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Drl1 is a putative lipase in Saccharomyces cerevisiae for which the exact role in the lipid metabolic pathways is still unknown. A previous genome-wide screening from our lab showed that Drl1 is involved in the regulation of diacylglycerol (DAG) localization, as normal DAG pools were disrupted in Drl1 knockout and catalytically dead strains. We aim to determine the role of Drl1 in lipid metabolism by studying its protein-protein interactions using TurboID, a more recent iteration of the BioID biotin proximity labelling technology, combined with an auxin-induced degron-fused Bpl1 to reduce endogenous biotinylation signal (https://doi.org/10.15252/msb.202211084). Streptavidin-HRP blots have shown that overnight incubation in auxin-enriched media greatly reduces signal from abundant endogenously biotinylated proteins, including Acc1, Hfa1 and Arc1. Furthermore, four-hour pulses of biotin combined with continued auxin incubation resulted in the appearance of additional bands on Streptavidin-HRP blots compared to controls, providing strong evidence that interactors of Drl1 are being biotinylated above background levels and would make promising candidates for Streptavidin-Sepharose affinity purification, with the end goal of sending enriched samples for proteomic analysis to identify likely interactors of Drl1. Different washes and elution conditions varying pH and denaturing agents, are currently being tested using Streptavidin-coated magnetic beads to determine optimum enrichment conditions for MS-MS analysis. This work has been financially supported by the Natural Sciences and Engineering Research Council of Canada (NSERC) grant to VZ.
Lalani et al. (Fri,) studied this question.