Figure 5.

Spotting growth assay of yeast STT3 point mutations. (A) Growth phenotype of yeast strains carrying point mutations in yeast STT3. Cells carrying wild type (WT) and mutations were spotted on −His+FOA plates. The growth of the colonies at three different temperatures was compared after 2 days. ‘t.s.' stands for temperature sensitive. Upper panel, WT and two mutations in the WWDYG motif; middle panel, alanine-scanning mutations in and near the DK motif; lower panel, two non-alanine mutations in the DK motif. Note that there was a single large colony at both 25 and 30°C for the K586R mutant. We confirmed that they were revertants, and thus K586R is regarded as a lethal phenotype. (B) The incorporation of mutated STT3 into the yeast OST complex. HA-tagged STT3 mutants in a yeast cell lysate containing 0.15% digitonin were immunoprecipitated under non-denaturing conditions, using an anti-HA antibody. The absorbed proteins were resolved by SDS–PAGE, followed by western blotting using anti-yeast STT3, anti-yeast WBP1, and anti-yeast SWP1 antibodies. Note that the underglycosylation of WBP1 resulted in three bands (labeled as 1, 2, and 3), whereas SWP1 lacks N-glycosylation sequons. STT3 migrated diffusely probably due to its many TM segments, irrespective of glycosylation.