Choudhary Vineet, Schneiter Roger
Department of Medicine, Division of Biochemistry, University of Fribourg, Fribourg, Switzerland.
Methods Mol Biol. 2009;580:221-32. doi: 10.1007/978-1-60761-325-1_12.
Sterols are essential lipid components of eukaryotic membranes. They are synthesized in the endoplasmatic reticulum (ER) from where they are efficiently transported to the plasma membrane, which harbors ~90% of the free sterol pool of the cell. The molecular mechanisms that govern this lipid transport, however, are not well characterized and are challenging to analyze. Saccharomyces cerevisiae offers the opportunity to circumvent some of the technical limitations associated with studying this forward transport of sterols from the ER to the plasma membrane, because the organism can also take up sterols from the environment, incorporate them into the plasma membrane and transport them back to the ER, where the free sterol is converted to steryl esters. This reverse sterol transport, however, occurs only under anaerobic conditions, where the cells become sterol auxotroph, or in mutant cells that cannot synthesize heme. The reverse sterol transport pathway, however, is more amenable to experimental studies, because arrival of the sterol in the ER membrane can be monitored unambiguously by following the formation of steryl esters. Apart from sterol acylation, we have recently described a reversible sterol acetylation cycle that is operating in the lumen of the ER. Acetylation occurs on both cholesterol and pregnenolone, a steroid precursor, and serves as a signal for export of the acetylated sterols into the culture media. The time-dependent appearance of acetylated sterols in the culture supernatant thus provides a new means to monitor the forward transport of chemically modified sterols out of the ER.
甾醇是真核细胞膜的重要脂质成分。它们在内质网（ER）中合成，然后从内质网被有效地转运到质膜，质膜中含有细胞约90%的游离甾醇库。然而，控制这种脂质转运的分子机制尚未得到充分表征，且分析起来具有挑战性。酿酒酵母提供了一个机会，可以规避一些与研究甾醇从内质网到质膜的正向转运相关的技术限制，因为该生物体还可以从环境中摄取甾醇，将它们整合到质膜中，并将它们运回内质网，在内质网中游离甾醇被转化为甾醇酯。然而，这种反向甾醇转运仅在厌氧条件下发生，此时细胞成为甾醇营养缺陷型，或者在不能合成血红素的突变细胞中发生。然而，反向甾醇转运途径更适合进行实验研究，因为可以通过追踪甾醇酯的形成来明确监测甾醇到达内质网膜的情况。除了甾醇酰化作用外，我们最近还描述了一种在内质网腔中运行的可逆甾醇乙酰化循环。乙酰化作用发生在胆固醇和孕烯醇酮（一种类固醇前体）上，并作为乙酰化甾醇输出到培养基中的信号。因此，培养上清液中乙酰化甾醇随时间的出现为监测化学修饰的甾醇从内质网的正向转运提供了一种新方法。
