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
Bibliographic record
Abstract
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 JA Postulated 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. Postulated 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.
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.002 | 0.001 |
| Meta-epidemiology (narrow) | 0.001 | 0.001 |
| Meta-epidemiology (broad) | 0.001 | 0.001 |
| Bibliometrics | 0.001 | 0.001 |
| Science and technology studies | 0.001 | 0.001 |
| Scholarly communication | 0.001 | 0.001 |
| Open science | 0.001 | 0.001 |
| Research integrity | 0.001 | 0.002 |
| Insufficient payload (model declined to judge) | 0.001 | 0.001 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".