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Identification of Env7 Phosphorylated Residues
Author(s) -
Garfinkle Elizabeth,
Gharakhanian Editte
Publication year - 2015
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.29.1_supplement.724.2
Subject(s) - phosphorylation , vacuole , biochemistry , serine , saccharomyces cerevisiae , lysosome , mutant , kinase , wild type , cleavage (geology) , microbiology and biotechnology , chemistry , biology , yeast , gene , enzyme , cytoplasm , paleontology , fracture (geology)
The baker's yeast, Saccharomyces cerevisiae , vacuole is functionally analogous to the human lysosome. A novel genomic screen was performed in our laboratory to identify genes involved in the last stage of trafficking to the lysosome. ENV7 is one of four novel genes identified, and its product is involved in defects at the late endosome and vacuole interface. We have shown that Env7 is a phosphorylated palmitoylated protein kinase that is also conserved in humans. Based on bioinformatic studies in our lab on Env7, we hypothesize that Env7 phosphorylation is occurring at the specific C‐terminus serine residues 236, 287, 300, and 331. The objective is to isolate the phosphorylated C‐terminus fragment and identify its phosphorylated residues. A protease inhibitor assay was performed to maximize partially degraded C‐terminal fragments of Env7‐HA without excess protein degradation. The developed protocol was used to generate fragments of wild type Env7 and mutant Env7 Cys13‐15Ser, which does not autophosphorylate in vitro . The partially degraded protein samples were separated using SDS‐polyacrylamide gel electrophoresis and visualized using a Western blot. The phosphorylated C‐terminal fragments traveled a shorter distance relative to non‐phosphorylated Env7 due to their heavy weight. Wild type fragments showed a doublet band, whereas the corresponding fragment for Cys13‐15Ser showed a strong, lower single band. This shows that the strong, top band of the doublet present in wild type may be the phosphorylated residue of interest. It also proves that the Cys13‐15 track is necessary for kinase activity of Env7. More protocols will be developed to compare the phosphorylated fragments. Identification of the phosphorylated residues will be performed with mass spectroscopy of the isolated fragments.