In vivo interaction between the polyprenol phosphate mannose synthase Ppm1 and the integral membrane protein Ppm2 from Mycobacterium smegmatis revealed by a bacterial two-hybrid system
In vivo interaction between the polyprenol phosphate mannose synthase Ppm1 and the integral membrane protein Ppm2 from Mycobacterium smegmatis revealed by a bacterial two-hybrid system
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DOI：
10.1074/jbc.m207922200
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发表时间：
2003-01-24
影响因子：
4.8
通讯作者：
Besra, GS
中科院分区：
文献类型：
--
作者：
Baulard, AR;Gurcha, SS;Besra, GS
Dolichol磷酸甘露糖（Dol-P-Man）是多种真核生物糖基化过程中的甘露糖供体。到目前为止，已经基于它们在内质网膜中稳定的方式表征了两组Dol-P-Man脱氢酶。属于第一组的酶，如酵母Dpm 1，是典型的整合膜蛋白，在其C末端具有跨膜片段（TMS）。相比之下，哺乳动物Dpm 1，第二组的酶，缺乏典型的TMS，并需要与小的疏水蛋白Dpm 3的协会，以适当地稳定在内质网膜。在结核分枝杆菌中，聚戊烯醇-P-Man合酶MtPpm 1参与细胞壁相关糖脂脂阿拉伯甘露聚糖的生物合成。MtPpm 1由两个结构域组成。C-末端催化结构域与真核Dol-P-Man酶同源。MtPpm 1的N-末端结构域包含6个TMS，其将酶锚在细胞质膜中。相比之下，在耻垢分枝杆菌中，MtPpm 1的两个结构域的直系同源物由两个不同的开放阅读框架Msppm 1和Msppm 2编码，其被组织为操纵子。没有TMS预测MsPpm 1，亚细胞分馏实验表明，这种酶是在大肠杆菌中产生时，胞质。计算机辅助拓扑预测和碱性磷酸酶插入表明MsPpm 2是一种完整的膜蛋白。利用最近开发的细菌双杂交系统，发现MsPpm 2与MsPpm 1相互作用以稳定细菌膜中的合酶MsPpm 1。这种相互作用使人联想到哺乳动物Dpm 1与Dpm 3的相互作用，并模拟了MtPpm 1的结构，如MtPpm 1的两个结构域在E.杆菌
Dolichol phosphate-mannose (Dol-P-Man) is a mannose donor in various eukaryotic glycosylation processes. So far, two groups of Dol-P-Man synthases have been characterized based on the way they are stabilized in the endoplasmic reticulum membrane. Enzymes belonging to the first group, such as the yeast Dpm1, are typical integral membrane proteins harboring a transmembrane segment (TMS) at their C terminus. In contrast, mammalian Dpm1, enzymes of the second group, lack the typical TMS and require the association with the small hydrophobic proteins Dpm3 to be properly stabilized in the endoplasmic reticulum membrane. In Mycobacterium tuberculosis, the Polyprenol-P-Man synthase MtPpm1 is involved in the biosynthesis of the cell wall-associated glycolipid lipoarabinomannan. MtPpm1 is composed of two domains. The C-terminal catalytic domain is homologous to eukaryotic Dol-P-Man synthases. The N-terminal domain of MtPpm1 contains six TMS that anchor the enzyme in the cytoplasmic membrane. In contrast, in Mycobacterium smegmatis, orthologs of the two domains of MtPpm1 are encoded by two distinct open reading frames, Msppm1 and Msppm2, organized as an operon. No TMS are predicted in MsPpm1, and subcellular fractionation experiments indicate that this enzyme is cytosolic when produced in Escherichia coli. Computer-assisted topology predictions and alkaline phosphatase insertions showed that MsPpm2 is an integral membrane protein. Using a recently developed bacterial two-hybrid system, it was found that MsPpm2 interacts with MsPpm1 to stabilize the synthase MsPpm1 in the bacterial membrane. This interaction is reminiscent of that of mammalian Dpm1 with Dpm3 and mimics the structure of MtPpm1 as demonstrated by the capacity of the two domains of MtPpm1 to spontaneously interact when co-expressed in E. coli.