Williams BCH 444 2014 Lectures 3-6

Williams room 5316 msb davidwilliamsutorontoca 19

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Unformatted text preview: alysts •  Protein quality control - ERAD and the UPR David B. Williams, Room 5316 MSB [email protected] 19 There s more than one way to find a translocon! -  Preprolactin mRNA encoding only 169 aa and NO STOP CODON. Yeast genetic approach Schekman Lab - Translate in vitro using wheat germ system in presence of SRP and ribosome-free ER microsomes. Include [35S] Met and LystRNA modified with photoactive cross-linker histidinol dehydrogenase - Nascent chain remains tethered to ribosome no stop codon (histidinol in growth medium) Classic biochemistry approach Rapoport Lab Fuse ER signal sequence to histidinol dehydrogenase *lys *lys *lys mutagenize engineered cells with ethyl methanesulfonate Gorlich et al. (1992) Cell 71:489 screen for mutants that can grow once more without added histidine UV obtained partially translocationdefective mutant (histidinol in growth medium) termed: sec61 transfect mutant cell clones with yeast cDNA plasmid library (only 1 plasmid per yeast cell) -purify protein of correct size from rough microsomes Deshaies and Schekman (1987) J. Cell Biol. 105:633 look for transfected yeast where (histidine required in the cDNA complements the mutant phenotype growth medium) i.e., it again dies without added histidine (functional translocation) -N-terminal sequence -screen cDNA library with degenerate oligos sec61 (histidine required in growth medium) cDNA sequence sequence cDNA in plasmid sec61 DNA sequence 20 Translocon composition Rapoport s group subsequently purified two other proteins co-purifying with sec61α: termed sec61β and sec61γ - sec61 complex Additional cross-linking experiments with shorter nascent chains identified additional components: - TRAM: translocating chain associated membrane protein - TRAP: translocon-associated protein complex Which components are essential for translocation? Reconstitution of minimal translocon in proteoliposomes: - add purified membrane proteins to mixture of purified phospholipids (PE, PS, PC and PI) - found that preprolactin synthesized in a wheat germ translation system (with added SRP) required only SRP receptor and the sec61 trimer. Therefore minimal translocon is Sec61 complex - addition of TRAM stimulated translocation up to 3-fold; other model proteins showed a stricter requirement for TRAM - [Gorlich and Rapoport (1993) Cell 75: 615] The translocon is highly conserved across species Mammalian cells Yeast Sec61α 40 kDa; 10 TM domains Ssh1p Sec61β 14 kDa; 1 TM domain Sbh1p Sec61γ 8 kDa; 1 TM domain Sss1p Bacteria TM = transmembrane (essential) SecY (essential) SecG (essential) SecE (essential) 21 Additional translocon-associated components TRAM: 36 kDa single polypeptide with 6 TM domains (enhances translocation of some proteins) TRAP: α, β, γ, δ subunits with a total of 7 TM domains (unclear function) Signal peptidase: 5 subunits with 7 TM domains (cleaves N-terminal signal sequences) Oligosaccharyl transferase (OST): 8-9 subunits with at least 21 TM domains (attaches oligosaccharide to Asn residues on nascent chains) The translocon environment - roughly to scale 22 Crystal structure of the SecY (Sec61) translocon Detergent-solubilized SecY complex from Metha...
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This note was uploaded on 03/27/2014 for the course BCH 444 taught by Professor Mccallan during the Spring '14 term at University of Toronto.

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