Figure 3. HCCS E159K is not able to complement S. cerevisiae CYC3-deficiency

A: Functional complementation of the S. cerevisiae strain B-8025. B-8025 was grown on minimal medium and transformed with human wild-type HCCS (HCCS WT), the HCCS mutant E159K (HCCS E159K), or yeast CYC3 (Cyc3p) expression constructs. Transformants were grown in liquid minimal medium, and aliquots of saturated (top row) and diluted cultures (middle and bottom rows) were spotted on glycerol medium containing copper (to induce expression of GST fusion proteins), and were incubated for 7 days at 30 °C. Dilution rates are indicated to the left of the figure. Note restoration of growth by ectopic expression of Cyc3p and HCCS WT, whereas no growth was observed for the untransformed strain and B-8025 expressing HCCS E159K. Strain B-7553 served as wild-type growth control. B: Expression of GST-HCCS fusion proteins in yeast strain B-8025. Expression of GST-HCCS E159K (left lane), GST-HCCS wild type (middle lane), and GST-Cyc3p (right lane) fusion proteins in the yeast mutant strain B-8025 grown in minimal medium was demonstrated by immunoblotting.