mutants for accumulation of the ERAD substrate CPY. In these screens we repeatedly isolated sec61 mutants in which Tipifarnib Transferase ligation had taken place without an insert. To our surprise, these sec61L7 mutants were viable. Here we describe the characterization of the de fects in sec61L7, and compare them to those of the yeast equivalent of the diabetes causing mutation in mouse SEC61. Results Yeast expressing sec61L7 are viable In order to be able to investigate functions of L7 of Sec61p, we generated a sec61 variant with AatII and BstZ17I restriction sites close to the luminal ends of TMDs 7 and 8. After mutagenesis, mutant L7 DNA was ligated into the AatII and BstZ17I sites of sec61pRS315 and transformed into KRY461 yeast which contained wildtype SEC61 on a URA3 plasmid.

Transfor mants were selected on minimal media without leucine, and the wildtype SEC61 plasmid was counterselected on plates containing 5 fluoroorotic acid. We identified L7 mutants of interest by colony blotting for cells that accumulated the ERAD substrate CPY intra cellularly. To our surprise we repeatedly isolated sec61 mutants in which the AatII and BstZ17I ends of our construct had religated without an insert. Compared to a deletion of DER1, an ER membrane protein involved specifically in ERAD of sol uble secretory proteins, the accumulation of CPY in sec61L7 was more modest, but still detectable in a screen. Upon sequencing we found that in the mutant amino acids 305 371 of Sec61p had been re placed with two amino acids, arginine and glutamate, only, which is equivalent to deletion of the entire L7 and the luminal ends of TMDs 7 and 8.

TMD7 is part of the lateral gate important for channel opening during secretory protein import into the ER, and deleting L7 should lead to a decreased flexibility of the channel, thus we expected dramatic translocation defects in sec61L7 cells. We found that nevertheless sec61L7 cells grew like wildtype cells on plates at 37 C and 30 C, the mutant cells were cold sensitive at 20 C. The doubling time for sec61L7 was increased by 50%. We con clude that L7 of Sec61p, although functionally import ant, is not essential. Interference with protein homeostasis in the ER leads to activation of the UPR and hypersensitivity to tunicamycin, which interferes with N linked glycosylation in the ER and hence with protein folding.

The Sec61 complex is subject to UPR regulation and translocation defective sec61 mutants are frequently UPR induced and tunicamycin sensitive. When we incubated sec61L7 yeast on YPD plates Batimastat with 0. 25 ug ml or 0. 5 ug ml http://www.selleckchem.com/products/Sorafenib-Tosylate.html tunicamycin we found strong tunicamycin sensitivity at 0. 5 ug ml. The sec61L7 strain was also sensitive to 0. 25 ug ml tunicamycin in contrast to sec61 32 cells, the sec61 mutant with the strongest ERAD defect reported to date. Tunicamycin sensitivity of yeast expressing sec61Y345H which is homologous to the diabetes causing sec61Y344H in M. musculus was similar to sec61 32. We conclude that sec61L7 causes strong hypersensiti