Saccharomyces cerevisiae Env7 is a novel serine/threonine kinase 16 (STK16)‐related protein kinase and negatively regulates organelle fusion at the lysosomal vacuole

Abstract

Membrane fusion depends on conserved components and is responsible for organelle biogenesis and vesicular trafficking. Yeast vacuoles are dynamic structures analogous to mammalian lysosomes. We report that yeast Env7 is a novel palmitoylated protein kinase ortholog and negatively regulates vacuolar membrane fusion. Microscopic and biochemical studies confirm localization of tagged Env7 at vacuolar membrane and implicate membrane association via palmitoylation of its N‐terminal Cys13–15. in vitro kinase assays establish Env7 as a protein kinase. Site‐directed mutagenesis of Env7 APE motif Glu269to alanine results in an unstable kinase‐dead allele which is stabilized and redistributed to detergent resistant fraction by interruption of the proteasome system in vivo. Palmitoylation‐deficient Env7C13–15S is also kinase‐dead and mislocalized to the cytoplasm. Microscopic studies establish that env7蜐 is defective in maintaining fragmented vacuoles during hyperosmotic response and in buds. ENV7 function is not redundant with a similar role of vacuolar membrane kinase Yck3 as the two do not share a common substrate, and ENV7 is not a suppressor of yck3蜐. Bayesian phylogenetic analyses strongly support ENV7 as an ortholog of human STK16, a Golgi protein kinase with undefined function. We propose that Env7 function in fusion/fission dynamics may be conserved within the endomembrane system.