S. cerevisiae ENV7 encodes a novel vacuolar membrane kinase involved in delivery or function at the vacuole and is an ortholog of human ser/thr kinase STK16

Abstract

Yeast vacuoles are functionally analogous to mammalian lysosomes. A genome wide screen by our lab has identified novel genes involved at endosome and vacuole interface (ENV). Here we present multifaceted characterization of the novel ENV7 gene product. A combination of confocal microscopic and biochemical studies confirm localization of tagged Env7p to vacuolar membranes. Bioinformatic analyses identified a highly conserved ser/thr kinase domain. We report that Env7p is a novel protein‐kinase capable of mediating both autophosphorylation and phosphorylation of exogenous substrates in vitro. Bayesian phylogenetic analysis strongly supports Env7p as an ortholog of human STK16, a ser/thr kinase localized to the endomembrane system with no known function. E269A site‐directed mutant of ENV7 is kinase‐dead. Interestingly, E269A mutant is unstable in vivo and shows aberrant patterns of membrane association. The mutant protein is significantly stabilized by chemical as well as genetic block of the proteasomal system. Based on these findings, we conclude that Env7p is a novel and conserved vacuolar membrane kinase whose phosphorylation and correct membrane association are essential for its in vivo stability. We also propose that misfolded or mislocalized Env7p is a substrate for proteasomal degradation.