A kinase cascade on the yeast lysosomal vacuole regulates its membrane dynamics: conserved kinase Env7 is phosphorylated by casein kinase Yck3

• Published in: The Journal of biological chemistry Vol. 295; no. 34; pp. 12262 - 12278
• Main Authors: Manandhar, Surya P., Siddiqah, Ikha M., Cocca, Stephanie M., Gharakhanian, Editte
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 21.08.2020; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Motifs; Casein Kinase I - genetics; Casein Kinase I - metabolism; Cell Biology; CK1 Yck3; Env7; Intracellular Membranes - enzymology; lysosomal vacuole; lysosome; Lysosomes - enzymology; Lysosomes - genetics; Membrane Fusion; protein phosphorylation; Protein Serine-Threonine Kinases - genetics; Protein Serine-Threonine Kinases - metabolism; regulation of membrane dynamics; Saccharomyces cerevisiae; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; serine/threonine protein kinase; Signal Transduction; subcellular organelle; vacuolar membrane dynamics; vacuole; Vacuoles - enzymology; Vacuoles - genetics; vacuolar membrane dynamics; vacuole; CK1 Yck3; serine/threonine protein kinase; regulation of membrane dynamics; subcellular organelle; Env7; protein phosphorylation; Saccharomyces cerevisiae; lysosome; lysosomal vacuole; membrane fusion
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA119.012346

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 (328SRFS331). 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.