Figure 2.

Cellular localization of WSC1. WSC1 was amplified by PCR and cloned into pMTS395 (39) to generate pMTS395-WSC1. This is a single-copy plasmid that contains a GFP (S65T) mutant gene, expressed under the control of a galactose-inducible promoter. The WSC1 gene was cloned 5′ to GFP. A functional test of this construct is shown in Fig. 6. (A) The strain KT626 was transformed with the GFP or the WSC1–GFP fusion construct. Transformants were selected on medium containing glucose and then grown for 12 h on galactose-containing medium. Cells were fixed in 4% paraformaldehyde followed by analysis using fluorescence confocal microscopy. (B) Cultures from A were diluted in glucose (glu)- or galactose (gal)-containing medium and grown for 12 h. Cells extracts were prepared and 20 μg of protein from membrane or cytosolic fractions was loaded unto a 10% gel. Immunoblot analysis was performed with a GFP monoclonal antibody (CLONTECH, product 8362–1).