Fig. 3: Ykl075cp protein localization and links to branched-chain amino acid (BCAA) metabolism.

a Localization of Ykl075cp and the actin cytoskeleton in cells containing either native YKL075C or YKL075C tagged with the Myc epitope of 3 trials. Ykl075cp-13Myc visualized by immunofluorescence using anti-Myc antibodies and Alexa488-tagged secondary antibodies. The actin cytoskeleton was visualized by staining with Alexa568-phalloidin. Scale bar, 2 µm. b Top 15 gene ontology (GO) terms of ykl075c∆ cells. Two-side multiple hypothetic correction test with Bonferroni correction statistical analysis from Yeastract+ database. c Table of BCAA proteins with altered transcript levels in ykl075c∆ cells. d, e Validation of mRNA transcript levels of BCAA transaminases BAT1 and BAT2 by quantitative PCR. Decreased BAT1 and increased BAT2 gene expression in ykl075c∆ cells are significantly different from WT cells (unpaired two-tailed t-test; p values: 2.9e⁻⁰³ (WT vs. bat1∆) and 3.3e⁻⁰³ (WT vs. bat2∆)). Data is representative of three trials. f Quantification of free intracellular BCAA levels in WT, ykl075c∆, bat1∆, and ykl075c∆ bat1∆ cells using Cell BioLabs BCAA colorimetric ELISA kit. p values: 2.43e⁻⁰³ (WT vs. ykl075c∆), 9.00e⁻⁰⁴ (WT vs. bat1∆), 5.00e⁻⁰⁴ (WT vs. ykl075c∆ bat1∆), 1.50e⁻⁰¹ (ykl075c∆ vs. bat1∆), 4.16e⁻⁰² (ykl075c∆ vs. ykl075c∆ bat1∆), and 9.10e⁻⁰¹ (bat1∆ vs. ykl075c∆ bat1∆). g Serial dilutions of WT, aan1(ykl075c)∆, tor1∆, and aan1∆ tor1∆ cells recovering from either DMSO or 200 nM rapamycin treatment on YPD plates. h Growth rate (OD₆₀₀/h for 72 h) of WT, aan1(ykl075c)∆, tor1∆, and aan1∆ tor1∆ cells during recovery from DMSO or 200 nM rapamycin treatment (RR). p values of two biological repeats: 8.49e⁻⁰¹ (WT_DMSO vs. aan1∆_DMSO), 9.96e⁻⁰¹ (WT_DMSO vs tor1∆_DMSO), 8.06e⁻⁰¹ (aan1∆_DMSO vs tor1∆_DMSO), 5.21e⁻⁰¹ (WT_RR vs. aan1∆_RR), 1.90e⁻⁰³ (WT_RR vs tor1∆_RR), and 8.00e⁻⁰⁴ (aan1∆_RR vs tor1∆_RR). i Representative western blot of the phosphorylation of ribosomal protein-S6 (Rps6p) in mid-log phase WT, aan1(ykl075c)∆, and tor1∆ cells in the presence or absence of rapamycin (200 µM). TCE total protein loading control. j Quantification of Rps6p phosphorylation of panel i of untreated or rapamycin-treated WT, aan1(ykl075c)∆, and tor1∆ cells. Data are representative of three trials. p value: 3.50e⁻⁰² (non-parametric Kruskal-Wallis test). Error bars: SEM for panels d-f, h and j.