Distinct Palmitoylation Events at the Amino-terminal Conserved Cysteines of Env7 Direct Its Stability, Localization, and Vacuolar Fusion Regulation in S. cerevisiae*

作者：Manandhar Surya P; Calle Erika N; Gharakhanian Editte^*

来源：Journal of Biological Chemistry, 2014, 289(16): 11431-11442.

DOI：10.1074/jbc.M113.524082

• Lysosomes
• Membrane Fusion
• Membrane Proteins
• Protein Kinases
• Protein Palmitoylation
• Protein Sorting
• Protein Stability
• Env7
• Yeast Lysosomal Vacuole
• Yeast Vacuolar Fusion

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 form of lipidation and has been implicated in protein membrane association as well as function. Many palmitoylated proteins have regulatory roles in dynamic cellular processes, including membrane fusion. Recently, we identified Env7 as a conserved and palmitoylated protein kinase involved in negative regulation of membrane fusion at the lysosomal vacuole. Env7 contains a palmitoylation consensus sequence, and substitution of its three consecutive cysteines (Cys(13)-Cys(15)) results in a non-palmitoylated and cytoplasmic Env7. In this study, we further dissect and define the role(s) of individual cysteines of the consensus sequence in various properties of Env7 in vivo. Our results indicate that more than one of the cysteines serve as palmitoylation substrates, and any pairwise combination is essential and sufficient for near wild type levels of Env7 palmitoylation, membrane localization, and phosphorylation. Furthermore, individually, each cysteine can serve as a minimum requirement for distinct aspects of Env7 behavior and function in cells. Cys(13) is sufficient for membrane association, Cys(15) is essential for the fusion regulatory function of membrane-bound Env7, and Cys(14) and Cys(15) are redundantly essential for protection of membrane-bound Env7 from proteasomal degradation. A role for Cys(14) and Cys(15) in correct sorting at the membrane is also discussed. Thus, palmitoylation at the N-terminal cysteines of Env7 directs not only its membrane association but also its stability, phosphorylation, and cellular function.

• 出版日期2014-4-18