We examined the effects of unsaturated fatty acid (UFA) species and their concentration on the expression of OLE1, which encodes the stearoyl CoA desaturase, in Saccharomyces cerevisiae. We controlled the amount of UFA taken up by the cell by varying the concentration of tergitol in the medium. When cultured with 1 mM fatty acid in 0.1% tergitol, cells took up much more fatty acid than when cultured with the same concentration of fatty acid at 1% tergitol, although the amount incorporated was dependent on UFA species. For each fatty acid tested, we found that the higher uptake (0.1% tergitol condition) had a stronger impact on OLE1 regulation. A principal product of the desaturase 16:1∆9, and the nonnative UFA 18:2∆9,12, most strongly repressed the reporter construct OLE1-lacZ transcription, while the other major product of the desaturase, 18:1∆9, and the nonnative UFA 17:1∆10 caused a more diminished response. Based on these results, our initial hypothesis was that OLE1 was regulated in response to membrane fluidity; however, subsequent work does not support that idea; we have found that conditions that affect membrane fluidity such as growth temperature and growth with saturated or trans fatty acid supplementation, do not regulate OLE1 in the direction predicted by fluidity changes. We conclude that at least one signal that regulates OLE1 transcriptional expression is most likely based on the fatty acids themselves.

The OLE1 gene in Saccharomyces cerevisiae encodes the sole fatty acyl desaturase in that species. OLE1 gene expression is controlled in part through transcriptional regulators Mga2p and Spt23p. These proteins reside in the ER, and when an insufficient supply of unsaturated fatty acids (UFAs) is detected, they are proteolytically cleaved and translocated into the nucleus, where they activate OLE1 expression. Recently our lab has isolated a mutant that is deficient in regulation of OLE1, called nro1 (no regulation of OLE1). The mechanism for the NRO1 protein's action is unknown. In this study, we report that growth tests using a reporter gene under control of the OLE1 promoter demonstrate that in wild type cells, normal regulation is observed with the UFAs 16:1Δ9 and 18:2Δ9, 12 but not with UFAs 18:1Δ9 or 17:1Δ9. Fatty acid profiles of nro1 mutant cells are similar to wildtype when cells are supplemented with the various UFAs, showing evidence of post‐transciptional regulation. Results examining the effect of nro1 on the proteolytic processing of Mga2p and Spt23p through western analysis are presented. Taken together, there is evidence that the NRO1 system helps to regulate OLE1 expression in response to fatty acids 16:1Δ9 and 18:2Δ9, 12, but not 18:1Δ9 or 17:1Δ9. Supported by a Summer Research Scholar Award from Hope College Biology Department

The OLE1 gene in Saccharomyces cerevisiae encodes the ∆9 desaturase, which inserts a double bond in saturated fatty acids to create unsaturated fatty acids (UFAs). OLE1 expression is controlled in part through the transcriptional regulators Mga2p and Spt23p in response to supply of UFAs. We investigated whether the regulation was uniform in response to different UFAs and at different concentrations. We found that in wild type cells, reporter gene assays show a stronger decrease in expression of OLE1 when fed 16:1∆9 or 18:2∆9, 12 as opposed to 18:1∆9 or 17:1∆10. Concentration of the fed fatty acid also impacted the regulation of OLE1 with higher levels of each UFA impacting expression to a greater degree. Fatty acid profiles of wild type cells show cells accumulate a higher concentration of 16:1∆9 and 18:2∆9, 12 than fed 18:1∆9 or 17:1∆10. This leads to the conclusion that the expression of OLE1 is dependent both on properties of fed fatty acids and the amount in the cell. While our initial hypothesis was that OLE1 is regulated in response to membrane fluidity, subsequent work does not support that idea. We have found that conditions that would affect membrane fluidity (besides UFA species and amount), such as growth temperature and saturated or trans fatty acid supplementation do not regulate OLE1 in the direction predicted by fluidity changes. Recently our lab has isolated a mutant that is deficient in regulation of OLE1, called nro1 (no regulation of OLE1). The signaling mechanism for the NRO1 protein’s action is unknown. Tests using the OLE1 promoter‐reporter gene constructs suggest that Nro1p responds more strongly to the fatty acids 16:1∆9 and 18:2∆9, 12, than 18:1∆9 and 17:1∆10. Characterization of NRO1 is discussed. Grant Funding Source: Supported by NSF‐REU DBI‐0754293

The OLE1 gene product, the delta‐9 desaturase, inserts a double bond in saturated fatty acids to create unsaturated fatty acids (UFAs). OLE1 expression is controlled in part through the transcription factors Mga2p and Spt23p in response to the supply of UFAs. These proteins reside in the endoplasmic reticulum, and when insufficient supply of UFAs is detected, they are proteolytically cleaved and translocated into the nucleus, where they activate OLE1 expression. Recently our lab has isolated a mutant that is deficient in regulation of OLE1, called nro1 (no regulation of OLE1). The mechanism for the NRO1 protein's action is unknown. In this study, growth tests and enzyme assays using reporter genes controlled by the OLE1 promoter region suggest that Nro1p responds more strongly to the fatty acids 16:1 Δ9 and 18:2 Δ9, 12, than 18:1 Δ9 and 17:1 Δ9. When wild type cells are fed 16:1 Δ9 and 18:2 Δ9, 12, fatty acid profiles revealed them present at a higher percentage than when fed other UFAs, probably because endogenous UFA production is more severely curtailed. The concentration of the fed fatty acid also impacted the regulation of OLE1, with higher concentrations bringing about more robust regulation. However, in nro1 cells, there was diminished regulation of expression in response to feeding certain UFAs as compared to wild type cells. Western blot analysis showed no evidence that Mga2p and Spt23p regulate OLE1 through the NRO1 signaling system. We conclude that the expression of OLE1 is dependent on properties of the fed fatty acid, the amount in the cell, and that the NRO1 gene product may be working through other regulatory networks besides Mga2p/Spt23p. Supported by an award from the National Science Foundation‐Research Experiences for Undergraduates.