Protein Modification and Vesicle Transport in Cells

Explain how the Endoplasmic Reticulum (ER) maintains quality control on proteins.
Describe how large molecules are transported across membranes via vesicles.
Describe and interpret results from techniques for studying secretory pathways.
Summarize how vesicles bud from membranes.

Overview: All polypeptides extruded into the ER lumen undergo one or more modifications, which include:
- Folding by chaperone proteins: Every protein requires assistance in folding to achieve its functional structure.
- Binding Immunoglobulin Protein (BiP): A significant chaperone protein that aids in the folding and assembly of multiple polypeptides. It plays a crucial role in the Unfolded-Protein Response (UPR), signaling when proteins are misfolded.

Folding by chaperone proteins: Essential for the initial folding of all proteins.
Signal Cleavage: If a polypeptide has an N-terminus signal sequence, it will undergo signal cleavage to release the mature protein.
Glycosylation:
- Definition: The addition of oligosaccharides to polypeptides, turning them into glycoproteins.
- Mechanism: Conducted by glycosyltransferases (GTs); this modification may enhance protein stability.
Disulfide Bridges:
- Performed by: Protein disulfide isomerase (PDI) which forms or transfers disulfide bonds between cysteine residues.
- Importance: These bonds contribute to protein stability. For example, they are pivotal in the structure of insulin.
Other Protein Processing: Additional modifications may include methylation, phosphorylation, and acetylation impacting protein functionality.

Review Refresher: Identifies various methods for tracking protein movement through cells, including:
- Immunofluorescence
- Fluorescent tags
- Ion-sensitive fluorescent dyes
- FISH (Fluorescent In Situ Hybridization)

Overview of Budding Process:
- GDP to GTP exchange in GTPase.
- Vesicle coat formation on the cytosolic side of the membrane.
- The neck of the vesicle is stretched and pinched by dynamin or similar proteins.
- GTP hydrolysis leads to the release of the vesicle from the parent membrane.
- The protein coat disassembles after vesicle fission.

GDP is swapped for GTP in GTPase for activation.
The vesicle coat forms on the cytosolic side of the membrane.
Through the action of dynamin or similar proteins, the neck of the vesicle is stretched and pinched off.
GTP hydrolysis facilitates the vesicle's release.
The coat proteins disassemble, preparing the vesicle for its transport functions.

Investigative study on vesicle fission was conducted by Wei et al. (2024), demonstrating SEM (Scanning Electron Microscopy) images of clathrin-mediated endocytosis and various results that contribute to understanding vesicle dynamics and protein transport.

Components Involved:
- LDL Particles: Composed of a phospholipid monolayer and the ApoB protein.
- LDL Receptor: Interacts with LDL particles.
- Clathrin and AP2 Complex: Engaged in forming coated pits and vesicles to facilitate the internalization of LDL.

Vesicle Types:
- COPI: Retrograde transportation (e.g., from Golgi to ER).
- Coat Proteins: COP proteins and ARF.
- COPII: Anterograde transportation (e.g., from ER to Golgi).
- Coat Proteins: Sec proteins; Sar1 involved.
- Clathrin-Coated: Involved in various transport events.

Ire1 Pathway:
- BiP exists in dynamic equilibrium. High levels of unfolded proteins prompt BiP to diffuse from the Ire1 protein, causing Ire1 to dimerize.
- This dimerization leads to splicing of a transcription factor's transcript, which is responsible for producing more chaperone proteins.
ATF6 Pathway:
- Involves BiP diffusing away from ATF6, enabling ATF6 to translocate to the cytosol.
- After cleavage in the Golgi, ATF6 acts as a transcription factor in the nucleus, facilitating the ER-associated degradation (ERAD) pathway, which identifies and dislocates misfolded proteins for proteasome degradation.
PERK Pathway:
- When BiP diffuses from Protein ER Kinase (PERK), it allows PERK to dimerize and autophosphorylate.
- PERK then phosphorylates Eukaryotic Initiation Factor (eIF2), inhibiting translation within the cell, decreasing polypeptide entry into the ER.

A mutation in huntingtin protein (HTT) results in elongated chains of glutamine (polyQ) that accumulate in the ER.
This accumulation triggers the UPR, resulting in excessive calcium ions in the cytosol, leading to neuronal excitability and symptoms of Huntington’s disease.

Morales et al. (2023) Study: Focused on the influence of membrane cholesterol on the binding of LDL packages to their receptors, which shows that increased cholesterol decreases binding affinity, thus affecting LDL endocytosis.