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. 2012 Apr 1;23(7):1146–1156. doi: 10.1091/mbc.E11-07-0624

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© 2012 De Smet et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

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FIGURE 2: — The expression level of catalytically active Sct1p determines the cellular content of C16:0 and affects cell growth. (A) Deletion of the SCT1 gene decreases the cellular C16:0 content. Cells from pct1, sct1pct1, and gpt2pct1 strains cultured in SL to mid-log phase were analyzed for fatty acid content by gas chromatography. (B) Overexpression of SCT1 results in a fourfold increase of C16:0 content dependent on the catalytic activity of Sct1p. Cells from sct1pct1 pYES2 (empty vector control), SCT1pct1 pYES2 (chromosomal expression), sct1pct1 pYES2-SCT1 (overexpression) and sct1pct1 pYES2-SCT1-HH (overexpression of tagged version), and sct1pct1 pYES2-SCT1[G253L]-HH (overexpression of a catalytically dead mutant) strains were cultured to mid-log phase in SGR. In A and B the relative abundance (mol%) of the six major fatty acids is shown, with the error bars representing the SD for sct1pct1 (n = 3), sct1pct1 pYES2 (n = 4), and pct1 pYES2 (n = 5) and the variation for the other strains (n = 2). (C) Western blots comparing the levels of HA-tagged Sct1p chromosomally expressed and episomally expressed from the GAL1 promotor in the pct1 background (left) and of overexpressed catalytically active and inactive Sct1p-HA in sct1pct1 cells (right). Dilution factors of the protein extracts are indicated (left); Sec61p served as loading control. (D) Serial dilutions of the indicated strains precultured in synthetic glucose medium were spotted on SGR agar plates and incubated at 30°C for 3 d. (E) Growth of sct1pct1 pYES2 and sct1pct1 pYES2-SCT1 in liquid SGR medium supplemented with 0.05% glucose. Data averaged from four independent experiments.