PM331 Lecture 1, ER Translocation 2020_2021 copy

Membrane Trafficking Overview

• Textbooks: Molecular Cell Biology, Lodish et al., 8th Edition; Cell Biology, Pollard and Earnshaw 2nd Edition.

• Key Concept: Focus on protein translocation across the Endoplasmic Reticulum (ER) membrane.

The Poem "The Road Not Taken" by Robert Frost (1916)

• Themes: Choices and their impacts on life.

• Notable Lines:

  • "Two roads diverged in a yellow wood"

  • "I took the one less traveled by, and that has made all the difference."

Membrane Trafficking

Definition

• Study of protein delivery to specific cellular locations.

• Investigates cellular mechanisms that govern this process and implications of breakdown (disease).

Regulation

• Proteins have alternate fates based on their path through the cell.

Organelle Distribution

• Cell Types: Various organelles in both animal and plant cells.

• Importance: Proteins must reach correct organelles for functionality.

The Signal Hypothesis

• Founders: Günter Blobel and Bernard Dobberstein (1975).

• Core Idea: Proteins contain signal sequences, acting as addresses directing them to specific organelles (e.g., nucleus).

Protein Signals

• Signals vary depending on the destination organelle.

"Necessary and Sufficient" Concept

• Signal sequences are both necessary and sufficient for organelle targeting.

• Evidence: Removing a signal sequence prevents correct targeting.

• Diverting Signals: Adding a signal can redirect a protein’s destination.

Experimental Verification

Emr et al. (1984)

• Demonstrated that an ER signal sequence could target a cytosolic protein (β-galactosidase) to the ER using a chimeric construct.

Pathways to Cellular Destinations

• Proteins can go to various locations: ER, Golgi apparatus, mitochondria, chloroplasts (plants), peroxisomes, nucleus, plasma membrane, endosomes.

• Secreted proteins pass through the ER first and are synthesized in rough ER.

Anatomy of the Endoplasmic Reticulum

Network Formation

• ER forms a continuous network throughout the cytosol; it extends into the nuclear envelope.

Types of ER

• Rough ER: Ribosome-bound, site of protein synthesis.

• Smooth ER: Lacks ribosomes, involved in lipid synthesis and detoxification.

Protein Translocation Mechanism

Cotranslational Import

1. Initiation: Ribosome synthesizes protein; if ER signal present, targeting begins.

2. Translocation: Protein threaded through the Sec61 channel while still being synthesized.

3. Outcome: Proteins destined for secretion enter the ER for processing.

Experimental Insights on Secretory Proteins

• Sabatini and Blobel (1970) showed secretory proteins are quickly localized to the ER using radiolabeled experiments.

• Proteins are protected within the microsomal membrane from proteolysis, indicating successful translocation.

Proteins and Gel Filtration Chromatography

• High molecular weight proteins are retained while smaller ones elute quickly; methods distinguish between translocated and non-translocated proteins.

The Signal Hypothesis Revisited

• Blobel and Dobberstein solidified the concept through their 1975 publication, observing that missing segments in nascent proteins (e.g., IgG) acted as targeting signals.

Translocon Structure

• The Sec61 channel opens to allow protein translocation into the ER; it consists of several critical components, including a gating mechanism that responds to the presence of the nascent chain.

Types of Membrane Proteins

Classifications

1. Type I: Cleavable signal sequence; N-terminus in the ER, C-terminus in the cytosol.

2. Type II: Internal signal anchor, no cleavable signal.

3. Type III: Orientation determined by charged residues.

4. Type IV: Polytopic; traverses the membrane multiple times.

5. GPI-linked Proteins: Attached to lipids after translocation.

Post-Translational Translocation

• In yeast, some secretory proteins enter the ER after translation, facilitated by chaperones like BiP to assist in protein folding within the ER lumen.

Protein Quality Control in the ER

Processes

1. Protein Folding Acceleration: Enhances the correct folding of new polypeptides.

2. Unfolded Protein Response (UPR): Activates stress responses when proteins misfold.

3. ER-Associated Degradation (ERAD): Responsible for eliminating misfolded proteins.

Conclusion

• Understanding ribosome function, membrane protein targeting, and the implications of ER quality control is vital in elucidating protein functionality and its broader impact on cellular health.