20235_Lecture 10_BIOC212_Membranes 4

Page 1: Overview of Lecture Content

  • Lecture Topic: Membrane Proteins

  • Course: BIOC 212 Winter 2025

  • Lecturer: Maria Vera Ugalde

  • References:

    • Alberts et al., Molecular Biology of the Cell – Chapter 12: ER associated degradation, UPR

    • Smith et al. (2011), "Road to ruin: targeting proteins for degradation in the endoplasmic reticulum" Science 334, 1086-1090

Page 2: Comparing SRP and HSP70

  • Key Components:

    • SRP (Signal Recognition Particle):

      • Recognizes signal sequence and signal anchor in polypeptides.

    • HSP70:

      • Recognizes incorrectly folded or nascent polypeptides.

  • Binding Competitions:

    • Determine if SRP and HSP70 compete for the same binding sites.

  • Binding Regulation:

    • Explore how binding is regulated for both proteins.

Page 3: Protein Quality Control in ER

  • Mechanisms:

    • Disulfide Bonds:

      • Important for protein stabilization and folding.

    • N-linked Glycosylation:

      • Affects protein folding and quality control.

Page 4: Secretory Pathway Overview

  • Key Structures:

    • Lysosome, Nuclear Envelope, Endoplasmic Reticulum (ER), Late Endosome, Early Endosome, Cytosol, Cisternae of Golgi Apparatus, Vesicles, Plasma Membrane.

  • Diagram referenced: Figure 13-3b from Molecular Biology of the Cell 5/e (© Garland Science 2008).

Page 5: Secretory Proteins in the ER

  • Protein Synthesis:

    • All secretory pathway proteins synthesized and folded in the ER.

  • Degradation of Misfolded Proteins:

    • Managed through the ubiquitin-proteasome system.

    • Only pathway for returning misfolded secretory proteins to cytosol.

  • Quality Control Checkpoint:

    • ER acts as the quality control checkpoint for all organelles in secretory pathway.

Page 6: ER Protein Homeostasis System

  • Key Components:

    • BiP (HSP70), ERdj Proteins (DNAJ co-chaperones), NEF Co-chaperones.

    • GRP94 (HSP90): No co-chaperones.

    • Thioredoxin Family: Includes PDI and ERp57.

    • Calnexin and Calreticulin: Important chaperones.

  • Key Processes:

    • Role of UGGT, glucosidases, mannosidases, and lectins in glycan binding and protein folding.

    • Presence of ER stress response, known as Unfolded Protein Response (UPR).

Page 7: Role of BiP and Co-chaperones

  • Folding Assistance:

    • Substrate-binding DNAJ (ERdj3) helps in protein folding.

  • Translocon Interaction:

    • Engages with essential proteins like signal peptidase and OST for proper folding and post-translational modification.

  • Folding and Modification Process:

    • Occurs concurrently during translocation in the ribosome.

Page 8: Function of Protein Disulfide Isomerase (PDI)

  • Oxidizing Environment of ER:

    • Facilitates formation of disulfide bonds crucial for protein folding.

  • Thioredoxins Role:

    • PDI and ERp57 catalyze correct disulfide formation in substrates, improving fold efficiency.

Page 9: Disulfide Isomerization Process

  • PDI Role in Catalysis:

    • Formation and Rearrangement of disulfide bonds during protein folding.

    • Mixed disulfide intermediates formation necessary for the correct native state.

Page 10: PDI Regeneration Steps

  • Chemical Cascade Mechanism:

    1. Reduction of PDI after oxidative functions.

    2. Oxidation by Ero1 protein; uses FAD as a cofactor.

    3. Ero1 regeneration by FAD.

    4. FAD Involvement in maintaining oxidation potential in the ER.

Page 11: Calnexin and Calreticulin Overview

  • Structural Characteristics:

    • Calnexin (CNX): Lumenal domain with a TM anchor.

    • Calreticulin (CRT): Similar but without TM helix, ER retention signal.

  • Functionality:

    • Both recognize glycan patterns on polypeptides, binding through thioredoxin (ERp57) interactions.

Page 12: Calnexin Binding Process

  • Binding Mechanism:

    • CNX binds N-linked glycan patterns - specifically recognizing glycan with 1 glucose that indicates incomplete folding.

  • Role in ER:

    • Ensures retention of polypeptides until properly folded; initiates degradation if not folded correctly.

Page 13: N-linked Glycosylation in Proteostasis

  • UGGT Function:

    • UDP-glucose:glycoprotein glucosyltransferase binds with non-native polypeptides and re-adds glucose.

  • Selection Process:

    • Native polypeptides are not recognized by UGGT, allowing only misfolded ones to be targeted for correction.

Page 14: Calnexin and Calreticulin Cycle Summary

  • Cycle Steps:

    1. CNX retains polypeptide in ER.

    2. Glucosidase removes terminal glucose.

    3. UGGT restores glucose on misfolded polypeptides.

    4. Properly folded polypeptides exit to Golgi.

    5. Mannosidase maintains glycan trimming process for quality control.

    6. Mannose-binding lectins (EDEM) direct short glycans for degradation.

Page 15: ER Associated Degradation (ERAD) Overview

  • Objective: Degrades both lumenal and transmembrane polypeptides.

