Transport of the intracisternal A-type particle Gag polyprotein to the endoplasmic reticulum is mediated by the signal recognition particle
Transport of the intracisternal A-type particle Gag polyprotein to the endoplasmic reticulum is mediated by the signal recognition particle
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DOI：
10.1128/jvi.77.11.6293-6304.2003
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发表时间：
2003-06-01
影响因子：
5.4
通讯作者：
Kräusslich, HG
中科院分区：
文献类型：
--
作者：
Fehrmann, F;Jung, M;Kräusslich, HG
脑池内A型颗粒（IAP）是有缺陷的内源性逆转录病毒，积聚在啮齿动物细胞的内质网（ER）中。包膜颗粒由MY Gag多蛋白在ER膜上组装和出芽产生。在这项研究中，我们分析了特异性ER转运的Gag多聚蛋白的UP元件MIA 14。为此，我们在微粒体膜或合成脂蛋白体存在下进行Gag的体外翻译，然后进行膜沉降或浮选。UP Gag的ER结合主要发生在细胞外，Gag多聚蛋白与仅含有信号识别颗粒（SRP）受体和Sec 61 p复合物的脂蛋白体特异性相互作用，形成最小的ER易位装置。在交联和免疫沉淀实验中证明了SRP直接参与MY Gag的ER靶向。MY多聚蛋白没有易位到ER中，它被发现与ER膜的细胞质侧紧密相关，但没有表现为一个完整的膜蛋白。用前催乳素的功能性信号肽取代IAP Gag N末端的疏水序列也没有导致嵌合蛋白易位到ER腔中，并且将MY疏水序列移植到前催乳素上也没有产生腔运输。这些结果表明，IAP Gag多聚蛋白的N-末端疏水区域作为介导SRP依赖性ER靶向的转运信号起作用，但多聚蛋白易位或整合到膜中被信号序列本身和Gag的额外区域阻止。
Intracisternal A-type particles (IAP) are defective endogenous retroviruses that accumulate in the endoplasmic reticulum (ER) of rodent cells. The enveloped particles are produced by assembly and budding of MY Gag polyproteins at the ER membrane. In this study, we analyzed the specific ER transport of the Gag polyprotein of the UP element MIA14. To this end, we performed in vitro translation of Gag in the presence of microsomal membranes or synthetic proteoliposomes followed by membrane sedimentation or flotation. ER binding of UP Gag occurred mostly cotranslationally, and Gag polyproteins interacted specifically with proteoliposomes containing only signal recognition particle (SRP) receptor and the Sec61p complex, which form the minimal ER translocation apparatus. The direct participation of SRP in ER targeting of MY Gag was demonstrated in cross-linking and immunoprecipitation experiments. The MY polyprotein was not translocated into the ER; it was found to be tightly associated with the cytoplasmic side of the ER membrane but did not behave as an integral membrane protein. Substituting the functional signal peptide of preprolactin for the hydrophobic sequence at the N terminus of IAP Gag also did not result in translocation of the chimeric protein into the ER lumen, and grafting the MY hydrophobic sequence onto preprolactin failed to yield luminal transport as well. These results suggest that the N-terminal hydrophobic region of the IAP Gag polyprotein functions as a transport signal which mediates SRP-dependent ER targeting, but polyprotein translocation or integration into the membrane is prevented by the signal sequence itself and by additional regions of Gag.