7 mutant protein suggests that structural changes are limited largely to the ER lumenal face of the Sec61 channel, and that the cytoplasmic surface of the channel remains similar to wildtype. Combined with our experimental data this indicates that ribosome binding to Sec61L7 channels can proceed normally and ribosome binding likely stabilizes trimeric Sec61L7 channels Oligomycin A structure such that subsequent channel opening can proceed in the absence of the lumenal end of the lateral gate and L7. Ribosomes and proteasomes bind to different regions of the cytoplasmic face of the Sec61 channel, but the largely unaltered cytoplasmic surface of the Sec61L7 channel likely also explains why proteasome binding was not reduced. We were suprised by this observation because we had found previously that a point mutation in L7, S353C, reduces proteasome affin ity for the Sec61 channel.

It therefore appears that when it is present the conformation of L7 is important for proteasome interaction with the channel, and that conformation of L7 can be transmitted through the transmembrane helices to the cytoplasmic face of the channel. Our data regarding proteasome binding to Sec61L7 channels suggest that the defect in soluble misfolded protein export in sec61L7 cells shown in Figure 3 is not due to reduced proteasome binding. The relative contributions of slow import and slow export to the profound ERAD defect in sec61L7 cells are difficult to differentiate for posttranslationally imported substrates.

We observed progressive accumulation of soluble CPY in the ER over time which suggests that export may be even slower than import, possibly because there is a direct competition of the two processes for common factors. This phenotype is similar to the result of overexpression of CPY where increasing the load on the ER to cytosol transport pathway causes cytosolic shown, and Figure 3D and had only a modest defect in ERAD of CPY. That sec61Y345H causes an ERAD defect in the absence of a secretory accumulation of secretory precursors which could be alleviated by increasing the expression of SEC61. Co translational membrane protein integration was barely Carfilzomib affected in sec61L7. The strong defects in soluble protein import and ex port through the Sec61L7 channel indicate that in the absence of L7 the channel can no longer open properly in the transverse direction.

While integration of membrane proteins via lateral channel opening to wards the lipid bilayer is still possible, and re entry of simple transmembrane ERAD substrates is only mod erately delayed, transport of soluble proteins through the channel despite in either direction is strongly impeded, and the general slowdown in transport might lead to competition of biosynthetic soluble protein import and misfolded soluble protein export for ERAD. Import of KHN mediated by the BiP signal peptide which can use both posttranslational and cotranslational import pathways was barely affected in sec61L7 cells. This suggests that BiP likewise will be translo