FIGURE 1.

Functional rescue of I278T CBS by chaperone manipulation. A, saturated culture of yeast strain WY35 expressing I278T CBS (WY35 pI278T) was diluted 1:1000 in SC–Cys media with the indicated amount of ethanol at 30 °C for 24 h. Growth was measured by A₆₀₀ (OD₆₀₀). All cultures were grown in triplicate with the error bars showing standard deviation. A control strain expressing wild-type human CBS is shown on the right. B, WY35 pI278T cells were grown in SC–Trp+Cys media with the indicated percent of ethanol added to the media. Cells were harvested, and CBS and α-tubulin were assessed by immunoblot. CBS enzyme activity was measured in triplicate with the average and standard deviation shown. The far right lane contains extract from WY35 with no plasmid. ND = not determined. C, heat shock rescue of I278T CBS activity. Yeast strain WY35 pI278T was grown overnight at either 25, 30, or 37 °C, and extracts were assessed for CBS activity and CBS protein by immunoblot as described under “Experimental Procedures.” The yeast labeled 45 °C were exposed to a 3-h heat shock after overnight growth at 37 °C. All experiments were done in triplicate, and standard deviation is shown. D, WY35 phCBS and WY35 pI278T strains were grown in SC–Trp+Cys media with the indicated amount of ethanol overnight. Total lysates were examined for Hsp26, Hsp70, Hsp104, and α-tubulin by immunoblot.