345H mutation or deletion of L7 had any effects on the interactio

345H mutation or deletion of L7 had any effects on the interaction of the Sec61 channel with proteasomes. We had observed previously that solubilization of yeast membranes and reconstitution of inhibitor Palbociclib total protein into proteo liposomes improved proteasome binding to the mem branes. We therefore prepared proteoliposomes from wildtype, sec61Y345H and sec61L7 puromycin high salt treated microsomes and performed binding experiments with purified yeast 19S proteasome particles as described. As shown in Figure 5, we found no differences in pro teasome binding between wildtype and sec61Y345H proteoliposomes. Binding of 19S particles to sec61L7 proteoliposomes consistently was slightly higher than to wildtype SEC61 proteoliposomes.

We conclude that the ERAD defects observed in sec61Y345H and sec61L7 yeast are not due to defects in proteasome interaction with the Sec61 channels in the ER membrane. Discussion In this paper we have characterized a new sec61 mutant, sec61L7, which lacks the functionally important ER lumenal loop 7 and the adjacent ends of TMDs 7 and 8. The deletion shortens TMD7 of Sec61p to 14 amino acids which on its own is too short to span a bilayer. In the context of a polytopic membrane protein, however, the hydrophobic mismatch of an individual short TMD during membrane integration can be compensated by the surrounding TMDs which stabilize the short segment in the membrane. Our data suggest that the topology of Sec61L7p was un altered as cells expressing sec61L7 as sole copy of SEC61 were alive and growing.

Sec61L7p was expressed only to about 70% of wildtype protein levels, and while the protein was stable in a cycloheximide chase our data cannot exclude a slight defect early in Sec61L7p biogenesis. In cells ex pressing SEC61 from a GAL promoter, however, protein levels need to be reduced well below 50% before trans location defects occur, and heterozygous diploids with only one functional copy of SEC61 do not have ER translocation defects. It therefore seems unlikely that the expression level of the mutant protein per se was the cause for the trans location defects observed. The sec61L7 mutant was more sensitive to cold and tunicamycin than sec61 32 cells, and displayed a stronger UPR induction suggesting a more severe disturbance of ER translocation and ER protein homeostasis than in the sec61 allele with the strongest ERAD defect identified previously.

Mutant sec61L7 cells strongly accumulated soluble posttranslationally trans located preproalpha factor in the cytosol, and displayed a profound import defect Brefeldin_A for soluble post translationally translocated pCPY in both cycloheximide chase and pulse chase experiments. Association of the Sec61L7 complex with the Sec63 complex was normal, however, so the defect in posttranslational import must be due to a functional defect in the heptameric read this complex. Although the solubilized Sec61L7 complex was unstable, cotranslational membrane integra tion of DPAPB was barely affected. Modelling of the Sec61L

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