FIGURE 7:

Growth analysis of yeast cells devoid of or with strong overexpression of Mcp2. (A) Strong overexpression of Mcp2 is toxic for WT yeast cells. WT cells were transformed with the empty plasmids pYX113 and pYX142 (∅): the empty plasmid pYX113 together with the pYX142-Mcp2 construct (TPIprMCP2 or TPIprMCP2-HA) under control of the TPI promoter or the empty plasmid pYX142 together with the pYX113-Mcp2 construct (GAL1prMCP2 or GAL1prMCP2-HA) under control of the GAL1 promoter. Cells were grown to logarithmic phase and spotted on SD-Ura-Leu or SGal-Ura-Leu plates in a 1:5 dilution series. Plates were incubated for growth at 30°C. (B) Expression levels of HA-tagged Mcp2 under control of the TPI and GAL1 promoter. Cells as in A were grown to midlogarithmic phase and crude mitochondrial fractions were analyzed by SDS–PAGE and immunodecoration with antibodies against the HA-tag or Tom40, a MOM protein, as loading control. (C) The toxic effect of Mcp2 overexpression depends on the conserved kinase residues of the protein. WT cells were transformed with the empty plasmid pYX113 (∅) or with pYX113 encoding the indicated variants under control of the GAL1 promoter. Cells were grown to logarithmic phase and spotted on SD-Ura and SGal-Ura plates in a 1:5 dilution series. Plates were incubated for growth at 30°C. (D) Increased sensitivity to oleate of yeast cells lacking Mcp2 at high temperatures. WT cells of different mating type (W303a and α), as well as mcp2Δ cells with different genetic background, were grown to logarithmic phase and spotted on synthetic medium containing glucose (SD) as well as on oleate-containing medium containing 0.1% glucose (YNBGO) in a 1:5 dilution series. Plates were incubated for growth at 37°C.