FIG. 5.

Effect of mutation of Tif6p at Ser-174 and Ser-175 on its in vivo phosphorylation. The haploid yeast strains described in the legend to Fig. 4C were each grown separately in a 25-ml low-phosphate medium. At A₆₀₀ of 0.5, the cells were treated with 5 mCi of [³²P]orthophosphate (28). Following incubation for 3 h at 30°C, Tif6p was immunoprecipitated (IP) from each cell lysate by using agarose-conjugated anti-HA antibodies. The immunocomplexes formed in each case were washed; subjected to SDS-PAGE, followed by transfer onto a polyvinylidene difluoride membrane; and probed with anti-HA antibodies to detect HA-tagged Tif6p (upper panel). The blot was also analyzed by using autoradiography (lower panel).