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 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.

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.

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, 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.

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.

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

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.

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.

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.

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.

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 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.