Max Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, Berlin, Germany.
Cell Mol Life Sci. 2010 Aug;67(15):2653-64. doi: 10.1007/s00018-010-0358-y. Epub 2010 Apr 6.
Bet3, a transport protein particle component involved in vesicular trafficking, contains a hydrophobic tunnel occupied by a fatty acid linked to cysteine 68. We reported that Bet3 has a unique self-palmitoylating activity. Here we show that mutation of arginine 67 reduced self-palmitoylation of Bet3, but the effect was compensated by increasing the pH. Thus, arginine helps to deprotonate cysteine such that it could function as a nucleophile in the acylation reaction which is supported by the structural analysis of non-acylated Bet3. Using fluorescence spectroscopy we show that long-chain acyl-CoAs bind with micromolar affinity to Bet3, whereas shorter-chain acyl-CoAs do not interact. Mutants with a deleted acylation site or a blocked tunnel bind to Pal-CoA, only the latter with slightly reduced affinity. Bet3 contains three binding sites for Pal-CoA, but their number was reduced to two in the mutant with an obstructed tunnel, indicating that Bet3 contains binding sites on its surface.
Bet3 是一种参与囊泡运输的转运蛋白颗粒成分，包含一个由脂肪酸连接到半胱氨酸 68 组成的疏水性隧道。我们报道过 Bet3 具有独特的自身棕榈酰化活性。在这里，我们发现精氨酸 67 的突变会降低 Bet3 的自身棕榈酰化，但这种影响可以通过提高 pH 值得到补偿。因此，精氨酸有助于使半胱氨酸去质子化，从而使其能够在酰化反应中充当亲核试剂，这一结论得到了非酰化 Bet3 的结构分析的支持。我们使用荧光光谱法表明，长链酰基辅酶 A 以微摩尔亲和力结合到 Bet3 上，而短链酰基辅酶 A 则不相互作用。具有缺失酰化位点或阻塞隧道的突变体与 Pal-CoA 结合，只有后者的亲和力略有降低。Bet3 含有三个 Pal-CoA 的结合位点，但在隧道阻塞的突变体中，其数量减少到两个，表明 Bet3 在其表面含有结合位点。
