Protein Trafficking (L11) Flashcards
Where are different types of proteins made?
Cytosolic proteins are made in the cytosol. Membrane proteins and secretory proteins are made in the rough endoplasmic reticulum. Nuclear proteins are made on the outer nuclear membrane.
What is the process of protein synthesis and trafficking?
Protein synthesis starts in the cytosol from mRNA. An mRNA encoding a cytosolic protein remains free in the cytosol. Ribosomes then join to it and are continually recycled during translation into the common pool of ribosomal subunits in the cytosol - known as the free ribosome cycle. In the rough ER, mRNA is bound to the membrane of the RER, and undergoes a membrane-bound ribosome cycle - these ribosomes are also added to the pool. The SRP cycle also occurs in the RER and takes ribosomal subunits from the cytosolic pool. mRNA molecules that have a specific ER signal sequence are trafficked to and associate with the ER. SRP (signal recognition particle) guides ribosomes to the right destination based on the signal sequence. SRP guides the ribosomes following binding to signal peptide and allows attachment of ribosomes to the translocator. RER makes secretory proteins like digestive enzymes and hormones like insulin. Following synthesis, proteins move into the smooth ER where lipids are made and vesicles form. ER-based protein removes signal peptides from secreted proteins e.g. insulin
What methods can be used to study protein trafficking?
You can use electron microscopy to look at the topography of ribosomes on ER membranes. They have subunits on the outside and protein translocator in the ER membrane. You can use differential centrifugation to separate heavy light vesicles - give you an insight into protein trafficking and membrane protein synthesis
How can proteins stay anchored in the membrane?
ER-based signal peptidase removed signal peptides from secretory proteins. Transmembrane proteins stay anchored in the ER membrane after their signal peptide is removed. Swapping the signal peptide for a lipid anchor in the ER can take place to continue membrane association (e.g. a GPI anchor)
How does protein maturation take place?
Protein maturation is a key function of the ER. The signal sequence is removed by a specific signal peptidase. This is followed by conformational maturation. Disulphide bridges are formed between cysteine residues to ‘solidify’ protein shape. The protein is glycosylated by a standard carbohydrate chain (quality control). ER-enzymes carry KDEL sequence (Lys-asp-Glu-leu) important for their return.
Addition of carbohydrates to proteins important for protein stability in the extracellular environment, cell-cell recognition (subtle changes have major effects), and cross-species separation (humans use beta galactose, animals use alpha).
What are the 2 types of glycosylation? How does this process happen?
- N-linked (uses asparginine)
- O-linked (uses threonine)
Initial addition of carbohydrates starts in the ER - by oligosaccharyltransferase. carbohydrate chains initially serve as quality control tags (if the protein isn’t folded properly the tag won’t be added properly). Tags are usually made up of glucose, mannose and N-acetylglucosamine.
The final addition of sugars and sorting takes place in the Golgi. Trimming and growth of carbohydrate chains proceed step-by-step in individual Golgi cisternae. This protein glycosylation is a multistep process involving the removal of things like glucose and mannose from the quality control signal. Starts off in the ER lumen then continues in the Golgi lumen. Each step requires separate Golgi cisternae to keep specific glycosylation enzymes away from each other.
How does the glycosylation of cells causes problems in clinical medicine?
Limitations in organ transplant are actually due to a simple sugar modification. Human cells make beta galactose which other anomals make alpha galactose. Humans will produce antibodies against alpha galactose and thus reject these transplants. Therefore, animal-derived organs are rejected by human organisms (e.g. using pig heart valves). However, you can genetically modify pigs lacking this galactose making transplantation possible.
What role do proteases play in protein maturation?
Proteases trim and activate hormones and enzymes during maturation of vesicles. Protein trimming takes place prior to secretion (e.g. for insulin a 24 amino acid sequence is removed from the middle section to make it functional). After trimming, proteins are packed into secretory vesicles. Immature secretory vesicles contain a clathrin coat, and when that is removed they become mature. Proteins can be constitutively secreted or regulated by receptors.
Explain what causes type 1 diabetes
Genetically determined, occurs due to a misfolding of insulin. protease in the secretory vesicle cannot cleave off the C-peptide. Secretion into the blood of the dysfunctional pro-insulin instead of insulin. This causes the generation of antibodies against the pancreatic cells (causes destruction of pancreas cells) This leads to a high conc. of blood glucose due to no working insulin. Normally, insulin triggers delivery of glucose transporters into the plasma membrane of muscle cells
