Saccharomyces cerevisiae Env7 Is a Novel Serine/Threonine Kinase 16-Related Protein Kinase and Negatively Regulates Organelle Fusion at the Lysosomal Vacuole
ABSTRACTMembrane 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 here that yeast Env7 is a novel palmitoylated protein kinase ortholog that negatively regulates vacuolar membrane fusion. Microscopic and biochemical studies confirmed the localization of tagged Env7 at the vacuolar membrane and implicated membrane association via the palmitoylation of its N-terminal Cys13 to -15.In vitrokinase assays established Env7 as a protein kinase. Site-directed mutagenesis of the Env7 alanine-proline-glutamic acid (APE) motif Glu269 to alanine results in an unstable kinase-dead allele that is stabilized and redistributed to the detergent-resistant fraction by interruption of the proteasome systemin vivo. Palmitoylation-deficient Env7C13-15S is also kinase dead and mislocalizes to the cytoplasm. Microscopy studies established thatenv7Δ is defective in maintaining fragmented vacuoles during hyperosmotic response and in buds.ENV7function is not redundant with a similar role of vacuolar membrane kinase Yck3, as the two do not share a substrate, andENV7is not a suppressor ofyck3Δ. Bayesian phylogenetic analyses strongly supportENV7as an ortholog of the gene encoding human STK16, a Golgi apparatus protein kinase with undefined function. We propose that Env7 function in fusion/fission dynamics may be conserved within the endomembrane system.