  • Regulation: Responds to signals for metabolic regulation and quality control prior to moving into the secretory pathway.

Page 16: ERAD Process Steps

  1. Substrate Recognition: Target substrates brought to E3 Ub ligase complexes.

  2. Polyubiquitination: Substrates marked for degradation and undergo retro-translocation.

  3. Deglycosylation and Degradation: Following translocation, substrates degraded by proteasome.

Page 17: BiP Role in ERAD

  • Function: Prevents aggregation of polypeptides that cannot fold properly.

  • Binding Complex: Involves specialized DNAJ (ERdj5) and lectin (EDEM) to target misfolded substrates.

Page 18: Substrate Targeting to E3 Ligases

  • Adaptors and E3 Ligases:

    • Involve transmembrane E3 ligases (e.g., HRD1, gp78) and various adaptors for recognition of misfolded proteins.

  • Interactions: Include derlins and other associated chaperones.

Page 19: Retro-Translocation Mechanism

  • Query on Mechanism:

    • Investigate whether retro-translocation requires a persistent pore or regulated opening mechanism.

  • Assistance from p97: Necessary to pull or unfold substrates for retro-translocation.

Page 20: p97 Mechanism in ERAD

  • Substrate Interaction:

    • Complexes with UB-binding adaptors to facilitate extraction of polyubiquitinated substrates.

  • Activities: Involves glycan removal and additional ubiquitination post-extraction.

Page 21: p97 and AAA Proteins Overview

  • AAA-Proteins: A large superfamily functioning as unfoldases and ATPases.

  • Structure and Function: p97 functions as a homo-hexamer with ATPase activity to extract proteins from membranes.

Page 22: ERAD Summary

  1. Recognition of misfolded proteins by lectins and chaperones.

  2. Disulfide bonds broken with DNJ action.

  3. Adaptors assist in guiding polypeptides to E3 ligases.

  4. E3 ligases carry out polyubiquitination and retro-translocation.

  5. p97 aids extraction from the membrane.

  6. Substrates undergo deglycosylation before proteasomal recognition.

Page 23: Stress Responses Overview

  • Cellular Adaptation: In response to stress causing misfolding, cells increase chaperone expression.

  • Heat Shock Response (HSR): Protects against cell death through cytosolic and nuclear protein responses.

  • UPR (Unfolded Protein Response): Specific to the ER, balancing recovery and cell death.

Page 24: UPR Mechanism

t- Activation Triggers: Result from unfolded protein accumulation in the ER, leading to an increased expression of chaperones and lipid synthesis.

  • Outcomes: Can lead to apoptosis if stress response is inadequate.

Page 25: Three Signaling Pathways of UPR

  • Components: Involves IRE1, PERK, and ATF6 as three sensors for misfolded proteins within the ER lumen.

  • Processes: Initiate responses offering regulated protein folding and degradation pathways.

Page 26: IRE1 and XBP1 Activation

  1. Functional Domains: Luminal, kinase, and RNase domains identified.

  2. Mechanics of Activation: Dimerization upon unfolded protein binding.

  3. Splicing of XBP1: Transforms unspliced (XBP1u) to spliced form (XBP1s) for transcription factor functionality.

Page 27: IRE1 Activation Process

  • Dimerization Trigger: Induced by direct binding of unfolded protein, releasing BiP from IRE1.

Page 28: Overview of PERK

  • Activation Processes: Dimerization and autophosphorylation upon stress trigger inhibition of translation with specific exceptions (e.g., ATF4).

  • ATF4 Role: Promotes further gene expression and apoptosis signaling.

Page 29: Integrated Stress Response (ISR)

  • Interaction of Kinases: Different stressors inhibit general translation while allowing certain mRNAs to be translated.

  • Relevance to ER Stress: Specific to PERK; other kinases respond to diverse stressors (e.g., proteasome inhibition).

Page 30: ATF6 Processing

  1. Localization: Naturally resides in the ER membrane; BiP masks exit signal.

  2. Signal Exposure: Occurs upon dissociation from BiP due to unfolded proteins.

  3. Function: Transcription regulation post-Golgi processing generates soluble ATF6(N) for UPR gene upregulation.

Page 32: UPR Summary Comparison

Component

Activation

Signaling

Transcription Factor

Output

IRE1

Binds unfolded proteins

RNase activates splicing

XBP1s

Chaperones, ERAD, lipid synthesis

PERK

Binds unfolded proteins

Inhibits translation

ATF4

XBP1, CHOP, cell death

ATF6

Ligand interaction frees Golgi signal

Processes gene transport

ATF6(N)

Chaperones, ERAD, lipid synthesis

Page 33: Substrate Binding Mechanisms

  • Comparative Analysis: Discuss how substrates are specifically bound by proteins such as BiP, GRP94, thioredoxins, calnexin, UGGT, HRD1, and gp78.