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Yck3 Dependent Phosphorylation of Env7 and its Regulation During Cell Cycle in Saccharomyces cerevisiae
Author(s) -
Manandhar Surya,
Valencia Sara Patrice,
Alvarado Carlos,
Mansoora Ikha,
Gharakhanian Editte
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.542.8
Subject(s) - phosphorylation , microbiology and biotechnology , vacuole , saccharomyces cerevisiae , biology , kinase , protein phosphorylation , subcellular localization , serine , yeast , protein kinase a , biochemistry , cytoplasm
Yeast vacuolar fusion and fission is a highly dynamic mechanism that responds to intracellular and extra‐cellular changes working to keep cells intact. This mechanism has been well conserved from yeast to humans. We discovered that Env7 is a conserved vacuolar membrane kinase that is involved in negative regulation of vacuolar fusion during budding and stress, similar to that of Yck3. We have shown that these two vacuolar kinases interact genetically and double delete shows the compromised phenotype [Manandhar, S; Gharakhanian, E., FEMS Yeast Res., 472–80, 2014] Here we also show that the double delete is defective in autophagy further supporting the possible interaction between these two genes. Based on gel shift assay, we have established that the phosphorylation of native Env7 is dependent on Yck3 and can be complemented by exogenously expressed Yck3 in an expression dependent manner. Here we also show that Yck3 directly interacts and phosphorylates Env7 at specific sites. By using site‐directed mutagenesis we have identified the Yck3 dependent phosphorylation sites of Env7. Our data strongly suggest that serine residues at positions 323 and 331 (S323 and S331) are the Yck3‐mediated phosphorylation sites of Env7 in consistent to the prediction by Bioinformatic analysis. Phosphorylation of Env7 is required not only for the stability of Env7 but also for regulating vacuolar morphology dynamics. Since we have shown that Env7 is essential for normal vacuoles dynamics during budding, studies in phosphorylation state and localization of Env7 during cell cycle arrest and progression using confocal microscopy are in progress. We are also currently working on exploring the possible downstream substrates that are likely involved in above mentioned cellular events. Support or Funding Information This project is funded by NIH‐SC1 GM112560‐01 grant to Dr. Editte Gharakhanian This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .