Goldshmidt Hanoch, Sheiner Lilach, Bütikofer Peter, Roditi Isabel, Uliel Shai, Günzel Mark, Engstler Markus, Michaeli Shulamit
The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel.
J Biol Chem. 2008 Nov 14;283(46):32085-98. doi: 10.1074/jbc.M801499200. Epub 2008 Sep 2.
The translocation of secretory and membrane proteins across the endoplasmic reticulum (ER) membrane is mediated by co-translational (via the signal recognition particle (SRP)) and post-translational mechanisms. In this study, we investigated the relative contributions of these two pathways in trypanosomes. A homologue of SEC71, which functions in the post-translocation chaperone pathway in yeast, was identified and silenced by RNA interference. This factor is essential for parasite viability. In SEC71-silenced cells, signal peptide (SP)-containing proteins traversed the ER, but several were mislocalized, whereas polytopic membrane protein biogenesis was unaffected. Surprisingly trypanosomes can interchangeably utilize two of the pathways to translocate SP-containing proteins except for glycosylphosphatidylinositol-anchored proteins, whose level was reduced in SEC71-silenced cells but not in cells depleted for SRP68, an SRP-binding protein. Entry of SP-containing proteins to the ER was significantly blocked only in cells co-silenced for the two translocation pathways (SEC71 and SRP68). SEC63, a factor essential for both translocation pathways in yeast, was identified and silenced by RNA interference. SEC63 silencing affected entry to the ER of both SP-containing proteins and polytopic membrane proteins, suggesting that, as in yeast, this factor is essential for both translocation pathways in vivo. This study suggests that, unlike bacteria or other eukaryotes, trypanosomes are generally promiscuous in their choice of mechanism for translocating SP-containing proteins to the ER, although the SRP-independent pathway is favored for glycosylphosphatidylinositol-anchored proteins, which are the most abundant surface proteins in these parasites.
分泌蛋白和膜蛋白穿过内质网（ER）膜的转运是由共翻译（通过信号识别颗粒（SRP））和翻译后机制介导的。在本研究中，我们调查了这两种途径在锥虫中的相对作用。我们鉴定出了酵母中参与转运后伴侣途径的SEC71的同源物，并通过RNA干扰使其沉默。该因子对寄生虫的生存能力至关重要。在SEC71沉默的细胞中，含信号肽（SP）的蛋白质穿过了内质网，但有几种蛋白质定位错误，而多跨膜蛋白的生物合成未受影响。令人惊讶的是，锥虫可以互换地利用其中两种途径来转运含SP的蛋白质，但糖基磷脂酰肌醇锚定蛋白除外，其水平在SEC71沉默的细胞中降低，但在缺乏SRP68（一种SRP结合蛋白）的细胞中未降低。仅在两种转运途径（SEC71和SRP68）共同沉默的细胞中，含SP的蛋白质进入内质网才被显著阻断。我们鉴定出了酵母中两种转运途径所必需的因子SEC63，并通过RNA干扰使其沉默。SEC63沉默影响了含SP的蛋白质和多跨膜蛋白进入内质网，这表明，与酵母一样，该因子在体内对两种转运途径都至关重要。这项研究表明，与细菌或其他真核生物不同，锥虫在选择将含SP的蛋白质转运到内质网的机制方面通常比较随意，尽管对于这些寄生虫中最丰富的表面蛋白——糖基磷脂酰肌醇锚定蛋白而言，不依赖SRP的途径更为有利。
