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MCB Lecture 3 – ER and Golgi What is co-translational translocation? o The ribosome transfers the growing polypeptide chain through ER membrane as it is still translating. What sequence does the polypeptide chain have that targets it for cotranslational translocation? o An ER Signal Sequence (usually at N-Terminus) What is the name of the receptor on the ER Membrane that recognizes the ER Signal Sequence? o SRP – Signal Recognition Particle Once the Polypeptide Chain is shuttled into the ER through the translocon, what protein helps it fold correctly? o BiP – Binding Protein What enzyme in the ER Lumen cleaves the N-Terminal Signal from the Polypeptide chain as it enters the lumen? o Signal Peptidase What is needed for a Single-Pass Trans-Membrane Protein? o Internal signal sequence (Start Transfer Signal) o An additional hydrophobic region to act as a stop-transfer signal What is needed for a Multi-Pass Trans-Membrane Protein? o Internal signal sequence (Start Transfer Signal) o Stop Transfer Sequence o Second Start Transfer Sequence o Second Stop Transfer Sequence o ETC… What three chaperones work to correctly fold proteins? o BiP o Calnexin o Calreticulin Once the protein is inside of the ER Lumen, an oligosaccharide is added. Answer the questions below regarding this process: o What enzyme adds this on? Dolichol is a membrane-bound lipid that assembles and transfers the oligonucleotide. o Via what type of glycosylation is the sugar chain added onto the protein? N-Linked Glycosylation o What amino acids is involved in the reaction between the sugar chain and the protein? Asparagine (Asn) o What specific molecule in Asparagine participates in the reaction? NH2 o What two types of sugar molecules are the ones added to the protein? What are the terminal sugar molecules? 9 Mannose Sugars 3 Glucose Sugars Later on, some of the sugars that were added to the new protein in the ER are removed. How many/what sugars are removed? Where does this happen? o 1 Mannose o 3 Glucose o Happens in the ER The processing of this sugar chain continues in the Golgi. How can this chain be further processed in the Golgi (in general)? o Further N-Linked trimming or additions o O-Linked Glycosylation (OH) of selected Serine or Threonine Residues If a protein enters the ER, and it is folded improperly, chaperones attempt to re-fold it. If this is also unsuccessful, what happens to the protein? o Deglycosylated o Ejected back through the translocon and into the cytosol o Poly-ubiquinated and degraded in the proteasome Cystic Fibrosis is an often-fatal disease that first presents in children and progresses until death at a very early age. Answer the following questions about Cystic Fibrosis: o What is the mode of inheritance for Cystic Fibrosis? Autosomal Recessive o What protein has the mutation? CFTR o What is the function of this protein? Cl- ion channel o What is the specific mutation called? ∆F508 o How does the mutation affect the protein? It causes the protein to fold incorrectly, so it is ejected out of the ER and degraded in the proteasome. Recent studies have shown that if the protein could make it to the plasma membrane, it could still function properly (even though it is misfolded) Familial Hypercholesterolemia can be caused by mutations in LDL-R. Answer the following questions regarding this concept: o What does an LDL-R Mutation cause? Misfolding in the LDL Receptor Protein o When the protein is misfolded, what is the result? The receptor does not make it to the plasma membrane, so the free LDL outside of the cell cannot come in. o What are the effects of there being no receptors in the plasma membrane? LDL and cholesterol build up outside of the cells leading to cardiovascular problems. o Xanthomata are a symptom of high cholesterol levels. What is this? Fatty, yellow cholesterol deposits in the body that present as fatty projections on the skin. o Xanthelasmata are also a symptom of high cholesterol levels. What is this? Fatty, yellow cholesterol deposits seen on the upper eyelid. o Corneal Arcus is another symptom of high cholesterol levels. What is this? Presents as a light ring around the iris of the eye. It is cholesterol deposits. Targeting Signals direct proteins to a particular cellular location. Answer the following questions about targeting signals: o What is the default protein path if the protein has no ER signal sequence? Stays in the cytosol o If the protein has a NLS Signal Sequence, where does it go? Nucleus o If the protein has a SKL Signal Sequence, where does it go? Peroxisome o If the protein has a KDEL Signal Sequence, where does it go? ER o If the protein has a KKXX Signal Sequence, where does it go? ER o If the protein has a M6P Signal Sequence, where does it go? Lysosome o If the protein has an rER Signal Sequence, but no additional Signal Sequences, where does it go by default? If it has an Internal Hydrophobic Sequence, it goes to the Plasma Membrane If it doesn’t, it gets secreted. o Is KDEL soluble or membrane bound? Soluble o Is KKXX soluble or membrane bound? Membrane Bound Which side of the Golgi faces the ER? o Cis-Golgi Which side of the Golgi faces the Plasma Membrane? o Trans-Golgi What type of coat is added to the vesicles leaving the ER and going to the CisGolgi? o COPII What type of coat is added to the vesicles that are recycled from the Golgi back to the ER? o COPI What is the ERGIC? o ER-Golgi Intermediate Compartment, which is the intermediate stack between the ER and Golgi that vesicles fuse with as the continue through the pathway If KDEL and KKXX are carried forward with the vesicles to the Golgi, what protein coat is added to bring them back to the ER? o COP I Because KDEL is soluble, what is needed to recycle it back to the ER from the Golgi? o KDEL Receptors in the membrane that bind and group them together to be transported back There is a unique mechanism for creating COP coated vesicles to move them along the ER and the Golgi. Answer the following questions about COP coated vesicles: o What is on the membrane that allows the cargo proteins to congregate in a particular area to form a vesicle? Cargo Receptors o What is the molecule that binds the receptor and the COP Coat Proteins to bring them together? Adapter o This process takes energy. What G-Protein is used for this to take place? ARF/SAR-GTP o After the vesicle buds off, what happens to the G-Protein? It is hydrolyzed by GAP and the coat protein falls off, exposing SNAREs used for fusion. As explained above, ARF and SAR are G-Proteins that promote coat protein assembly. What is the difference between ARF and SAR? When is one used over the other? o ARF: COP I o SAR: COP II What exchanges GDP for GTP on ARF and SAR? o GEF What hydrolyzes GTP to GDP on ARF and SAR? o GAP Where are GAGs (and other carbohydrates) synthesized? o Golgi Explain the difference between the Vesicular Transport Model and the Cisternal Maturation Model. Which model is correct? o Vesicular Transport Model: The vesicles exocytose and endocytose from Golgi Stack to Golgi Stack until it gets from the Cis-Stack to the Trans-Stack. o Cisternal Maturation Model: The stacks move from Cis to Trans, with the vesicles inside of them and then recycle themselves. o **It is believed that the correct model is a blend between the two. Proteins that are destined for lysosomes are tagged with what? In what location are they tagged with this? o They are tagged with Mannose-6-Phosphate o They are tagged in the Cis-Golgi