| In Saccharomyces cerevisiae (baker's yeast), the cell vacuole is analogous to the mammalian lysosome. The simple eukaryotic system of yeast has served as a model system for investigating trafficking mechanisms of lysosomal hydrolases. Several mutants isolated in our laboratory, internally accumulate the precursor form of carboxypepetidase Y (p2CPY) and have been hypothesized to have a transport defect in the CPY pathway between the late endosome and the v&barbelow;acuole (env mutants). In this study, functional complementation was used to clone the defective gene in env3. Caffeine had been previously established as an agent that inhibited growth of the env3 strain. The inhibited growth of env3 on caffeine medium was also established to cosegregate with its mutant phenotype of p2CPY accumulation. Therefore, complementation of caffeine sensitivity was assessed for gene cloning. The env3 strain was transformed with a yeast genomic library and transformed env3 cells were selected for the complementation of caffeine growth defect. Clones viable on caffeine medium were subjected to immunodetection analyses resulting in the identification of 3 putative clones showing rescue of the p2CPY accumulation phenotype. This suggested that the three putative clones harbor a plasmid that encodes a wild-type allele of the gene defective in env3. The plasmid of one putative clone was isolated and sequencing of its genomic insert revealed three possible genes: NFS1, DCC1, and BUD3. These 3 genes will be further investigated to confirm the identity of ENV3. |
