Hsp70 interacts with I278T CBS. A, yeast strains deleted for the indicated genes were grown in the presence of 4% ethanol, and total lysates were examined for Hsp70 and α-tubulin by immunoblot. B, yeast strain LS3 (α leu2 ura3 ade2 trp1 cys4::LEU2 SSA2::KanMX) was transformed with pI278T or phCBS, and saturated cultures were diluted 1:1000 in SC–Trp–Cys+G418 media and grown with aeration at 30 °C for 24 h in the absence and presence of ethanol. Growth was measured by A₆₀₀ (OD₆₀₀). C, WY35 or LS3 cells transformed with either phCBS or pI278T were grown in SC–Trp+G418+Glu media in the presence or absence of 4% ethanol as indicated. Cells were harvested, and CBS, Hsp70, and α-tubulin were assessed by immunoblot. D, yeast strain WY35 phCBS, WY35 pI278T, LS1 pI278T, and LS1 phCBS were grown in SC–Trp+Cys media with or without ethanol. Two hours before harvest, 50 μm MG132 was added, and cell 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.

Ethanol and MG132 rescue of I278T. WY35 phCBS and WY 35 pI278T were grown in SC–Trp+Cys media in the absence and presence of 4% ethanol. Where indicated, 50 μm MG132 was added 3 h before cells were harvested. Total cell lysates were then examined for CBS, Hsp26, and α-tubulin by immunoblot. CBS enzyme activity was measured for each extract in triplicate with the mean ± S.D. for each sample indicated.

Ubiquitin-dependent degradation of I278T and Hsp26. A, yeast strain LS4pI278TΔ was grown in SC–His–Leu–Trp+Glu at 30 °C to an A₆₀₀ of 0.7 and then either shifted to 37 °C or maintained at the current temperature as indicated. At the indicated times, total lysates were prepared and analyzed for CBS and Hsp26 by immunoblot. B, yeast strain LS3 transformed with either pI278T or phCBS was grown in SC–Trp+G418 to an A₆₀₀ of 0.7 and then treated with 50 μm MG132 for 3 h. Extracts were then subjected to immunoprecipitation (IP) with either Hsp26 and CBS antibodies. Immune complexes were analyzed for ubiquitin (Ub), CBS, and Hsp26 by immunoblot.

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.

Loss of Hsp104 does not affect I278T function. A, yeast strain LS2 (α leu2 ura3 ade2 trp1 cys4::LEU2 Hsp104::KanMX) was transformed with a plasmid expressing either I278T CBS (pI278T) wild-type human CBS (phCBS). Saturated cultures of the yeast were diluted 1:1000 in SC–Trp–Cys+G418 media and grown with aeration at 30 °C for 24 h in the absence and presence of 4% ethanol. Growth was measured by A₆₀₀ (OD₆₀₀). Experiment was done in triplicate, and error bars indicate standard deviation. B, LS2 cells transformed with either phCBS or pI278T were grown in SC–Trp+G418+Glu media. Cells were harvested, and CBS, Hsp104, and α-tubulin were assessed by immunoblot. Extracts were also examined for CBS activity in triplicate.

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.

Equilibrium model of Hsp26 acting as quality control chaperone. Missense mutant proteins are acted on by two competing systems. If bound by Hsp26, missense mutant proteins are ubiquitinated and directed to the proteosome for degradation. If bound by Hsp70, mutant proteins are refolded into an active conformation. Ub, ubiquitin.