Determination of growth-phase dependent influences exerted by prions on yeast lipid content using HPTLC—densitometry

Abstract

Summary Prions of the baker’s yeast Saccharomyces cerevisiae allow for the inheritance of complex traits based solely on the acquisition of cytoplasmic protein aggregates and confer distinctive phenotypes to the cells which harbor them, creating heterogeneity within an otherwise clonal cell population. These phenotypes typically arise from a loss-of-function of the prion-forming protein that is unable to perform its normal cellular function(s) while sequestered in prion amyloid aggregates, but the specific biochemical consequences of prion infection are poorly understood. To begin to address this issue, we initiated a direct investigation into the potential control that yeast prions exert over fungal lipid content by utilizing the prions [URE3] and [PSI+], the first two prions discovered in yeast. We utilized silica gel high-performance thin-layer chromatography (HPTLC)–densitometry to conduct pair-wise quantifications of the relative levels of free sterols, free fatty acids, and triacylglycerols [petroleum ether–diethyl ether–acetic acid (80:20:1) mobile phase, phosphomolybdic acid (PMA) detection reagent]; steryl esters and squalene (hexane–petroleum ether–diethyl ether–acetic acid (50:20;5:1), PMA]; and phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol (chloroform–diethyl ether–acetic acid (65:25:4.5), cupric sulfate–phosphoric acid) in otherwise clonal prion-infected ([PSI+] or [URE3]) and prion-free ([psi−] or [ure-o]) cells in two growth phases: log-phase and stationary phase. Our analysis revealed multiple statistically significant differences (p < 0.00625) between prion-infected and prion-free cells. Interestingly, prion-induced changes varied dramatically by growth phase, indicating that prions exert differential influences on cell physiology between log and stationary growth. Further experimental replication and extension of the analysis to other prions is expected to resolve additional physiological effects of prion infection. This investigation demonstrates that HPTLC–densitometry is an effective method for studying prion-induced alterations in lipid content in yeast.

Using High Performance Thin Layer Chromatography-Densitometry to Study the Influence of the Prion [RNQ+] and Its Determinant Prion Protein Rnq1 on Yeast Lipid Profiles

“…Direct comparison between the [ RNQ + ] strain vs. the wild-type [ rnq − ] strain would be expected to reveal any effects on lipid profile due solely to the presence of [ RNQ + ] aggregates, while comparison of the Δ rnq1 strain and the wild-type [ rnq − ] strain should reveal effects due to the loss of the normal function of the Rnq1 protein itself. Prior to this investigation no phenotypes related to lipids have been observed either for the presence of [ RNQ + ] or due to the deletion of RNQ1 , so this investigation also tested the hypothesis that the observed changes found in Bui et al [ 18 ] in the [ URE3 ]/[ RNQ + ] strain were due solely to the presence of [ URE3 ]. This hypothesis was nullified; our results indicated significant changes in lipid content between strains possessing or lacking [ RNQ + ].…”

“…On the other hand, it is much easier to detect physiological changes empirically. Since lipids serve in a wide variety of functions in yeast cells (structural, energy storage, and signaling), in the past, we predicted that it might be likely that a change in lipid profile could be observed when a prion is present in S. cerevisiae [ 18 ]. A set of efficient lipid extraction procedures for yeast cells was previously described by Schneiter and Daum [ 19 ], and several analytical methods have been utilized for quantification of lipids in yeast cells, most notably high performance liquid chromatography [ 20 ] and gas chromatography-mass spectrometry [ 21 ].…”

“…HPTLC (high performance TLC) has been utilized extensively in lipid quantification in biological samples (reviewed by Fried [ 23 ] and Bui et al [ 24 ]). Recently, we showed that HPTLC was a robust method of quantifying lipids in yeast cells, allowing highly reproducible and accurate measurements [ 18 ].…”

“…Previously, we investigated the differences in lipid profiles of prion-free yeast cells ([ psi − ] and [ ure-o ]) against their respective clonal prion-infected strains ([ PSI + ] and [ URE3 ]) in both logarithmic phase and stationary phase [ 18 ]. The presence of these prions was found to exert significant changes to lipid profiles of yeast cells in both growth phases.…”

“…The presence of these prions was found to exert significant changes to lipid profiles of yeast cells in both growth phases. In addition, we also saw that the influence of prions on the yeast lipid profile varied between the growth phases being tested [ 18 ]. However, the interpretation of these results was complicated because the [ URE3 ] yeast strain used also harbored the [ RNQ + ] prion.…”