The vacuole of the fungus Saccharomyces cerevisiae has been a model for lysosomal trafficking, biogenesis, and function. It is hypothesized that mutants in non-essential yeast genes involved in protein trafficking at the late endosome to vacuole interface (ENV) would display an internal accumulation of immature proCPY at the late endosome, with secretion and late endosome/Golgi trafficking intact and functional. A genomic screen of the yeast haploid MAT-α deletion strain library of non-essential genes has yielded 15 candidate ENV genes, each confirmed to be deleted in the corresponding mutant. These mutants were shown to exhibit intact and functional secretion and trafficking at the Golgi and late endosome interface. The pro fragment of p2CPY was not detected. These results suggest each candidate env mutant has a defect in vacuolar trafficking and/or function at the post-endosomal stage. Based on the results of this study, we can now call these identified ORFs, ENV genes.