One of the major new findings emanating from this study is that cells have a mechanism to sense and react to changes in their membrane sterol composition by modifying their membrane lipid compositions affecting mainly sphingolipids. The mechanism of the adaptation of sphingolipid levels is not understood yet, but is most likely not uniquely due to differences in transcript levels of the different genes involved in the pathway as most of the genes were unchanged. The mechanism used to detect the changes in sterol composition is currently under study and genetic approaches are possible. It is not the absence of ergosterol that is sensed, because each erg
mutant shows a different pattern of sphingolipids. The pathway leading to changes in sphingolipid composition is most likely fundamentally different from previously described sterol-sensing mechanisms where different amounts of cholesterol are sensed by the SREBP pathway or other sterol sensors and then control gene expression (Goldstein et al., 2006
), because sterol intermediates, rather than the total quantity of sterols, seem to affect sphingolipid composition in different ways and the changes are unlikely brought about directly through changes in gene expression. Recently, an SREBP-dependent pathway has been described in Schizosaccharomyces pombe
, which has several features in common with what we have seen here, because there is a coordinate regulation of anaerobically expressed genes with ergosterol biosynthesis genes (Todd et al., 2006
; ) and because sterol biosynthesis intermediates seem to be important (Hughes et al., 2007
), but no specific effects on sphingolipids have been described. The pathway cannot be identical in S. cerevisiae
because this yeast does not have an SREBP homolog, although they do have oxysterol-binding proteins that have been proposed to be involved in intracellular sterol transport (Raychaudhuri et al., 2006
An interesting question is the physiological significance of the adaptation mechanism. First, it is likely that natural intermediates in the ergosterol biosynthesis pathway are being recognized. They are present in significant amounts under normal growth conditions and are found in larger amounts in the erg
mutants (Supplementary Table S2). Sterol biosynthetic intermediates have been shown to be localized preferentially to the ER membrane (Zinser et al., 1993
), and their amounts could be important in regulating adaptation mechanisms. Second, yeast may frequently encounter azole and other inhibitors of ergosterol biosynthesis in their natural environment and therefore require adaptation mechanisms to adjust their sphingolipid composition when azole compounds are present and inhibit sterol biosynthesis at various steps.
The control of sphingolipid species is unlikely to be solely the result of transcriptional changes because few differences in sphingolipid metabolic enzymes were observed. In particular, some changes in sphingolipid content are likely to be due to head group turnover via Isc1p (Supplementary Figure S2). The mode of activation of Isc1p in this case is unknown, but one possibility is regulation by localization because its localization has been shown to change as a result of changes in culture conditions in yeast (Vaena de Avalos et al., 2004
In contrast to the effect of sterol mutations on sphingolipid composition, mutations affecting hydroxylation and head group turnover of sphingolipids do not seem to affect the sterol composition. Therefore, this process is not reciprocal. There could be different explanations for this situation. First, the species of sphingolipids that change in the sterol mutants are sphingolipid species that are normally present in substantial quantities, and only their amount changes. They cannot all be considered as intermediates, because each of these sphingolipid species might be functional. Known antifungals that target the sphingolipid biosynthesis pathway act at or before the step of inositolphosphorylceramide (IPC) synthesis (Horvath et al., 1994
; Mandala and Harris, 2000
; Sugimoto et al., 2004
). In contrast, the major sterol species is ergosterol, and other species are probably just intermediates in its biosynthesis. Second, during evolution some metazoans either never had or lost the ability to synthesize sterols and are sterol auxotrophs (Vinci et al., 2008
). Two particularly well-studied systems are Drosophila
and Caenorhabditis elegans
(Silberkang et al., 1983
; Matyash et al., 2001
). Experiments with Drosophila
did not detect any major changes in phospholipid head groups or acyl group composition when grown with different amounts of cholesterol (Silberkang et al., 1983
). Sphingolipids were not examined in this study. No information is published yet from either system about the lipid composition of these organisms when they are grown using different sterols. It is possible that these organisms have a mechanism similar to that described here for yeast that could be used to adjust membrane composition in response to which sterols they ingest.
The specific and major changes in sphingolipid species when sterol intermediates are present in the membrane show a dependence of sphingolipid metabolism on sterol composition, but also suggest that sterols and sphingolipids function together. The second major finding from our study stems from our genetic analysis of double mutants in sterol and sphingolipid metabolism and provides proof for their functional interaction. This study revealed a number of novel synthetic and suppression phenotypes between sterol and sphingolipid biosynthesis mutants. Two aspects of this systematic and unbiased analysis are particularly convincing. First is the penetration of phenotypes, with 13 of the 15 possible double mutants showing synthetic or suppression phenotypes. Second is the astounding specificity of the combinations required to affect each phenotype and the strict specificity for the site of hydroxylation on the sphingolipid molecule and its combination with a particular sterol mutation. Synthetic phenotypes can occur for different reasons: when there are two parallel pathways and one mutation is in each pathway or when two mutations affect the same pathway, quite often at the same step. Each mutation would affect the step partially, but the double mutant would have a much stronger phenotype. Classically, this can result when two proteins work together as a complex. In a similar manner it is possible to interpret our data by suggesting that sterols and sphingolipids might function in a variety of pathways as a sterol–sphingolipid complex. Sterols and sphingolipids are clearly capable of forming condensed complexes in artificial membranes (Radhakrishnan et al., 2000
), and there is no reason to expect that they should not be able to do the same in biological membranes.
