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Creation of a real‐time fluorescence assay of the yeast PI4‐kinase, Pik1, to test the role of the PI/PC transfer protein Sec14 in the biosynthesis of phosphatidylinositol‐4‐phosphate
Author(s) -
Meehan Kailey,
Panagabko Candace,
Atkinson Jeffrey
Publication year - 2019
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.2019.33.1_supplement.480.2
Subject(s) - phosphatidylinositol , biochemistry , kinase , diacylglycerol kinase , protein kinase a , phospholipid transfer protein , phospholipid , myristoylation , microbiology and biotechnology , chemistry , biology , protein kinase c , membrane
The yeast phosphatidylinositol‐4 kinase Pik1 is critical for successful vesicular traffic from the trans‐Golgi to the plasma membrane and endosomes. Although a soluble enzyme, the small myristoylated, Ca2+‐binding, EF hand protein, frequenin (Frq1) facilitates its membrane localization. It has been suggested that the PI/PC transfer protein Sec14p assists Pik1 in locating its substrate by presenting membrane embedded PI to the kinase. In order to test this hypothesis, we have employed a fluorescence‐based kinase assay (BellBrook Labs) that reports production of ADP following the lipid kinase's consumption of ATP. We used this kit to create a liposome‐based real‐time assay for the measurement of enzyme activity. The assay was developed and validated with a commercial lipid kinase to optimize signal from phospholipid vesicles prior to application to Pik1. A yeast expression system for Pik1 was created to preserve the stability of the 125 kDa enzyme by co‐expressing both Frq1 and the heat shock protein, Cdc37, producing the Pik1‐Frq1 complex with a removable 10xhistidine tag on Pik1. Following purification on metal affinity resin, our goal was to measure the purified kinase activity under different supporting lipid conditions and in the presence and absence of Sec14p, a protein hypothesized to act as a presenter of PI to the kinase enzyme. Support or Funding Information Natural Sciences and Engineering Research Council of Canada, Discovery Grant (RGPIN‐155187 and RGPIN‐2017‐06149) to JAPostulated mechanism for the substrate presentation model of Sec14 which partially extracts phosphatidylinositol from a lipid bilayer and raises it to the membrane‐resident PI4‐kinase Pik1.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .