FIG. 5.
Conserved cysteine residues are critical for Isa1p and Isa2p function. (A) The isa1Δ haploid strain was transformed either with a control vector (pLJ100) or with the pLJ100-derived plasmids that express the wild type or the indicated mutant alleles of Isa1-HA; transformants were tested for complementation of the lysine and glutamate growth defects, as for Fig. 1B. (B) The wild-type strain BY4741 was transformed with plasmids for GAL1-driven expression of the wild type or the indicated mutant variants of Isa1-HA. Cells were initially grown in the presence of galactose, followed by the addition of glucose for the indicated time points to repress gene expression. Cell lysates were prepared and analyzed by Western blotting using an anti-HA antibody. (C) The isa2Δ strain was transformed either with a control vector (pLJ200) or with pLJ200-derived plasmids that express the wild type or the indicated mutant variants of Isa2-HA; complementation of the isa2Δ growth defect was monitored as shown in panel A. (D) Western blot analysis of Isa2-HA expression was monitored as shown in panel B.