Volume 8 issue 10 October 2007
Regulation of copper-dependent endocytosis and vacuolar degradation of the yeast copper transporter, Ctr1p, by the Rsp5 ubiquitin ligase
J Liu, A Sitaram, and CG Burd

Supplemental Figure 1: Vacuolar targeting of Ctr1p(M260A)-GFP is blocked in the end4-1 mutant. Ctr1p(M260A)-GFP was expressed from the native CTR1 locus in an end4-1 mutant and vacuolar targeting and degradation was monitored before and 120 min after addition of copper sulfate (50 µM) to the growth media. Cells were grown at 26° C.

Figure 1 (.jpg)

Supplemental Figure 2: Ctr1p(M260A)-GFP copper transport-defective protein is ubiquitylated in a copper-dependent manner. Wild-type Ctr1p-GFP or Ctr1p(M260A)-GFP was expressed from the native CTR1 locus in an end4-1 mutant. At the indicated times after the addition of copper, cell extracts were prepared exactly as described in the legend to figure 5 and GFP-tagged Ctr1p was immunopurified using an affinity-purified anti-GFP antiserum. The immunopurified material was then probed with antibodies to ubiquitin and bound antibodies were visualized by enhanced chemiluminesence. Cells were grown at 26° C.

Figure 2 (.jpg)