Unit 04 Pt3

Unit Overview

  • Unit 04: Intracellular Trafficking & Coats

  • Primary Reference: Molecular Biology of the Cell, 6th edition, by Alberts B, Johnson A, Lewis J, et al. (Garland Science; 2022)

  • Covers topics from Chapter 13 (Pages 749-807)

Outline of Topics

  • SNAREs Mediate Membrane Fusion

  • Interaction of SNAREs and the need for disassembly before reuse

  • Membrane Fusion Proteins from Viruses for Cell Entry

  • Transport Mechanisms from the Endoplasmic Reticulum (ER) through the Golgi Apparatus

    • COPII-coated Transport Vesicles

    • Proper folding and Assembly for ER Exit

    • Vesicular Tubular Clusters Role

    • Retrieval Pathway using Sorting Signals

    • Selective retention of proteins in their functional compartments

    • Structure of the Golgi Apparatus as a sorting station

SNAREs: Mechanism & Structure

  • SNAREs (Soluble NSF Attachment Protein Receptors) facilitate membrane fusion:

    • Transport vesicles must be tethered and then fuse with target membranes.

    • Require close proximity (approx. 1.5 nm) and exclusion of water for fusion.

  • v-SNAREs (vesicular SNAREs) and t-SNAREs (target SNAREs):

    • 35 different organelle-specific SNAREs

      • v-SNAREs: Made of a single polypeptide on vesicles

      • t-SNAREs: Composed of 2-3 proteins on target membranes

    • These proteins have helical domains that form a trans-SNARE complex during interaction.

Fusion Process

  • Energy from helices interaction drives membrane fusion:

    • Lipids flow between membranes to create a fusion stalk.

    • Completion of fusion leads to the formation of a new bilayer.

  • Regulatory aspects of fusion include:

    • Delayed fusion during regulated exocytosis requiring signaling processes.

Neurotransmitter Release

  • In neurons, neurotransmitter release occurs via synaptic vesicle fusion:

    • SNAREs play a critical role in vesicle fusion at synaptic junctions.

    • Toxins from bacteria like tetanus/botulism can interfere with SNARE function, leading to motor reflex issues.

SNARE Reuse and Regulation

  • Post-fusion disassembly of SNARE complexes requires:

    • ATP, NSF protein, and accessory proteins.

    • In Drosophila, NSF mutants (like comatose) affect vesicle transport.

  • Specificity in vesicle transport relies on correct Rabs and SNAREs, regulation mechanisms are still under investigation.

Cellular Fusion Reactions

  • Fusion is required in several biological processes:

    • Examples include fertilization and muscle fiber formation.

    • Viruses utilize fusion mechanisms:

      • Enveloped viruses (e.g., HIV) fuse with cell membranes after receptor binding.

      • Influenza enters via receptor-mediated endocytosis, utilizing low pH for fusion activation.

Transport from ER to Golgi

  • Golgi apparatus functions:

    • Major site for carbohydrate synthesis and enzymatic modifications (protein/lipid glycosylation).

    • Sorts cargo from the ER for further processing.

  • ER exit is selective and involves COPII-coated vesicles targeting correctly folded proteins.

Mechanisms of Exit from the ER

  • Proteins sorted for exit must have recognized exit signals:

    • Selectivity based on COPII coat components and folding state of proteins.

    • Specific receptors exist for key proteins like Factor V, related to blood clotting.

  • Proteins lacking exit signals can result in slow leakage to the Golgi.

Protein Quality Control

  • Misfolded/incompletely-folded proteins are retained in the ER, with chaperones masking exit signals.

  • Degradation pathways for defective proteins are critical:

    • Quality control ensures functional throughput and can impact disease (e.g., CFTR involvement in cystic fibrosis).

Vesicular Tubular Clusters

  • Homotypic fusion of transport vesicles from the ER leads to the formation of tubular clusters.

  • Requires matching v-SNAREs and t-SNAREs.

Retrograde Transport Mechanism

  • Vesicular tubular clusters facilitate COPI-coated vesicles for retrieval of ER proteins:

    • This process is known as retrograde transport, performing continual maturation as they approach the Golgi.

Sorting Signals in Retrieval to the ER

  • ER retrieval involves specific signal sequences binding to COPI coats:

    • C-terminal signals (e.g., KKXX for membrane proteins, KDEL for soluble proteins) manage packaging for retrograde transport.

Golgi Apparatus Structure

  • Comprised of stacked cisternae organized next to the nucleus:

    • Structures can be disrupted by microtubule alterations, showcasing dependence on cytoskeleton.

Golgi Functional Regions

  • The Golgi exhibits 'sided-ness' with distinct entry (cis) and exit (trans) faces for molecular processing:

    • Transport through the Golgi is associated with modification events crucial for function.

Research Questions

  • How do vesicles fuse with target membranes?

  • Compare intracellular vesicle transport and viral entry.

  • Discuss mechanisms returning ER-bound proteins to the ER, including coated vesicle interactions.

  • Distinguish Golgi complex regions and their unique functions.