Lecture 14: Vesicular Traffic, Secretion, and Endocytosis, Part 1

Major Topics Covered in Lecture

  • Techniques for Studying the Secretory Pathway

  • Molecular Mechanisms of Vesicle Budding and Fusion

  • Early Stages of the Secretory Pathway

Learning Objectives

  • Describe the basic structure and function of the Golgi complex.

  • Discuss the two proposed mechanisms of Golgi complex formation.

  • Describe different types of coated vesicles, their movement, and their roles in the endomembrane system.

  • Discuss the mechanisms of vesicle formation and vesicle fusion with a membrane.

  • Elucidate the structure, function, and polarization of the Golgi complex.

  • Explain the signals used to target proteins to their appropriate cellular location.

  • Describe the mechanisms of protein transport to basolateral or apical membrane surfaces.

Overview of the Secretory and Endocytic Pathways of Protein Sorting

  • The endomembrane system includes:

    • Transport vesicles

    • Endoplasmic reticulum (ER)

    • Golgi complex

    • Nuclear envelope

    • Endosomes

    • Lysosomes

  • Secretory and endocytic pathway protein trafficking is based on the principle that:

    • Transport vesicles transport membrane and soluble proteins from one membrane-bounded compartment to another.

  • Transport vesicles:

    • Collect cargo proteins in membrane budding from a donor compartment.

    • Deliver cargo proteins to the next compartment by fusing with the target membrane.

Secretory Pathway

  • Definition: A pathway for the distribution of soluble and membrane proteins synthesized by the rough ER to final destinations at the cell surface or in lysosomes.

  • Stages of Secretory Pathway:

    • Stage 1: Rough Endoplasmic Reticulum

    • Step 1: Proteins bearing an ER signal/targeting sequence are synthesized and cotranslationally inserted into the ER membrane or lumen.

    • Stage 2: Protein Trafficking

    • Step 2: Proteins are packaged into transport vesicles that bud from the ER and fuse to form new cis-Golgi cisternae.

    • Step 3: ER enzymes or structural proteins are retained in the ER or retrieved by vesicles that bud from the cis-Golgi and fuse with the ER.

    • Step 4: Each cis-Golgi cisterna and its contents move from the cis to the trans face of the Golgi complex by non-vesicular cisternal maturation.

    • Step 5: Retrograde transport vesicles move Golgi-resident proteins back to the previous Golgi compartment.

    • Step 6: Constitutive secretion involves continuous transport of vesicles that fuse with the plasma membrane, leading to:

      • Continuous secretion of soluble proteins.

      • Membrane proteins becoming plasma membrane proteins.

    • Step 7: Regulated secretion occurs in certain cells where proteins are stored in regulated secretory vesicles and only secreted upon receiving a neuronal or hormonal signal.

    • Step 8: Lysosome-destined proteins are transported in vesicles that bud from the trans-Golgi and fuse with late endosomes for delivery to lysosomes.

Endocytic Pathway

  • This pathway includes:

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

Molecular Mechanisms of Vesicle Budding and Fusion

  • Key Concepts:

    • Three types of coated vesicles facilitate protein transport:

    • COPII-coated vesicles (anterograde movement from ER to Golgi)

    • COPI-coated vesicles (retrograde movement from Golgi to ER)

    • Clathrin-coated vesicles (from TGN to endosomes or lysosomes)

    • Small GTPase proteins promote coat protein polymerization on donor membranes to bud off vesicles carrying cargo.

    • Coat shedding reveals Rab and SNARE proteins essential for vesicle fusion with target membranes.

GTPase Switch

  • GTPase proteins exist in two forms: GTP-bound (active) and GDP-bound (inactive).

  • GTPases hydrolyze GTP to GDP.

  • Mechanisms:

    • GEF (Guanine nucleotide exchange factor): stimulates GDP exchange for GTP.

    • GAP (GTPase-activating protein): stimulates hydrolysis of GTP to GDP.

Overview of Vesicle Budding and Fusion with a Target Membrane

  • Vesicle Budding:

    • Vesicles bud from donor membranes.

    • Cytosolic coat protein complexes bind to the cytosolic domain of membrane cargo proteins, causing membrane evagination.

    • Some cargo proteins act as receptors for soluble proteins, pulling them into the budding vesicles.

    • Donor membrane-specific SNARE proteins (v-SNARES) are incorporated into the vesicle membrane during budding.

  • Vesicle Fusion:

    • Targeting involves the interaction between specific v-SNAREs on the vesicle and t-SNAREs on the target membrane.

    • Twisting interactions lead to the fusion of vesicle and target membranes.

Coated Vesicles Involved in Protein Trafficking

  • Types of Coated Vesicles:

    • COPII-coated vesicles: transport from ER to Golgi.

    • COPI-coated vesicles: retrograde transport from Golgi to ER and between Golgi cisternae.

    • Clathrin-coated vesicles: transport from the TGN to endosomes, lysosomes, and plant vacuoles.

Mechanism of COPII Coat Assembly and Disassembly

  • Role of Sar1 GTPase in COPII coat dynamics:

    • Step 1: Sar-GDP binds to Sec12 ER integral membrane protein, activating GEF activity.

    • Step 2: Sar1-GTP recruits the Sec23/Sec24 coat protein complex, completing coat assembly.

    • Step 3: Sec23 GAP activity promotes GTP hydrolysis, leading to Sar1-GDP release, thus disassembling the coat.

Role of Sorting Signals

  • Targeting sequences on cargo proteins bind to specific coat proteins, facilitating their incorporation into transport vesicles:

  • Notable signals include:

    • KDEL (Lys-Asp-Glu-Leu) for ER-resident soluble proteins.

    • Mannose-6-phosphate (M6P) for soluble lysosomal enzymes.

Docking and Fusion Mechanism of Transport Vesicles

  • Docking Steps:

    1. Transport vesicle docks onto target membrane via Rab GTPases.

    2. v-SNARE (VAMP) interacts with t-SNAREs (syntaxin and SNAP-25) stabilizing the vesicle.

    3. Membrane fusion dissociates SNARE complexes, allowing for vesicle content release.

    4. SNARE complex dissociation requires assistance from NSF and α-SNAP.

Structural Features of SNARE Complex

  • The SNARE complex involves four long helices, crucial for membrane fusion.

  • Formation of a four-helix bundle helps overcome membrane electrostatic repulsion and facilitates lipid bilayer merging.

Late Stages of the Secretory Pathway

  • Proteins sorted into vesicles at the trans-Golgi network (TGN) are targeted to:

    • Lysosomes

    • Secretory granules (regulated or constitutive secretion)

    • Other cellular locations based on sorting signals and protein modifications.

Additional Concepts

  • The KDEL retrieval system maintains ER luminal protein levels essential for protein folding and function.

  • Golgi polarity: Cis face receives cargo from the ER, while the Trans face directs proteins to various destinations.

Summary of Golgi Dynamics

  • The cisternal maturation model describes how cisternae mature and move through the Golgi stack.

  • Enzymatic activities and transport mechanisms ensure proper protein processing and sorting.

Reading Assignment and Next Steps

  • Next Lecture: Vesicular Traffic, Secretion, and Endocytosis, Part 2

  • Reading Assignment: Chapter 14: pages 642-660

  • The next quiz is available on the Achieve site until Tuesday (October 14th) at 8:00 am.