FIG. 9.
SLM1 overexpression partially corrects growth and actin polarization defects of pkh1-ts pkh2Δ mutant cells. (A) Vector alone or plasmids containing PKH1, SLM1, SLM1S659D, or SLM1ΔCN, under the control of the galactose-inducible GAL1 promoter, were transformed into the temperature-sensitive pkh1-ts pkh2Δ mutant strain. Growth of transformants was tested on synthetic complete medium containing galactose as the carbon source and supplemented with 1 M sorbitol (SG, Sorb). Plates were incubated at the indicated temperatures and photographed after 3 to 4 days. (B) pkh1-ts pkh2Δ cells containing the indicated plasmids were grown at 26°C in medium supplemented with 1 M sorbitol and galactose. Cells were then shifted to 38°C for 2 h and fixed, and the actin cytoskeleton was visualized with Alexa594-phalloidin. (C) Small- to medium-budded pkh1-ts pkh2Δ mutants cells transformed with the indicated plasmids from panel B were scored for their actin polarization state. Cells were classified as having an actin cytoskeleton that was polarized (containing cables and polarized actin patches), partially polarized (containing cables and partially polarized patches), or depolarized (containing no cables and depolarized patches). One hundred fifty cells per sample were counted.