FIG. 5.

Newly synthesized GFP-Ras2 colocalizes with mitochondria in class C vps mutants. (A) GFP-Ras2 was expressed from the galactose-inducible promoter in wild-type (LBR938) and vps33Δ mutant (RJY1654) cells harboring Cox4-RFP. The localization of Cox4-RFP and GFP-Ras2 was examined by fluorescence microscopy, and representative images are shown. In each case, at least five fields and approximately 100 cells were examined. (B) The wild-type (LRB938) and vps33Δ mutant (RJY1654) strains expressing GFP-Ras2 were lysed, fractionated by a sucrose gradient as described in Materials and Methods, and immunoblotted with anti-GFP antibody. The plasma membrane fractions were identified by blotting with a polyclonal antibody to Pma1, the mitochondrial fractions were identified using anti-Por1 and anti-Cox2 antibodies, and an antibody to Dpm1 was used for the ER fractions. The immunoblots were quantified by densitometry and plotted as a function of fraction number, with fraction 1 being the top of the gradient.