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

Membrane fusion/fission is a highly dynamic and conserved process that responds to intra- and extracellular signals. While 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 down-regulates vacuolar membrane fusion. Additionally, we have previously reported that Env7 phosphorylation in vivo is dependent on YCK3, a gene that encodes a vacuolar membrane casein kinase I (CKI) homolog that non-redundantly 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 phospho-mimetic 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.