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tra12117-sup-0001-TableS1.docWord document42KTable S1: Yeast strains used in this study.
tra12117-sup-0002-TableS2.docWord document124KTable S2: SFA-dysregulated genes.
tra12117-sup-0003-TableS3.docWord document47KTable S3: List of the genes involved in sphingolipid pathway used for hierarchical clustering.
tra12117-sup-0004-FigureS1.tifTIFF image392KFigure S1: A yeast model for lipotoxicity. A) hem1Δ cells can synthesize haem only if the medium is supplemented with Δ-aminolevulinate (ALA). Since haem is used as the prosthetic group of Ole1p, the fatty acid desaturase, and of several enzymes of the ergosterol pathway, hem1Δ cells accumulate saturated fatty acids (SFA) and suffer ergosterol depletion when grown in the absence of ALA (YPD). As with pancreatic β-cells, SFA accumulation is associated with ER-swelling and induction of the unfolded protein response (UPR) in this organelle, but also with the diversion of selective plasma membrane proteins, such as the uracil permease Fur4p, from the Golgi apparatus to the vacuole for degradation. Full growth can be recovered if the cells are grown in the presence of exogenous sources of ergosterol and unsaturated fatty acids (UFA), such as the mono-unsaturated species oleic acid. B) On a biophysical point of view, SFA accumulation correlates with an increase in the saturation rate of phospholipids which results, in turn, in an ordering of the membrane bilayers. See text for details.
tra12117-sup-0005-FigureS2.tifTIFF image221KFigure S2: Ceramide quantification. hem1Δ cells were grown in the media indicated for 7 h before lipid extraction and MS analysis in the positive ion mode. The relative quantities of the phytoceramide species t18:0/26:0-B (m/z = 696.7 Da) and t18:0/26:0-C (m/z = 712.7 Da), which were the most represented ceramides in our samples and differ from each other by one hydroxyl group, were estimated by comparison to N-octadecanoyl-phytosphingosine (Avanti Polar Lipids, Inc.), used as a standard. Their schematic structures are also displayed. These species were unambiguously identified by scanning for the positive ion precursors of m/z 282.3 (2). A.U.: Arbitrary Units.
tra12117-sup-0006-FigureS3.tifTIFF image2589KFigure S3: Gcs1p recruitment to liposomes of defined composition and various radiuses. To determine the role of unsaturated lipids on Gcs1p recruitment, purified Gcs1p was incubated with either pure DOPC (dioleoyl phosphatidylcholine), pure POPC (palmitoyl-oleyl PC) or pure DMPC (dimyristoyl PC) liposomes of different radius [i.e. extruded through polycarbonate filters of respective pore size 0.4 µm (T2), 0.1 µm (T3) and 0.03 µm (T3), or no added liposomes (T1)], then loaded at the bottom of a three steps sucrose gradient before centrifugation as described in Figure 6. Gcs1p recruitment to liposome was quantified by analysis of top fractions on SDS–PAGE and comparison to 100% protein fraction (total loaded protein volume diluted into 100 μL final top volume).

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