FIGURE 2.

Mutational analyses of the Bud13p-Snu17p interaction. a, interactions between Snu17p, Bud13p, and Pml1p were assayed by one-step Ni-NTA purification of wild type or mutant proteins co-expressed in E. coli. Supernatant (S) and pellet (P) fractions from bacterial lysates as well as flow-through (F) and elution (E1, E2, and E3) fractions from the Ni-NTA purifications are shown after Coomassie staining of the gels. M indicates the molecular mass marker with size indicated on the left in kDa. Note that Pml1p is less well stained than Snu17p and Bud13p. b, complementation of Δbud13 strains with an empty vector, or plasmids expressing wild type or mutant Bud13p was monitored through the growth of serial dilutions of transformants on selective plates at the indicated temperatures. c, Western blot showing the level of wild type and mutant Bud13p is detected through the fused TAP tag. d, complementation of a Δsnu17 strain was performed as for bud13 in b. e, the levels of wild type and mutant Snu17p were monitored by detection of the TAP tag by Western blotting.