1. A kinase cascade on the yeast lysosomal vacuole regulates its membrane dynamics: conserved kinase Env7 is phosphorylated by casein kinase Yck3.
- Author
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Manandhar SP, Siddiqah IM, Cocca SM, and Gharakhanian E
- Subjects
- Amino Acid Motifs, Casein Kinase I genetics, Lysosomes genetics, Membrane Fusion, Protein Serine-Threonine Kinases genetics, Saccharomyces cerevisiae genetics, Saccharomyces cerevisiae Proteins genetics, Signal Transduction, Vacuoles genetics, Casein Kinase I metabolism, Intracellular Membranes enzymology, Lysosomes enzymology, Protein Serine-Threonine Kinases metabolism, Saccharomyces cerevisiae enzymology, Saccharomyces cerevisiae Proteins metabolism, Vacuoles enzymology
- Abstract
Membrane fusion/fission is a highly dynamic and conserved process that responds to intra- and extracellular signals. Whereas the molecular machineries involved in membrane fusion/fission have been dissected, regulation of membrane dynamics remains poorly understood. The lysosomal vacuole of budding yeast ( Saccharomyces cerevisiae ) has served as a seminal model in studies of membrane dynamics. We have previously established that yeast ENV7 encodes an ortholog of STK16-related kinases that localizes to the vacuolar membrane and downregulates vacuolar membrane fusion. Additionally, we have previously reported that Env7 phosphorylation in vivo depends on YCK3 , a gene that encodes a vacuolar membrane casein kinase I (CKI) homolog that nonredundantly functions in fusion regulation. Here, we report that Env7 physically interacts with and is directly phosphorylated by Yck3. We also establish that Env7 vacuole fusion/fission regulation and vacuolar localization are mediated through its Yck3-dependent phosphorylation. Through extensive site-directed mutagenesis, we map phosphorylation to the Env7 C terminus and confirm that Ser-331 is a primary and preferred phosphorylation site. Phospho-deficient Env7 mutants were defective in negative regulation of membrane fusion, increasing the number of prominent vacuoles, whereas a phosphomimetic substitution at Ser-331 increased the number of fragmented vacuoles. Bioinformatics approaches confirmed that Env7 Ser-331 is within a motif that is highly conserved in STK16-related kinases and that it also anchors an SXXS CKI phosphorylation motif (
328 SRFS331 ). This study represents the first report on the regulatory mechanism of an STK16-related kinase. It also points to regulation of vacuolar membrane dynamics via a novel Yck3-Env7 kinase cascade., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Manandhar et al.)- Published
- 2020
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