Protein Mobilization and Membrane Proteins

Protein Mobilization

• Continuation of protein mobilization discussion.
• Introduction to membrane proteins and their role in establishing electric potential across the cell membrane.
• Brief mention of neural signaling, action potential, and synaptic transmission, now covered in detail in course 120.
• Focus on the foundational aspects of membrane potential.

Review of Protein Secretion and Membrane Insertion

• Review of receptor and secreted protein concepts.
• Discussion on how proteins are secreted out of the cell and how receptors are positioned in the membrane to transmit signals.
• The cell membrane acts as a barrier to maintain cell organization and prevent unwanted substances from entering.
• The hydrophobic middle layer of the cell membrane restricts the passage of proteins.
• Only small, uncharged molecules can diffuse through the cell membrane.
• Implications for drug development, where drugs must penetrate the cell membrane to reach intracellular targets.
• The process of inserting receptors into the membrane is similar to protein secretion.
• Ribosome translation of mRNA occurs in the cytosol.
• Proteins need to be directed to specific locations (nucleus, membrane, mitochondria, or for secretion) via signal sequences.

ER Signal Sequence and Protein Targeting

• The endoplasmic reticulum (ER) signal sequence directs proteins to the ER.
• Proteins destined for the cell membrane or secretion must first enter the ER.
• Signal recognition particle (SRP) recognizes and binds to the signal sequence.
• SRP transports the protein to the ER membrane.
• SRP receptor on the ER membrane binds to SRP.
• The signal sequence is transferred to a translocation channel, a tunnel-like protein in the ER membrane.
• Proteins are threaded through the translocation channel into the ER lumen.
• The signal sequence is hydrophobic and stays within the membrane.
• Ribosomes continue translation, threading the protein into the ER, sometimes with multiple ribosomes on a single mRNA.

Stop Transfer Sequence

• The stop transfer sequence is another hydrophobic sequence found further down the protein.
• It halts the threading process, stopping the protein in the middle of the membrane.
• The ribosome completes translation, leaving the C-terminus of the protein dangling.
• The N-terminus of the protein initially points into the cytosol due to how SRP loads the signal sequence into the translocation channel.
• The ER lumen is the space inside the ER membrane.

Signal Peptidase and Transmembrane Protein Formation

• Signal peptidase, an ER membrane protein, cleaves off the signal sequence.
• The signal sequence remains in the membrane, while the rest of the protein dangles inside the ER lumen.
• The N-terminus of the remaining protein now contains an NH_2 head.
• This process results in a single-pass transmembrane protein.

Protein Secretion vs. Transmembrane Protein Formation

• If a protein lacks a stop transfer sequence, the entire protein is threaded into the ER lumen.
• Signal peptidase cleaves off the signal sequence, allowing the protein to float freely in the ER lumen.
• Proteins without a stop transfer sequence are secreted out of the cell, e.g., epidermal growth factor (EGF).
• Proteins with a stop transfer sequence become transmembrane proteins, such as cell receptors like tyrosine kinase receptors.

Protein Sorting and Secretory Pathway

• Proteins move from the ER to the Golgi apparatus via vesicles, and then from the Golgi to the cell membrane via more vesicles.

Experimental Techniques to Study Protein Trafficking

• Green fluorescent protein (GFP) is used to track protein movement in cells.
• GFP was discovered in glowing jellyfish and can be fused to proteins of interest.
• GFP allows visualization of protein localization using fluorescence microscopy.
• Mutants are used to disrupt protein trafficking pathways.
• Mutations can cause proteins to get stuck in the ER, Golgi, or secretory vesicles.
• Epistasis analysis is used to determine the order of events in the protein trafficking pathway.
• Mutations that disrupt the ER-to-membrane pathway are often lethal, similar to cell cycle mutants.
• Conditional mutants (e.g., temperature-sensitive mutants) are used to study lethal mutations.
• At permissive temperatures (e.g., 25°C), the pathway functions normally, while at restrictive temperatures (e.g., 37°C), the pathway is disrupted.

Key Proteins in Vesicular Transport

• COPII: Involved in pinching off vesicles from the ER.
• Rab: A protein that tethers the vesicle and pulls it to the target membrane.
• SNARE: Proteins on the vesicle and target membrane that bind to each other to facilitate fusion.
• Clathrin: Involved in pinching off vesicles from the Golgi.

Protein Orientation During Trafficking

• The orientation of transmembrane proteins is established in the ER and maintained throughout the trafficking process.
• The intracellular signaling domain (e.g., tyrosine kinase) always faces the cytosol.
• The receptor domain faces the ER lumen initially, which eventually becomes the extracellular space.

Designing Proteins for Specific Purposes

• To engineer a receptor: Include a signal sequence, receptor domain, stop transfer sequence, and signaling domain.
• To make a secreted ligand: Include a signal sequence and the ligand sequence, but exclude the stop transfer sequence.

Multi-Pass Transmembrane Proteins

• Moving the signal sequence from the front of the sequence will turn it into a start transfer sequence.
• Proteins can span the membrane multiple times by incorporating multiple start and stop transfer sequences.
• The orientation of multi-pass transmembrane proteins depends on the arrangement of start and stop transfer sequences.

Car T-cell therapy

• Car T-cell therapy is a type of immunotherapy that uses genetically engineered T cells to target and destroy cancer cells.
• The T cells are engineered to express a chimeric antigen receptor (CAR) that recognizes a specific antigen on the surface of the cancer cells.
• When the CAR T cells bind to the cancer cells, they are activated and kill the cancer cells.