Crosslinking of wildtype membranes resulted in a single prominent crosslinked band which was about 10 kD larger than Sec61p. Immunoblotting on the crosslinked material with anti bodies against Sbh1p and Sss1p revealed that this band contained primarily Sec61p Sss1p heterodimers, selleckbio but a very modest amount of Sec61p Sbh1p heterodimers was also detected. In sec61L7 microsomes, the crosslink was at least 5 fold weaker com pared to wildtype membranes confirming changes in the interactions of Sec61L7p with Sss1p. We con clude that L7 of Sec61p is essential for hetero oligomeric stability of the Sec61 complex, and thus for stability of the Sec61 channel. Loss of L7 does not affect Sec61 complex interaction with the Sec63 complex The heptameric Sec complex consists of the trimeric Sec61 complex associated with the Sec63 complex com prising Sec62p, Sec63p, Sec71p and Sec72p.

Sec71p is the only glycosylated Sec complex subunit, association of the Sec61 complex with the Sec63 complex can there fore be demonstrated by co precipitation of Sec61p with the lectin ConcanavalinA. The heptameric Sec com plex is stable in digitonin. To ask whether L7 deletion in Sec61p had any effect on formation of the Sec complex, we solubilized wildtype and sec61L7 microsomes in digitonin and removed ribosome bound Sec61 complexes by ultracentrifugation. From the lysate, we precipitated the heptameric Sec complex using ConcanavalinA Sepharose and analysed both the amount of free Sec61 complex in the supernatant and the amount of ConcanavalinA associated Sec61 complex by Western Blotting.

Saturation of the precipitation was controlled by a second ConcanavalinA precipitation from the supernatant. In lysates from SEC61 wildtype membranes, the amount of Sec61p in the free fraction was 25 30%, and the remainder was found with the hepta meric Sec complex in the ConcanavalinA bound fraction. The amount of digitonin solubilized Sec61L7p was substantially lower than that of the wild type protein, and its distribution was also different, al most all detectable Sec61L7p was found in the ConcanavalinA bound fraction, and little if any in the free fraction. Inspection of the upper part of the gel showed that Sec61L7p forms SDS resistant aggregates in digitonin, in contrast to wildtype Sec61p.

The ratios of wildtype or mu tant Sec61p to Sec62p, however, were similar in the ConcanavalinA bound fractions suggesting no dramatic effects of the L7 deletion on heptameric Sec complex formation. Cilengitide Loss of L7 does not interfere with binding of proteasomes to the Sec61 complex Numerous mutations in SEC61 affect export of misfolded proteins from the ER to the cytosol for degradation by pro teasomes. In addition, proteasomes can bind directly to the Sec61 channel, and a specific mu tation in L7 affects proteasome binding.