Department of Biological Sciences, Tata Institute of Fundamental Research, Colaba, Mumbai, India.
PLoS One. 2013 Aug 23;8(8):e72687. doi: 10.1371/journal.pone.0072687. eCollection 2013.
Plasmodium enolase localizes to several sub-cellular compartments viz. cytosol, nucleus, cell membrane, food vacuole (FV) and cytoskeleton, without having any organelle targeting signal sequences. This enzyme has been shown to undergo multiple post-translational modifications (PTMs) giving rise to several variants that show organelle specific localization. It is likely that these PTMs may be responsible for its diverse distribution and moonlighting functions. While most variants have a MW of 50 kDa and are likely to arise due to changes in pI, food vacuole (FV) associated enolase showed three forms with MW50, 65 and 75 kDa. Evidence from immuno-precipitation and western analysis indicates that the 65 and 75 kDa forms of FV associated enolase are ubiquitinated. Using mass spectrometry (MS), definitive evidence is obtained for the nature of PTMs in FV associated variants of enolase. Results showed several modifications, viz. ubiquitination at K147, phosphorylation at Y148 and acetylation at K142 and K384. MS data also revealed the conjugation of three ubiquitin (Ub) molecules to enolase through K147. Trimeric ubiquitin has a linear peptide linkage between the NH2-terminal methionine of the first ubiquitin (Ub1) and the C-terminal G76 of the second (Ub2). Ub2 and third ubiquitin (Ub3) were linked through an atypical isopeptide linkage between K6 of Ub2 and G76 of Ub3, respectively. Further, the tri-ubiquitinated form was found to be largely associated with hemozoin while the 50 and 65 kDa forms were present in the NP-40 soluble fraction of FV. Mass spectrometry results also showed phosphorylation of S42 in the cytosolic enolase from P. falciparum and T337 in the cytoskeleton associated enolase from P. yoelii. The composition of food vacuolar proteome and likely interactors of enolase are also being reported.
疟原虫烯醇酶定位于几个亚细胞区室，如细胞质、核、细胞膜、食物泡（FV）和细胞骨架，而没有任何细胞器靶向信号序列。该酶已被证明经历多种翻译后修饰（PTM），产生几种显示细胞器特异性定位的变体。这些 PTM 可能负责其多样化的分布和兼职功能。虽然大多数变体的 MW 约为 50 kDa，并且可能由于 pI 的变化而产生，但与食物泡（FV）相关的烯醇酶显示出三种形式，MW~50、65 和 75 kDa。免疫沉淀和 Western 分析的证据表明，FV 相关烯醇酶的 65 和 75 kDa 形式是泛素化的。使用质谱（MS），获得了 FV 相关烯醇酶变体中 PTM 性质的明确证据。结果表明存在几种修饰，即 K147 的泛素化、Y148 的磷酸化以及 K142 和 K384 的乙酰化。MS 数据还揭示了通过 K147 将三个泛素（Ub）分子连接到烯醇酶上。三聚体泛素在第一个泛素（Ub1）的 NH2-末端甲硫氨酸和第二个（Ub2）的 C-末端 G76 之间具有线性肽键。Ub2 和第三个泛素（Ub3）通过 Ub2 的 K6 和 Ub3 的 G76 之间的非典型异肽键连接。此外，发现三泛素化形式主要与血晶蛋白相关，而 50 和 65 kDa 形式存在于 FV 的 NP-40 可溶性部分中。质谱结果还显示，恶性疟原虫细胞质烯醇酶的 S42 磷酸化和约氏疟原虫细胞骨架相关烯醇酶的 T337 磷酸化。还报告了食物泡蛋白质组的组成和烯醇酶的可能相互作用物。
