering protein complexes (e.g., HOPS).

Membrane Fusion Mechanism

  • Mechanism involves docking of vesicle SNAREs to target SNAREs, forming a trans-SNARE complex that brings membranes close together for fusion, possibly involving ATP hydrolysis for disassembly of SNARE complexes.

Autophagy

  • Autophagy is a degradation pathway involving the formation of double-membrane autophagosomes which capture cytosolic components or damaged organelles, later fusing with lysosomes for degradation.

  • Cargo selection is managed through autophagy receptors identifying polyubiquitylated proteins, connecting them to the Atg8/LC3 protein on the autophagosomal membrane for transport.

Key Concepts to Consider

  • How do clathrin-coated vesicles form, and what roles do COPI and COPII play?

  • The significance of Rab GTPases in vesicle transport regulation and mechanisms of vesicle tethering and fusion.

  • Understanding endocytic pathways and the role of ESCRT complexes in intraluminal vesicle formation.

  • Importance of cytoskeletal elements like actin and microtubules in vesicle transport and stability.

Questions for Review

  • What are the main functions and mechanisms of COPI and COPII vesicles?

  • How do Rab GTPases facilitate vesicle identity and transport?

  • Discuss endocytosis and the significance of ESCRT complexes in managing cellular receptors and proteins.

Course Information

  • Instructor: Dr. David Tumbarello

  • Email: D.A.Tumbarello@soton.ac.uk

Reading List

  • Core Chapters:

    • Chapter 13 and 16 (General Principles of Protein Sorting and Membrane Trafficking)

    • Chapter 14: Sections 14.2 (ER to Golgi), 14.3 (Golgi Export), 14.4 (Lysosomes), 14.5 (Endocytosis), 14.6 (Exocytosis)

    • Chapter 17: Sections 17.1 (Cytoskeleton Intro), 17.2 (Intermediate Filaments), 17.3 (Microtubules), 17.5 (Actin)

    • Chapter 18: Sections 18.1 (Motor Proteins), 18.2 (Cell Shape), 18.4 (Vesicle Transport Factors)

  • Supplementary Material:

    • Advanced reviews on vesicle coat proteins (Clathrin, COPI, COPII) and the Rab GTPase cycle available on Blackboard.

Learning Outcomes

  • Biophysical Requirements: Understand the energetic and structural requirements for membrane curvature and fusion.

  • Coating Diversity: Compare and contrast the molecular architecture of Clathrin, COPI, and COPII coats.

  • Clathrin Mechanics: Detail the recruitment of adaptors like AP2 and the role of phosphoinositides like PI(4,5)P2PI(4,5)P_2.

  • Fission Dynamics: Explain the mechanochemical role of Dynamin in pinching off vesicles.

  • Regulatory GTPases: Analyze the Rab cycle and SNARE-mediated fusion mechanisms.

The Endomembrane System and Vesicle Transport

  • The endomembrane system is a dynamic, interconnected network of organelles that regulates protein flux and lipid metabolism.

  • Compartmentalization: Allows for distinct chemical environments (e.g., low pH in lysosomes) to facilitate specific enzymatic reactions.

  • Organelles Involved:

    1. Nuclear Envelope: Continuous with the Rough ER.

    2. Endoplasmic Reticulum (ER): The entry point for the secretory pathway; site of folding and glycosylation.

    3. Golgi Apparatus: Composed of cis, medial, and trans cisternae; serves as the central sorting hub.

    4. Endosomes: Sort internalized cargo to lysosomes or back to the plasma membrane.

    5. Lysosomes: Acidic compartments (pH≈4.5−5.0pH \approx 4.5 - 5.0) for hydrolytic degradation.

    6. Plasma Membrane: The final destination for exocytosis and initiation point for endocytosis.

Mechanics of Vesicle Transport

  • Budding: Curvature is induced by the assembly of coat proteins which deform the donor membrane.

  • Cargo Selection: Sorting signals (e.g., KDEL, Di-leucine motifs) are recognized by adaptor proteins.

  • Motility: Vesicles are transported along microtubules by kinesin (plus-end directed) or dynein (minus-end directed) motors.

  • Tethering and Fusion: Effector proteins ensure the vesicle hits the correct target before SNAREs facilitate lipid bilayer merger.

