Hsp26 interacts with I278T CBS. A, yeast strain LS1 (α leu2 ura3 ade2 trp1 cys4::LEU2 hsp26::KanMX) was grown in SC–Trp+Cys, and total lysates were examined for CBS and Hsp26 protein by immunoblot. Extracts were also examined for CBS activity in triplicate. CR indicates cross-reacting band that acts as an internal control. B, yeast strain LS1 was transformed with either phCBS or pI278T. Yeast were grown in SC–Trp–Cys in the absence and presence of 4% ethanol, and growth was measured after 24 h using A600 (OD). C, indicated strains were grown in SC+Cys media, and total soluble extracts were prepared. Extracts were then subjected to native gel electrophoresis followed by immunoblot with native CBS antiserum. D, yeast strains WY35 phCBS and WY35 pI278T were transformed with a plasmid overexpressing Hsp26 (pHsp26). Strains were grown in SC–Trp–Ura+Cys media, and total lysates were examined for CBS, Hsp26, and α-tubulin by immunoblot. E, yeast strain WY35 phCBS, WY35 pI278T, LS1 pI278T, and WY35 were grown in SC–Trp+Cys media with or without ethanol. At this time, 50 μm MG132 (to prevent I278T degradation) was added, and cells were incubated an additional 2 h before lysates were prepared. Immunoprecipitation (IP) was performed with anti-Hsp26 antibody, and CBS present in the immune complexes was analyzed by immunoblot. Lane labeled control shows extract without immunoprecipitation.