FIGURE 3.
Levels of VTC subunits in vtc mutants. A, influence of Vtc5 on proteins on isolated vacuoles. 30 μg of vacuolar protein from BY4741, vtc5Δ, and BY4741 PGPD1-Vtc5 were analyzed by SDS-PAGE and Western blotting with antibodies against the indicated proteins or the HA tag. B, influence of Vtc5 on proteins in whole-cell extracts. Whole-cell extracts were prepared from cells expressing Vtc4 from its endogenous promoter with an HA tag, grown in YPD to logarithmic phase. Proteins were analyzed as in A. Note that we used Vtc4-HA in this case because Vtc4 is difficult to detect in whole-cell extracts with our antibodies. C, quantification of the Vtc4 levels from A (white bars) and B (black bars) from three independent preparations each. n.s., not significant. D, influence of Vtc2, Vtc3, and Vtc4 on the level of Vtc5. Vacuoles were isolated from the indicated BY4741 strains expressing Vtc5 with or without a GFP tag. Vacuolar proteins were analyzed as in A. Vam3 was used as a loading control. The migration of relevant molecular mass markers (size in kDa) is indicated on the right-hand side of the panels.