2/12/26 Protein Trafficking and ER Function

Class Summary: Protein Synthesis, Folding, and Trafficking

Introduction

  • Initiated with a clicker question summarizing the previous class topics.

Complex Concepts of Translocon

  • Focus on the mechanism of how membrane protein orientation in the membrane is accomplished.

    • Key Points:

    • Determined during protein synthesis (translation) while the protein is still translocating into the endoplasmic reticulum (ER).

    • The translocon, a protein channel, facilitates proper orientation.

    • The translocon has positively charged and negatively charged portions which aid in orienting the incoming peptide.

    • Example: The positive portion of the protein faces the cytosolic side.

Juxtamembrane Residues

  • Definition: Refers to both the charges in the translocon and the charges of the incoming peptide, impacting the protein orientation.

Protein Insertion into Membrane

  • Integral membrane proteins insert into the membrane at the beginning of the secretory pathway, specifically during their entry into the ER.

  • Once inserted correctly and oriented, they remain anchored as membranes curve into vesicles for transport towards the Golgi complex.

  • The established orientation remains constant throughout the transport to the plasma membrane.

Orientation and Topology

  • The topology of membrane proteins is defined:

    • Parts protruding into the ER lumen will face the cell exterior when presented on the plasma membrane.

    • Modification: Glycosylation occurs to ensure proper orientation, which includes sugar markers that indicate their intended destinations.

Charges Within Protein Orientation

  • Positive charges face the cytosol, and negative charges face the ER lumen, ensuring the extracellular side is presented properly.

Protein Folding Mechanism

  • After translocation into the ER, proteins must undergo several critical processes:

    • Hydrophobic regions need to fold correctly for function, achieved with chaperone proteins:

    • Types:

      • Hsp70, Hsp40, Hsp90

      • Calnexin and Calreticulin

    • Chaperones assist in protein folding within the ER, which is crucial for their functionality.

    • Misfolded proteins must undergo the process known as ER associated degradation (ERAD).

Glycosylation

  • Appropriate glycosylation signals help in correct protein folding and stability:

    • N-glycosylation motif: N-X-S/T

    • The attached sugar chains (glucose and mannose) are covalently bonded to the nitrogen atom of the asparagine side chains indicating proper folding.

Sugar Modification Role

  • Serves to stabilize proteins during long transport processes through multiple vesicles.

  • Sugar chains contribute to proper folding that indicates the protein’s state.

ER-Associated Degradation (ERAD)

  • Misfolded proteins must be exported and degraded:

    • Requires removal of glycosylation before entering the cytosol to undergo degradation via ubiquitin marking for proteasome action.

  • Accumulation of misfolded proteins can signal the unfolded protein response, potentially leading to cellular death.

Mechanism of Vesicular Transport

  • Understanding specialized vesicle transport between the ER and Golgi:

    • Proteins cannot diffuse freely; they must be encapsulated in vesicles for movement.

    • The mechanism involves the budding and fusion processes of vesicles; proteins destined for different locations are recognized and sorted.

Evidence of Vesicle Formation

  • Experiments by Randy Schekman demonstrated critical insights into vesicular trafficking regulation using budding yeast as a model organism.

  • Observed that specific genetic mutations revealed critical components involved in vesicular structures and pathways.

Protein Coat Variability

  • Types of Coat Proteins:

    • Clathrin coat

    • COPI coat

    • COPII coat

    • COPII is primarily responsible for transporting proteins from the ER to the Golgi complex.

COPII Assembly Process

  • SAR1 Protein: Required for COPII coat assembly, converting from GDP to GTP before insertion into the membrane. Interaction with guanine exchange factors assists in this process.

    • Sec23/Sec24 Dimer: Binds to SAR1 GTP and helps select cargo proteins.

    • Sec13/Sec31 Oligomers: Form the outer scaffold of the vesicle.

Cargo Selection Process

  • Soluble cargo proteins must have exit signals recognized by receptors for their inclusion during transport, engaging in specific recognition events with coat proteins.

Vesicle Fusion and Recycling

  • Once at the Golgi, the vesicle membrane fuses, stripping off the COPII coat proteins.

  • Timing regulated via the RAB family proteins allows for specific docking and fusion.

  • All coat proteins and cargo receptors maintain a cycle, ensuring efficient recycling and proper delivery processes.

ER Resident Proteins Retrieval

  • Proteins meant to remain in the ER carry motifs (e.g., KDEL). These proteins can be retrieved if mistakenly transported to the Golgi through specific receptors, which also operate via a distinct COPI coat for retrograde transport.