Phospholipid Analysis in Genetically Modified Yeast by Fourier Transform Mass Spectrometry
by Wilkins, Charles L.; Batoy, S. Marricor Andresa; Borgmann, Sabine; Flick, Karin; Kaiser, Peter; Jones, Jeffrey J.
Phospholipid profiles of genetically modified yeast cells related to the SCFMet30 system were analyzed using MALDI FTMS. The main function of the ubiquitin ligase SCFMet30 is to regulate the activity of Met4 transcription factor. Met4 is the transcriptional activator responsible for regulation of sulfur amino acid metab. To elucidate the potential role of Met4 in lipid metab., wild type and mutant yeast strains lacking different components of the Met4-contg. transcription factor complex were generated by deleting the genes indicated by std. procedures. Both wild type and mutant yeast were grown in YEPD medium at 30C, harvested by filtration, washed in water and lysed in isopropanol. For MALDI FTMS, a satd. soln. of DHB in 70 % methanol was used as matrix. When histogram plots derived from the averaged spectra of wild type and met4D mutants were cross-correlated, the correlation coeff. was 0.85. Significant differences are obsd. in the phosphatidic acid (PA), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) contents between the two samples. For yeast strains lacking genes encoding the cofactors Cbf1 (cbf1?), Met28 (met28?), or both redundant Met31 and Met32 (met31? met32?), none of them showed similar changes in lipid profiles as was obsd. for met4? mutants. These results suggest that Met4 requires a different set of cofactors for regulation of lipid metab., or that Met4 has functions independent from its role as a transcription factor. This anal. demonstrates that application of the MALDI FTMS strategy to a genetic model system is effective in linking gene function to lipid homeostasis.