van Roermund Carlo W T, de Jong Mark, IJlst Lodewijk, van Marle Jan, Dansen Tobias B, Wanders Ronald J A, Waterham Hans R
Department of Clinical Chemistry, University of Amsterdam, Academic Medical Centre, PO Box 22700, 1100 DE, Amsterdam, The Netherlands.
J Cell Sci. 2004 Aug 15;117(Pt 18):4231-7. doi: 10.1242/jcs.01305.
Peroxisomes have a central function in lipid metabolism, including the beta-oxidation of various fatty acids. The products and substrates involved in the beta-oxidation have to cross the peroxisomal membrane, which previously has been demonstrated to constitute a closed barrier, implying the existence of specific transport mechanisms. Fatty acid transport across the yeast peroxisomal membrane may follow two routes: one for activated fatty acids, dependent on the peroxisomal ABC half transporter proteins Pxa1p and Pxa2p, and one for free fatty acids, which depends on the peroxisomal acyl-CoA synthetase Faa2p and the ATP transporter Ant1p. A proton gradient across the peroxisomal membrane as part of a proton motive force has been proposed to be required for proper peroxisomal function, but the nature of the peroxisomal pH has remained inconclusive and little is known about its generation. To determine the pH of Sacharomyces cerevisiae peroxisomes in vivo, we have used two different pH-sensitive yellow fluorescent proteins targeted to the peroxisome by virtue of a C-terminal SKL and found the peroxisomal matrix in wild-type cells to be alkaline (pH(per) 8.2), while the cytosolic pH was neutral (pH(cyt) 7.0). No Delta pH was present in ant1 Delta cells, indicating that the peroxisomal pH is regulated in an ATP-dependent way and suggesting that Ant1p activity is directly involved in maintenance of the peroxisomal pH. Moreover, we found a high peroxisomal pH of >8.6 in faa2 Delta cells, while the peroxisomal pH remained 8.1+/-0.2 in pxa2 Delta cells. Our combined results suggest that the proton gradient across the peroxisomal membrane is dependent on Ant1p activity and required for the beta-oxidation of medium chain fatty acids.
过氧化物酶体在脂质代谢中发挥着核心作用，包括各种脂肪酸的β-氧化。β-氧化过程中涉及的产物和底物必须穿过过氧化物酶体膜，此前已证明该膜构成一个封闭屏障，这意味着存在特定的转运机制。脂肪酸穿过酵母过氧化物酶体膜可能有两条途径：一条是针对活化脂肪酸的途径，依赖于过氧化物酶体ABC半转运蛋白Pxa1p和Pxa2p；另一条是针对游离脂肪酸的途径，依赖于过氧化物酶体酰基辅酶A合成酶Faa2p和ATP转运蛋白Ant1p。有人提出，作为质子动力一部分的跨膜过氧化物酶体质子梯度是过氧化物酶体正常功能所必需的，但过氧化物酶体pH的性质仍无定论，对其产生机制也知之甚少。为了测定酿酒酵母过氧化物酶体在体内的pH值，我们使用了两种不同的对pH敏感的黄色荧光蛋白，它们通过C端的SKL靶向过氧化物酶体，发现野生型细胞中的过氧化物酶体基质呈碱性（pH(per) 8.2），而细胞质pH呈中性（pH(cyt) 7.0）。在ant1Δ细胞中不存在ΔpH，这表明过氧化物酶体pH是以ATP依赖的方式调节的，提示Ant1p活性直接参与过氧化物酶体pH的维持。此外，我们发现faa2Δ细胞中的过氧化物酶体pH值>8.6，而pxa2Δ细胞中的过氧化物酶体pH值保持在8.1±0.2。我们的综合结果表明，跨膜过氧化物酶体质子梯度依赖于Ant1p活性，是中链脂肪酸β-氧化所必需的。
