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

Abstract

<jats:p>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 (<jats:italic>Saccharomyces cerevisiae</jats:italic>) has served as a seminal model in studies of membrane dynamics. We have previously established that yeast <jats:italic>ENV7</jats:italic> 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<jats:italic> in vivo</jats:italic> is dependent on <jats:italic>YCK3</jats:italic>, 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 (<jats:sub>328</jats:sub>SRFS<jats:sub>331</jats:sub>). 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.</jats:p>

Yeast cell death pathway requiring AP-3 vesicle trafficking leads to vacuole/lysosome membrane permeabilization

“…Proteins that are phosphorylated by Yck3 kinase are also candidate effectors of cell death, including Vps41 in the HOPS complex that tethers AP-3 vesicles to the vacuole and orchestrates subsequent fusion (Cabrera et al, 2010;LaGrassa and Ungermann, 2005). Other targets of Yck3 include the vacuole membrane kinase Env7 (Manandhar et al, 2020); the guanine nucleotide exchange factor (GEF) for Ypt7/Rab7, Mon1-Ccz1 (Lawrence et al, 2014); and the SNARE Vam3 (Brett et al, 2008). Our cell death screen identified the RAG-like GTPases Gtr1 and Gtr2 (Figure 2B), which are known to modulate the activity of the target of rapamycin complex 1 (TORC1) in response to nutrients and are anchored to the vacuole membrane by Ego1/ Meh1, a cargo protein of AP-3 (Hatakeyama et al, 2019).…”

Gene-dependent yeast cell death pathway requires AP-3 vesicle trafficking leading to vacuole membrane permeabilization

“…In this scenario, Yck3 could promote cell death by suppressing turnover of misfolded proteins and damaged membranes. Other proteins phosphorylated by Yck3, such as Env7 (vacuole membrane kinase) (Manandhar et al, 2020), Mon1-Ccz1 (GTP exchange factor/GEF for Ypt7/Rab7) (Lawrence et al, 2014), and the SNARE Vam3 (Brett et al, 2008) are also potential death effectors.…”

The nutrient-responsive CDK Pho85 primes the Sch9 kinase for its activation by TORC1

“…While at the moment this is only speculative, it is important to realize that the opposing roles of Pcl6 and Pcl7 were only observed in the pho80Δ background in which the phosphorylation of the Fab1 kinase and its regulator Vac7 are compromised [ 50 ]. Importantly, there are many other players involved in vacuolar fission/fusion, such as Env7 [ 94 ], the HOPS subunit Vps41 [ 95 ] or the I-BAR signature protein Ivy1 that seems to control the availability of PI3P for Fab1 at signaling endosomes [ 24 , 96 ]. Hence, further research is needed to fully understand the roles of Pho85 and the aforementioned cyclins in controlling the endosome-vacuole dynamics.…”