Figures S1-S16 and Table S1 and S2, which depict additional results (PDF) The authors declare the following competing financial interest(s): W.Z. is a co-founder of Velox Biosystems Inc., Baylx Inc., and Amberstone Biosciences Inc. J.L. has equity in Codiak BioSciences and holds the rights to multiple extracellular vesicle diagnostics and therapeutics patents.
Background: Env7 is a conserved palmitoylated kinase that regulates vacuolar fusion. Results: Specific Env7 palmitoylated cysteines direct its membrane localization, stability, and function. Conclusion: Distinct palmitoylation events direct Env7 membrane localization as well as its stability and function at the membrane. Significance: This may represent the largest set of functions attributed to palmitoylated cysteines in a single protein.
Palmitoylation at cysteine residues is the only known reversible lipid modification and has been implicated in protein membrane association as well as function. There is growing evidence that many palmitoylated proteins have regulatory roles in dynamic cellular functions including membrane fusion. Recently, we identified Env7 as a palmitoylated vacuolar membrane protein‐kinase involved in negative regulation of vacuolar fusion. Our phylogenetic analyses have established Env7 as an ortholog of human STK16, an endomembrane kinase with no known function. Env7 contains a conserved palmitoylation consensus sequence and substitution of the three consecutive cysteines (Cys13‐15) within that domain renders the protein non‐palmitoylated and cytoplasmic. In this study, we further dissect and define the role of individual cysteines of the consensus sequence in vivo. Our results indicate that all three cysteines serve as palmitoylation substrates, and any pairwise combination is essential and sufficient for near wild type levels of Env7 palmitoylation, membrane association, phosphorylation, and fusion regulatory function. Furthermore, individually, each can serve as a minimum requirement for distinct properties of Env7 in cells. Cys13 is sufficient for membrane association, Cys15 is essential for the fusion/fission regulatory function, and Cys14 or Cys15 is essential for protection of membrane bounded Env7 from proteasomal degradation. A role for Cys14 and Cys15 in correct sorting at the membrane is discussed. Thus, Env7 palmitoylation at the N‐terminal triple cysteines directs not only its membrane association, but also its stability, phosphorylation, and cellular function.
Grant Funding Source: Supported by National Science Foundation
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