Saccharomyces cerevisiae Env7 Is a Novel Serine/Threonine Kinase 16-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 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 vitro kinase 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 system in vivo. Palmitoylation-deficient Env7C13-15S is also kinase dead and mislocalizes to the cytoplasm. Microscopy studies established that env7Δ 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 substrate, and ENV7 is not a suppressor of yck3Δ. Bayesian phylogenetic analyses strongly support ENV7 as 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.

ACKNOWLEDGMENTS

This project is funded by NSF-RUI research grant 0843569 to E.G. and NSF-MRI grant DBI0722757 for confocal microscopy. S.M. was supported by the NSF-RUI grant.

We are grateful to Greg Payne (UCLA) and his laboratory members for continuous support and reagents and to Simon Malcomber (CSULB) for guidance with the phylogenetic analyses. We thank Walter Schmidt (University of Georgia) for plasmids, Dieter Wolf (University of Stuttgart, Stuttgart, Germany) and Daniel Klionsky (University of Michigan) for yeast strains, Christian Ungermann (University of Osnabrück, Osnabrück, Germany) for the gift of anti-Vps41 antibodies, and Stephane Lefrancois (University of Montreal, Montreal, Canada) for advice on palmitoylation experiments. We also thank Maribeth Seranilla for construction of the 2μm ENV7-carrying plasmid and assistance with the ENV7-GFP strain experiments.