1Y345H mutation causes a delay in the initiation of ERAD. Deletion of L7 effects on transmembrane protein ERAD Since we had detected a profound defect in soluble pro tein transport across the ER membrane in both directions in cells BTB06584? lacking L7 of Sec61p, but none in cotranslational import of transmembrane proteins, we decided to also investigate the fate of two transmembrane ERAD substrates in the sec61L7 strain. We first used pulse chase experiments to determine the half life of the single spanning transmembrane ERAD substrate KWW, and for comparison that of its soluble counterpart KHN. KHN consists of the yeast Kar2p signal peptide fused to the simian virus 5 HA neuraminidase ectodomain, and is imported into the ER using both the co and the post translational pathway.
As expected, it therefore was imported more efficiently into the ER of sec61L7 cells than preproCPY. Nevertheless we observed a dramatic increase in half life for soluble KHN, confirming the ERAD defect for soluble substrates in sec61L7 yeast. In the transmem brane ERAD substrate KWW the simian virus 5 HA neuraminidase ectodomain is fused to the single membrane spanning domain of the type I membrane protein Wsc1p. In wildtype cells KWW was degraded with a t1 2 of about 30 min comparable to its re ported t1 2 of 35 min. While the t1 2 of KWW was slightly increased in sec61L7 cells to approximately 50 min, the effect of the absence of L7 was modest compared to that on ERAD of soluble substrates. We next investigated the fate of Deg1,Sec62p, an ERAD substrate with two transmembrane domains and both termini in the cytoplasm, using cycloheximide chase experiments.
The cytosolic N terminus of Deg1,Sec62p contains an N glycosylation acceptor site which during ERAD is translocated into the ER lumen and modified. Unfortunately, the protein was poorly expressed in our strain background so the determination of its exact half life was problematic, and although we repeated the experiment several times, expression could not be improved. What can be seen on the blot, how ever, is that the glycosylated form of Deg1,Sec62p, for which ERAD had been already initiated by translocation of the N terminus into the ER Brefeldin_A lumen, was degraded with similar kinetics in SEC61 wildtype and sec61L7 cells. While in wildtype cells this glycosylated form was dominant, in sec61L7 cells the unglycosylated lower band was more prominent.
This lower band was largely stable in sec61L7 cells, dem onstrating again that L7 is essential for initiation of ERAD processes that require translocation of a soluble domain across the ER membrane. In contrast entry of TMDs into the lateral inhibitor Tofacitinib gate of the Sec61 channel during ERAD appears to be only moderately dependent on the presence of L7. Stability of Sec61L7p Deletion of 66 amino acids resulted in Sec61L7p migrat ing faster in SDS gels than wildtype Sec61p. The amount of Sec61L7p detected by immunoblotting with an N terminal antibody was only 70% compared to wildtype and sec61 32 cells
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