Institut für Biochemie und Molekularbiologie, ZBMZ, Universität Freiburg, 79104 Freiburg, Germany.
BMC Biol. 2009 Nov 13;7:76. doi: 10.1186/1741-7007-7-76.
The signal recognition particle (SRP) receptor plays a vital role in co-translational protein targeting, because it connects the soluble SRP-ribosome-nascent chain complex (SRP-RNCs) to the membrane bound Sec translocon. The eukaryotic SRP receptor (SR) is a heterodimeric protein complex, consisting of two unrelated GTPases. The SRbeta subunit is an integral membrane protein, which tethers the SRP-interacting SRalpha subunit permanently to the endoplasmic reticulum membrane. The prokaryotic SR lacks the SRbeta subunit and consists of only the SRalpha homologue FtsY. Strikingly, although FtsY requires membrane contact for functionality, cell fractionation studies have localized FtsY predominantly to the cytosolic fraction of Escherichia coli. So far, the exact function of the soluble SR in E. coli is unknown, but it has been suggested that, in contrast to eukaryotes, the prokaryotic SR might bind SRP-RNCs already in the cytosol and only then initiates membrane targeting.
In the current study we have determined the contribution of soluble FtsY to co-translational targeting in vitro and have re-analysed the localization of FtsY in vivo by fluorescence microscopy. Our data show that FtsY can bind to SRP-ribosome nascent chains (RNCs) in the absence of membranes. However, these soluble FtsY-SRP-RNC complexes are not efficiently targeted to the membrane. In contrast, we observed effective targeting of SRP-RNCs to membrane-bond FtsY. These data show that soluble FtsY does not contribute significantly to cotranslational targeting in E. coli. In agreement with this observation, our in vivo analyses of FtsY localization in bacterial cells by fluorescence microscopy revealed that the vast majority of FtsY was localized to the inner membrane and that soluble FtsY constituted only a negligible species in vivo.
The exact function of the SRP receptor (SR) in bacteria has so far been enigmatic. Our data show that the bacterial SR is almost exclusively membrane-bound in vivo, indicating that the presence of a soluble SR is probably an artefact of cell fractionation. Thus, co-translational targeting in bacteria does not involve the formation of a soluble SR-signal recognition particle (SRP)-ribosome nascent chain (RNC) intermediate but requires membrane contact of FtsY for efficient SRP-RNC recruitment.
信号识别颗粒（SRP）受体在共翻译蛋白靶向中起着至关重要的作用，因为它将可溶性 SRP-核糖体-新生链复合物（SRP-RNCs）连接到膜结合的 Sec 易位酶上。真核 SRP 受体（SR）是一种异二聚体蛋白复合物，由两个不相关的 GTP 酶组成。SRβ亚基是一种完整的膜蛋白，将 SRP 相互作用的 SRα亚基永久地连接到内质网膜上。原核 SR 缺乏 SRβ亚基，仅由 SRα同源物 FtsY 组成。引人注目的是，尽管 FtsY 需要膜接触才能发挥功能，但细胞分级分离研究将 FtsY 主要定位在大肠杆菌的胞质部分。到目前为止，可溶性 SR 在大肠杆菌中的确切功能尚不清楚，但有人认为，与真核生物不同，原核 SR 可能已经在细胞质中结合了 SRP-RNCs，然后才开始进行膜靶向。
在本研究中，我们确定了可溶性 FtsY 在体外共翻译靶向中的贡献，并通过荧光显微镜重新分析了 FtsY 的体内定位。我们的数据表明，FtsY 可以在没有膜的情况下结合 SRP-核糖体新生链（RNCs）。然而，这些可溶性 FtsY-SRP-RNC 复合物不能有效地靶向到膜上。相比之下，我们观察到 SRP-RNC 有效地靶向到膜结合的 FtsY。这些数据表明，可溶性 FtsY 对大肠杆菌中的共翻译靶向没有显著贡献。与这一观察结果一致，我们通过荧光显微镜对细菌细胞中 FtsY 定位的体内分析表明，绝大多数 FtsY 定位于内膜，而可溶性 FtsY 在体内仅构成微不足道的物种。
SRP 受体（SR）在细菌中的确切功能迄今仍是个谜。我们的数据表明，细菌 SR 在体内几乎完全是膜结合的，这表明可溶性 SR 的存在可能是细胞分级分离的一个假象。因此，细菌中的共翻译靶向不涉及形成可溶性 SR-信号识别颗粒（SRP）-核糖体新生链（RNC）中间物，而是需要 FtsY 的膜接触才能有效地招募 SRP-RNC。
