Extended Data Fig. 5. yArf1 regulates LD-associated functions and ß-oxidation.
(A) Growth test of yARF1 and yarf1-11 strains, deprived of the LRO1/DGA1 (∆lro1 ∆dga1) or of PEX34 (∆pex34), on rich media (YPD), rich media lacking saturated fatty acids (SFA) and containing cerulenin (Cer) (non-sup), containing SFA or SFA and cerulenin at 23 °C. (B) Co-localization of yArf1-GFP and yArf1-11-GFP with Lro1-3xmCherry grown at 23 °C or shifted to 37 °C for 30 min. Arrows indicate yArf1/yArf1-11 co-localizing or juxtaposed to Lro1. Scale bar: 5 µm. (C, D) In vivo TAG mobilization assay performed on yARF1 and yarf1-11 strains. Cellular TAG levels evidenced by thin-liquid chromatography extracted from cells grown in the presence of cerulenin or DMSO (C) were measured (D). Mean and standard deviation from at least n = 3 biological replicates are shown. (E) Peroxisome numbers evaluated by microscopy using Pex11-mScarlet in the yARF1- and yarf1-11-GFP strains grown at 23 °C or shifted to 37 °C. Peroxisome numbers per cell were quantified in each strain and conditions. Mean and standard deviation are shown; yArf1+Pex11-mScarlet 23 °C = 289 cells, yArf1+Pex11-mScarlet 37 °C = 431 cells, yArf1-11+Pex11-mScarlet 23 °C = 200 cells, yArf1-11+Pex11-mScarlet 37 °C = 304 cells from n = 3 biological replicates. Scale bar: 5 µm. (F) Co-immunoprecipitation of yArf1-GFP and yArf1-11-GFP with Pex13-6xHA. Strains were grown at 23 °C or shifted to 37 °C. (G) High-resolution microscopy of yARF1- and yarf1-11-GFP strains expressing Pex11-mScarlet grown at 23 °C or shifted to 37 °C. Arrows indicate Arf1 partially co-localizing with, or juxtaposed to peroxisomes. Scale bars: 5 µm. (H) mRNA levels measurement of POX1, PXA1 and PXA2 in yarf1-11 compared to yARF1 grown at 37 °C by qRT-PCR. Fold changes were measured by the 2−∆∆Ct method. Mean and standard deviation from n = 3 biological replicates are shown. (I) Growth test of yARF1, yarf1-11, ∆lro1 ∆dga1 or ∆pex34 strains, on YPD or media supplemented with 0.3 M sodium acetate at 23 °C and 30 °C. Source numerical data and unprocessed blots are available in source data.