Visualization Techniques

  • VSV-G GFP Assay: Utilizing a temperature-sensitive mutant of the Vesicular Stomatitis Virus Glycoprotein. At 40∘C40^{\circ}C, protein is misfolded and retained in the ER. Shifting to 32∘C32^{\circ}C allows synchronized folding and transport, which can be tracked via fluorescence microscopy.

Detailed Vesicle Coat Proteins

  • Coats serve two primary functions:

    1. Concentrating specific membrane proteins and lumenal cargo into a specialized patch.

    2. Molding the forming vesicle into a sphere or tube.

  • Types of Coated Vesicles:

    • COPII: Mediate Anterograde transport (ER to Golgi). Formation is initiated by the GTPase Sar1Sar1.

    • COPI: Mediate Retrograde transport (Golgi to ER) and intra-Golgi transport. Triggered by ARF1ARF1 GTPase.

    • Clathrin: Mediate transport from the TGN to endosomes and endocytosis from the plasma membrane.

Clathrin-Coated Vesicles (CCVs)

  • Structure: The clathrin triskelion consists of 3 heavy chains (≈190kDa≈ 190 kDa each) and 3 light chains (≈25kDa≈ 25 kDa each).

  • Adaptor Proteins (APs): Link clathrin to the membrane and cargo. AP2 is the major adaptor at the plasma membrane.

    • AP2 Configuration: Heterotetramer ( ̑, ̒2, ̓2, ̔2 subunits).

    • Activation: ̔2 and ̓2 bind to PI(4,5)P2PI(4,5)P_2 in the membrane, causing a conformational change that exposes cargo-binding sites.

Role of Dynamin

  • Dynamin is a large GTPase that oligomerizes at the neck of the budding vesicle.

  • Mechanism: GTP hydrolysis causes a conformational shift (constriction) that physically severs the vesicle from the membrane.

  • Mutation: The Shibire mutant in flies prevents GTP hydrolysis at high temperatures, leading to paralyzed flies due to a lack of synaptic vesicle recycling.

COP I and COP II Molecular Machinery

  • COPII Formation Sequence:

    1. Activation: Sec12 (a membrane GEF) exchanges GDP for GTP on Sar1Sar1.

    2. Insertion: Sar1−GTPSar1-GTP extends an amphipathic helix into the ER membrane.

    3. Inner Coat: Recruitment of Sec23/Sec24. Sec24 specifically binds to cargo exit signals.

    4. Outer Coat: Recruitment of Sec13/Sec31 to crosslink the complexes and drive budding.

  • COPI Retrieval:

    • Soluble ER resident proteins contain a C-terminal KDEL sequence. If they escape to the Golgi, KDEL receptors bind them in the low-pH environment of the Golgi and package them into COPI vesicles for return to the ER.

Rab GTPases: The 'Address' System

  • Rabs are small monomeric GTPases that act as molecular switches (GTP−bound=On;GDP−bound=OffGTP-bound = On; GDP-bound = Off).

  • Cycle:

    • GDI: Rab-GDP is kept soluble in the cytosol by Guanine Nucleotide Dissociation Inhibitor.

    • GDF: GDI Displacement Factor helps Rab associate with the correct organelle membrane.

    • GEF: Exchanges GDP for GTP to activate Rab.

    • GAP: GTPase Activating Protein stimulates GTP hydrolysis to turn Rab off.

  • Specific Rabs: Rab5 marks early endosomes; Rab7 marks late endosomes.

SNARE-Mediated Fusion

  • v-SNAREs (Vesicle) and t-SNAREs (Target) interact to form a stable 4-helix bundle (trans-SNARE complex).

  • Energy: The formation of the bundle is highly exergonic, providing the force to displace water and merge the lipid bilayers.

  • Recycling: After fusion, the adapter ̑-SNAP and the ATPase NSF use ATP hydrolysis to pry the SNAREs apart for reuse.

Autophagy: Cellular Self-Eating

  • Process: A crescent-shaped membrane (phagophore) engulfs cargo, closing to form a double-membrane autophagosome.

  • Key Marker: LC3-II (Atg8) is conjugated to phosphatidylethanolamine (PE) on the autophagosome membrane.

  • Lysosomal Fusion: Facilitated by HOPS complex (tether) and SNAREs. The inner membrane and cargo are degraded by acid hydrolases.