Fusion of Ura3 mutants to membrane proteins.

<p>A. Fluorescence image of cells expressing Ura3-2 fused to GFP-Sso1, after incubation for two hours at 37°C. B. Growth curves of wild-type cells and those expressing soluble Ura3-2 or the same protein fused to GFP-Sso1. Mean and standard deviation of triplicate samples are shown. Note optical density is log scale. Cells were shifted to 37°C at time zero but there is a lag before growth slows, presumably due to pools of uracil. The membrane tethered form remained ts, though with a slightly longer lag. Similar results were obtained with Ura3-3. C. Cycloheximide chase of the Sso1 fusions at 37°C. D. Cycloheximide chase of equivalent fusions of Ura3-2 to Snc1 and Vam3. E. Cycloheximide chase of GFP-Use1 with and without Ura3-2 attached to it. Numbers to the left of the blots indicate the actual molecular weights of the proteins (kDa). SNARE fusions typically migrate more slowly than standard molecular weight markers, appearing about 10 kDa larger than they are.