(a) GARP knockouts and LEM3 knockouts show highly correlating profiles in chemical genomics datasets. Correlation coefficients (CCs) between the profile of LEM3 and each other profile in the chemogenetic screen (Hoepfner et al., 2014) are plotted on the x-axis. Plotted on the y-axis are the similar sets of values for the VPS52 profile with all other profiles. (b) Depletion of VPS53 or LEM3 results in resistance to the cytotoxic PC analog miltefosine. WT cells, OsTir cells, Vps53-AID OsTir cells, vps53Δ cells, OsTir TEF_LEM3 cells, Vps53-AID OsTir TEF_LEM3 and lem3Δ cells were spotted on control plates (top left panel), plates containing 500 µM IAA (top right panel), plates containing miltefosine (lower left panel) or a combination of IAA and miltefosine (lower right panel). (c) GARP depletion results in changes of the cellular phospholipid composition. The lipidomic analysis of phosphoglycerolipids and sphingolipid intermediates from IAA treated OsTir Vps53-AID-6HA (black bars) or OsTir Vps53−6 HA cells are shown. Long chain bases (LCB), ceramides (CER) phosphatidic acid (PA), phosphatidyl-serine (PS), phosphatidyl-inositol (PI), phosphatidyl-ethanolamine (PE) phosphatidylcholine (PC) and phosphatidylglycerol (PG). (d) GARP depletion has a minor effect on the vacuolar lipidome. Same as c) except that lipids were extracted from enriched vacuoles. Error bars represent standard deviations from three different experiments. (e) Co-localization of mNeon-tagged Lem3 with mCherry-tagged Vph1 in a strain harboring OsTir Vps53-AID-6HA are shown in mock treated (control) and IAA treated cells (IAA). Quantification of 3 different experiments shows co-localization of vacuoles and Lem3 in approximately 15–20% of cells after 90 min IAA treatment compared to control cells (white bars). Additional addition of myriocin prevents Lem3 localization to the vacuole in IAA treated compared to control cells (black bars) (error bars show standard deviations).