2. BIO201A Cell Biology Lecture 29 Wednesday 04/04/07

3. Important Announcements: – Next exam will be the Final given on May 8th. 34 questions over section 3 and 14 lab questions. – Final exam is 11:45AM to 2PM in four rooms: NSC 201, NSC 218, Norton 112 and OBrian 109. Information later on which room you will need to use. – Exam 2 scores are posted to UBLearns. – Exam 2 key and responses will be posted soon.

4. Synthesis of proteins on RER Signal sequence: Leads nascent protein to the RER and through the translocon 1.It is represented by 6-10 non-polar amino acids near the end of a nascent protein 2.It targets nascent proteins for the RER 3.It is required for cotranslational translocation. This process requires: A. Signal recognition particle (SRP) B. Translocon channel C. GTP hydrolysis to provide the energy for translocation 4.In the RER, the completed protein is modified by chaperones and other factors to put it in its proper conformation and structure

5. Synthesis of integral membrane proteins on RER Note: Steps 1 through 3 are the same as in fig. 8-12, but the SRP and SRP receptor aren’t shown. See fig. 8-12 for descriptions of steps 1-3. 4. If the nascent protein has a hydrophobic STOP TRANSFER SEQUENCE it will stop further translocation during translation and make it an integral membrane protein. If it doesn’t have a stop-transfer sequence, it will be a soluble protein instead of an integral membrane protein 5. Some translation may continue until complete 6. The translocon opens, leaving the integral membrane protein stuck in the membrane Two amino acid sequences are necessary for the synthesis of an integral membrane protein: Signal sequence and stop-transfer sequence

6. Directed movements of transport vesicles by COPI, COPII and Clathrin Clathrin: Moves materials from TGN to lysosomes, endosomes and plant vacuoles. Also from plasma membranes to endosomes COPI: coat proteins for retrograde movement toward RER COPII: Anterograde from RER VTC: Vesicular Tubular Clusters (not vesicles) ERGIC: Endoplasmic Reticulum Golgi Intermediate Compartment

7. What do the COP proteins and Clathrin do? 1.Help to form vesicles by causing membrane curvature and budding 1.Help provide a mechanism for selecting proteins to go into vesicles 3. Help provide a mechanism for vesicle identification so they can go to the right place

8. Concept for Transmembrane Receptor binding Transmembrane receptor in its native conformation. COP can’t bind Cytoplasm Lumen membrane Soluble cargo Cargo binds to receptor. Conformation of receptor changes -- - -- - +++ +++ Now COP can bind COP

9. Coat Protein Trafficking of soluble proteins in vesicles from RER 1. A transmembrane cargo receptor, facing the lumen of RER, binds specific cargo only inside the RER 2. The other end of the receptor protein, the part facing the cytoplasm, binds a specific coat protein. This labels the outside of the vesicle to tell anything in the cytoplasm what is inside 3. The coat protein can help the membrane to bud off and may help to traffic the vesicle to its appropriate destination 1. 2. 3.

10. How to sort and retain proteins from Golgi into COPII coated vesicles 1.Soluble proteins (cargo) in the lumen bind to specific transmembrane cargo receptors which face the lumen. This targets them to go into the vesicles. 2.COPII proteins bind to the cytoplasmic part of the cargo receptor 3.The exterior COPII targets the vesicle to move in an anterior direction 1. 2. 3.

12. membrane Clathrin Adaptor Lysosomal enzyme (cargo) A transmembrane receptor for clathrin binding and cargo trafficking

13. One way for a vesicle to know where to go V-SNARE: Protein on vesicle T-SNARE: Protein on the target membrane

14. Retrieval of RER enzymes that get into Golgi RER proteins that contain the amino acid sequence KDEL (lys, asp,glu,leu) should stay in RER Some RER enzymes accidentally “escape” to Golgi in COPII vesicles These enzymes can be retrieved by KDEL receptors in COPI vesicles and returned to RER Could a lysosomal enzyme be “fooled” to go to RER by adding a KDEL sequence to it? Could an RER protein be forced to leave by changing the KDEL to something else?