Lecture 11-Vesicular Transport I- 2023

Lecture 11: Vesicular Transport

Overview

  • Chapter 15 dedicated to vesicular transport, crucial for cellular function.

Vesicular Transport Basics

Key Functions

  • Transport vesicles:

    • Bud from one membrane and fuse with another.

    • Key role of the Endoplasmic Reticulum (ER) as an entry point into various cellular destinations (secretory pathway).

Pathways for Cargo Movement

  • Cargo travels in membrane-bound intermediates through:

    • ER ➜ Golgi ➜ Vesicles ➜ Endosomes ➜ Lysosome ➜ Plasma Membrane.

  • Cargo destinations include:

    1. Secreted proteins in the extracellular space.

    2. Membrane proteins are delivered to the plasma membrane.

    3. Golgi complex for further processing.

    4. Lysosome for degradation.

Mechanism of Vesicular Transport

Exocytosis and Endocytosis

  • Exocytosis: Vesicles transport proteins and molecules out from the cell.

  • Endocytosis: Inward transport where extracellular molecules are imported into the cell.

  • Cargo can include both transmembrane and soluble proteins.

Vesicle Budding Process

Clathrin Coating

  • Clathrin forms a triskelion, cage-like structure for vesicle budding.

  • Budding occurs from:

    • Golgi apparatus (outward secretory pathway);

    • Plasma membrane (inward endocytic pathway).

Challenges in Vesicular Transport

  • Key logistical challenges include:

    • Vesicle budding

    • Vesicle docking/fusion

    • Vesicle transport

    • Recycling of components

    • Cargo selection.

Cargo Selection Mechanisms

Clathrin-Coated Vesicles

  • Clathrin alone does not select cargo.

  • Adaptor proteins (adaptins) assist in:

    • Cargo selection

    • Connecting the coat to the membrane.

  • Cargo has specific transport signals recognized by specific cargo receptors.

  • Interaction between adaptins and cargo receptors enables the selection of different cargo types.

Coats and Their Functions

Regulation of Coated Vesicle Formation

  • Initiated by small GTPases activated by GEF.

  • Types of coats:

    • COPII (Sar1 involved)

    • COPI and Clathrin (ARFs involved).

Vesicle Docking Mechanism

Importance of Tethers and SNAREs

  • Docking requires:

    • Recognition of specific organelle by incoming vesicle.

    • Rab GTPases and tethering proteins play significant roles.

    • Different Rab isoforms participate in varying transport events.

Role of Rab GTPases

Membrane Sorting

  • Over 60 Rab proteins present in humans, alternating between GTP/GDP states.

  • Distinct Rabs determine various transport stages, ensuring specificity in transport processes.

SNARE Protein Functionality

Fusion Mechanism

  • SNARE proteins mediate docking and fusion:

    • v-SNAREs on vesicles;

    • t-SNAREs on target membranes.

  • Fusion achieves:

    • Cargo delivery;

    • Membrane integration into the target compartment.

Lipid Bilayer Orientation Post-Fusion

Orientation Outcomes

  • Two possible orientations: a) Maintained orientation (exoplasmic face remains exoplasmic). b) Inverted orientation (exoplasmic face becomes cytosolic).

Protein Modification in the ER

Covalent Modifications

  • Majority of proteins entering the ER undergo chemical modifications:

    • Disulfide bonds enhance folding and stability.

    • Glycosylation aids in folding and provides transport signals.

    • Minimal glycosylation occurs in cytosol.

    • Preformed branched intermediates are added to asparagine residues during translocation.

Glycosylation Details

Mechanism

  • Glycosylation involves:

    • Signal consisting of three amino acids (e.g., Asn).

    • Sugar chain added to NH2 in Asn (N-linked glycosylation).

    • Initiates a series of modifications extending into the Golgi apparatus.

ER Exit Signals

Protein Retention in the ER

  • Enzymes within ER lumen encode specific signals:

    • Signal sequence

    • ER retention signal

    • Start transfer sequence for functionality.

Ensuring Protein Quality

Control Mechanisms

  • Exit from the ER is tightly regulated to ensure quality:

    • Misfolded proteins retained within the ER and targeted for degradation.

    • Chaperones assist in protein folding.

  • Unfolded Protein Response (UPR) activated during accumulation of misfolded proteins:

    • Stimulates production of additional chaperones;

    • Inhibits protein synthesis;

    • Adjusts ER volume.

Golgi Apparatus Functionality

Modification and Sorting

  • The Golgi apparatus:

    • Located near the cell nucleus and centrosome.

    • Composed of flattened membrane-enclosed sacs called cisternae.

    • Contains two sides:

      • cis-Golgi adjacent to ER;

      • trans-Golgi facing plasma membrane.

Exocytosis of Secretory Proteins

Exocytosis Pathway

  • Secretory proteins released through vesicles from the trans-Golgi network fusing to the plasma membrane.

  • Types of secretion:

    • Constitutive secretion: Unregulated release.

    • Regulated secretion: Restricted to specialized cells (e.g., hormones, digestive enzymes).

Secretory Vesicle Function

Storage and Release

  • Secretory vesicles store and release concentrated proteins:

    • Involve processes of docking and fusion with the plasma membrane.

    • Show examples in neurons (e.g., neurotransmitter release).

Cargo Selection Signals

Sorting Mechanisms

  • Specific amino acid sequences play crucial roles in cargo sorting:

    • Di-acidic sorting signal (Asp-X-Glu, DXE).

    • Interaction with coat proteins, membrane cargo receptors, and v-SNAREs.

Vesicle Trafficking Dynamics

Early Secretory Pathway

  • Retrograde transport: Retrieves v-SNAREs and membrane components, correcting sorting errors.

  • Anterograde transport: Moves proteins along the secretory pathway.

    • Involves COPI vesicles retrieving missorted ER resident proteins that possess the C-terminal KDEL sequence.

Retrograde Transport Mechanism

KDEL System

  • Utilizing the KDEL receptor:

    • Operates in the cis-Golgi network at acidic pH, enhancing receptor-protein binding.

    • Integrates ER resident proteins into COPI vesicles for transport back to the ER where a higher pH promotes their release from KDEL receptors.

Golgi Transport Dynamics

Non-vesicular Transport

  • Known as cisternal maturation, not mediated by vesicular traffic.

  • Modifying enzymes are transported forward through the Golgi:

    • Modifying enzymes from trans-Golgi migrate to medial-Golgi; medial-Golgi enzymes move to cis-Golgi by retrograde vesicular transport.

Summary of Golgi Operations

Cisternal Maturation Model

  • This model emphasizes the dynamic nature of Golgi function, with modification, sorting, and transport occurring concurrently to facilitate protein maturation and sorting.