We tested many different phenotypes including growth in presence of cell wall disturbances, weak organic acid sensitivity, different carbon sources, temperatures, various classes of inhibitors affecting a number of different pathways as well as a systematic analysis of transcript levels. It is virtually impossible that sterols and sphingolipids happen to act in parallel pathways to carry out each of these functions where synthetic phenotypes have been uncovered. Therefore, we are obliged to conclude that sterols and sphingolipids function together to carry out a wide variety of cellular functions. The conclusion that sterols and sphingolipids function together is consistent with one of the tenets of the raft hypothesis. The other main tenet of the raft hypothesis is that the increased order produced by sterol–sphingolipid interactions is important for function. To begin to test this, we measured plasma membrane anisotropy as an indicator of membrane order. Even though there were substantial and significant differences in anisotropy, indicating a decrease in membrane order in several of the mutants, we were not able to correlate the measurements of membrane anisotropy with any of the phenotypes we assayed, and therefore we conclude that membrane order is not likely to have a great influence on the functions we examined. Therefore, our results do not provide support for this part of the raft hypothesis.
The raft hypothesis has been invoked to explain a large number of membrane trafficking events, which prompted us to examine the localization of two plasma membrane proteins that have been shown to be localized in microdomains of the plasma membrane. Here again, the raft hypothesis does not easily help to explain the results of steady-state localization of Tat2p and Can1p in the various single and double mutants. The two transporters colocalize to the same plasma membrane patches, but they show completely different dependence on sterols and sphingolipids for their localization. Localization of Tat2p, which was affected by sphingolipid and sterol mutations, did not correlate with anisotropy measurements. It could be that anisotropy measurements of the plasma membrane do not reflect fluidity of the compartment where Tat2p sorting occurs (Golgi or endosomal compartment) or that measuring the overall fluidity of the membrane does not indicate the fluidity in rafts, so we cannot disprove involvement of liquid order phases. On the other hand, there is no reason from our studies or others in the literature at this time to invoke a role for liquid order phases in vivo.
In what ways could sterol–sphingolipid interactions influence function with such a complex pattern of phenotypes? A trivial explanation would be that addition of sterol mutations with sphingolipid mutations cause an indirect effect by generally increasing the stress to the cells, which affects many functions. This is not the case for the hypersensitivities seen here to caffeine or sorbic acid, which do not correlate with stress gene induction. Indirect effects, although certainly occurring in some cases, are unlikely to be a common explanation for the synthetic effects we see because of the specificity of the position of hydroxylation of the sphingolipid and because different combinations of specific sterol and sphingolipid mutations affect only specific cellular pathways rather than having highly pleiotropic effects.
Biophysical (Ahmed et al., 1997
; Radhakrishnan et al., 2001
; Feigenson, 2007
) and molecular dynamics (Aittoniemi et al., 2007
) experiments have clearly demonstrated that sterols and sphingolipids can interact preferentially in artificial membranes. This means that these lipids exist in at least two forms in the membrane, free sterol and sphingolipid, as well as sterol–sphingolipid complexes. Our results suggest that this interaction will also be seen in vivo if suitable techniques become available. We postulate that most of the phenotypes seen here are mainly the result of defects in direct protein–lipid interactions and not changes in the fluidity properties of the membrane or membrane domains. Any integral or peripheral membrane protein could interact with either a sterol, a sphingolipid, or a sphingolipid-sterol complex, helping to explain the complexity of the phenotypic analysis. Membrane proteins might require the sterols and/sphingolipids for proper folding, assembly, targeting, regulation, or activity. Eukaryotic membrane proteins have had a long time to optimize these interactions during evolution because most cells have sterols and all have sphingolipids. Direct sterol–sphingolipid interactions could also help to explain why it is so difficult to express, purify, and crystallize eukaryotic membrane proteins in bacteria.
There are various ways to interpret the enhancement of phenotypes we have seen. For example, in erg4
single mutant cells Pdr12p shows substantial activity, but in erg4 sur2
double mutants activity is lost. Pdr12p might require interaction with sterol–sphingolipid complexes for full activity. The interaction could be reduced because of a stronger change in shape of the complex due to the combination. The complex might adapt a different tilt in the membrane because of the two mutations. Molecular dynamics studies suggest that sphingomyelin binding to cholesterol controls its tilt in the lipid bilayer (Aittoniemi et al., 2006
). Alternatively, the combination of the erg4
mutations could affect the equilibrium between the free lipids with sterol–sphingolipid complexes affecting Pdr12p activity. We propose that this paradigm—of protein interaction with sterols, sphingolipids, or their complexes—could explain the majority of phenotypes we see and that have been shown to depend upon sterols or sphingolipids in the literature without invoking the need for order in the membranes.
In order for sterols and sphingolipids to function together, their structural compatibility had to be maintained through evolution. Strikingly, there was one outlier from the lipid analysis of the double erg mutants (). The sphingolipid pattern in the erg5 erg6 double mutant was more similar to wild type than the corresponding single mutants. The double mutation is “sphingolipid neutral.” The ERG5 and ERG6 genes are responsible for changes in the sterol side chain, the principle difference in the biosynthetic pathways of cholesterol and ergosterol. The two genes are linked in S. cerevisiae. This suggests that the ERG5 and ERG6 genes could have been inherited or lost together during evolution without causing major sphingolipid changes, allowing a coevolution of sphingolipids and sterols.
This study also demonstrates that changes in sterol and sphingolipid composition can affect how a cell responds to a drug or inhibitor. Several examples are shown here, most prominently, the hypersensitivity of the erg2 scs7 double mutant to caffeine and rapamycin. Our data show that the reduction in activity of TORC2, not TORC1, the direct target of caffeine and rapamycin, is the reason for the hypersensitivity. The extent to which this paradigm can be extended to humans is not presently clear, but it is evident that genetic factors and diet affect sensitivity and effects of certain drugs. Sterols and sphingolipid composition could play an important role in pharmacological variations in the population.