In the present study, we have evaluated the impact of ergosterol or UFA depletion on the biogenesis of the model polytopic membrane protein Fur4p. In the dynamic system used, Fur4p fails to be delivered to the plasma membrane when sterol amounts decrease below a critical concentration of ~4.5 ± 1.2 μg ergosterol/109 cells (B and A). Under these conditions, Fur4p falls prey to a quality control system that displays all the characteristics of the Golgi-based quality control process: 1) Fur4p is sorted to the endosomal pathway without prior targeting to the plasma membrane (D and A), ) Fur4p is protected from degradation when accumulated in the Golgi apparatus (B), and ) is internalized in the vacuolar lumen in a Rsp5p-dependent manner (C). This is a new example of the influence of sterols on the biogenesis of a membrane-associated protein.
Another important finding of this study is that the fatty acyl content of phospholipids influences Fur4p biogenesis. Indeed, as observed upon ergosterol scarcity, heme-induced UFA shortage resulted in Fur4p sorting to the endosomal pathway. To our knowledge, this is the first time that such an effect of cellular fatty acyl composition on the biogenesis of an integral protein is reported. ESI-MS analysis of phospholipid species revealed that UFA starvation induced two distinct responses on the fatty acyl content, i.e., a decrease in the unsaturation ratio and a shortening of the chain length (). These effects are not related to an indirect consequence of heme depletion, but they are rather directly connected to the impaired fatty acid desaturation. Indeed, all three phenomena, i.e., Fur4p failure to reach the cell surface, a decrease in the unsaturation ratio, and the shortening of fatty acyl chains, were observed in an ole1Δ strain when grown without supplementation with an exogenous UFA source (unpublished data).
Shortening of the fatty acyl chains in phospholipids may represent an adaptation process to impaired desaturation. Yeast has already been reported to adjust its unsaturation level and the length of its phospholipid fatty acyl chains in response to various stresses such as temperature variations (Suutari et al., 1997
) or conditions of PC depletion (Boumann et al., 2006
). If one considers the potential effects of saturated fatty acid (SFA) accumulation in phospholipids under haem deficiency, one may expect a dramatic decrease in membrane fluidity, which is not compatible with most of the cellular processes that require high membrane dynamics. Shortening the acyl chains in phospholipids under these conditions may contribute to maintaining the membranes in the Ld state. This may explain why essential cellular processes such as secretion are still fully functional under lipid depletion, as judged by invertase secretion assays and Pma1p biogenesis studies ().
A major question that arose from our observations was how depleting the cells of ergosterol or UFA, which were found to differently impact the overall lipid composition ( and ), could have the same effects on the biogenesis of a plasma membrane protein, such as Fur4p. The most reasonable hypothesis is that they influence a specific physical parameter of the lipid bilayer in the same way, e.g., domain formation, such as Lo/DRMs/raft domains, membrane fluidity, thickness, or intrinsic curvature.
Based on recent literature, we initially hypothesized that the disruption of raft domains could account for Fur4p recognition by the Golgi quality control process. However, this is obviously not the case, because the pattern of Fur4p association to DRMs was identical under aerobic-like and lipid-depleted conditions (D). This was confirmed for individual ergosterol and UFA depletions (our unpublished data). Moreover, the association of another protein, Pma1p, to DRMs was also shown not to be impaired under the various lipid depletions (D; our unpublished data). Pma1p association to DRMs has been proposed to be a prerequisite for plasma membrane delivery and stability (Bagnat et al., 2001
; Pizzirusso and Chang, 2004
; Gaigg et al., 2005
). Accordingly, Pma1p targeting to the cell periphery was not abolished under ergosterol and UFA shortage ().
If ergosterol scarcity and short saturated fatty acyl chain accumulation in phospholipids upon UFA depletion were not compatible with unidirectional changes in membrane fluidity, they may influence membrane width in the same way, by reducing bilayer thickness. Thickness of the hydrophobic membrane-spanning regions of an integral protein should match the thickness of the bilayer to avoid exposure of hydrophobic residues to water, a phenomenon known as hydrophobic-mismatch (for review, see Lee, 2003
). However, such a phenomenon cannot account for our results with Fur4p. Indeed, we could show that the permease escapes the Golgi quality control process when grown in the presence of a short unsaturated fatty acid, myristoleic acid (, + Erg + C14:1). Such an addition restored the phospholipid unsaturation ratio to a level equivalent to what was observed under aerobiosis-like conditions (B), but it maintained a low average fatty acyl chain length (C). Therefore, the unsaturation ratio of phospholipids seems to be a more critical parameter for Fur4p cell surface delivery than membrane width.
A model that fully accounts for our results is membrane curvature (van den Brink-van der Laan et al., 2004
; de Kroon, 2007
). Some phospholipids such as PC display an overall cylindrical shape and tend to organize themselves in bilayers. In contrast, other phospholipids such as PE display conical shapes (type II lipids), and they tend to form nonlamellar phases with a negative curvature such as the hexagonal phase HII
. When present in phospholipid bilayers, hexagonal phase-promoting lipids result in curvature stress, an overall property of the membrane that can influence the structure and activity of membrane-anchored proteins (for reviews, see Booth, 2003
; van den Brink-van der Laan et al., 2004
). It has been proposed that the presence of nonbilayer lipids may result in increased lateral pressure in the acyl chain region that could favor the packing of transmembrane helices (van den Brink-van der Laan et al., 2004
). Interestingly, increasing the amount of diunsaturated PC species and the relative cholesterol concentration augments the propensity of PC/cholesterol mixtures to form an HII
phase in model membranes (Epand et al., 2003
; Tenchov et al., 2006
). Moreover, the accumulation of short saturated fatty acid chains in PE in yeast has been shown to remarkably decrease its HII
phase propensity due to a reduction of its overall conical shape (Boumann et al., 2006
). Therefore, based on these observations, one may expect that ergosterol and UFA shortage, through a decrease in diunsaturated PC (D) and PE (unpublished data) species in favor of saturated forms, induce unidirectional changes in membrane curvature, i.e., a decrease in curvature stress. The hypothesis for such a direct connection between curvature stress and optimal Fur4p delivery to the plasma membrane is strengthened by the observed deleterious effects of PE depletion on Fur4p biogenesis (), effects that can be rescued in part by replacing PE with the HII
hexagonal phase promoter phosphatidylpropanolamine (; Storey et al., 2001
Finally, the results presented in this study point to the Golgi apparatus being a major compartment for lipid-mediated controlled cell surface delivery of plasma membrane proteins. In this respect, the fact that Pma1p trafficking to the cell periphery is not abolished under lipid depletion () suggests that Fur4p and Pma1p are not equally sensitive to changes in the lipid environment. This may be related to unique immediate lipid surroundings that may account for their different sensitivities to Triton X-100 solubilization (D), or a different oligomerization status of these two proteins. Lipid-mediated early segregation at the Golgi level could account, at least in part, for Fur4p and Pma1p localizations to distinct plasma membrane subdomains (Malinska et al., 2004
; Grossmann et al., 2007
). In this context, modifying curvature stress may be a way to regulate the delivery of selective plasma membrane proteins to their final destination.