Spatial distribution of protein translation and of protein translocation in the yeast Saccharomyces cerevisiae

酿酒酵母中蛋白质翻译和蛋白质转位的空间分布蛋白质是合成的，并可能在它们后来起作用的地方转位，这一概念仍然是一个相当新的概念，但适用于越来越多的蛋白质。最为人所知和最了解的局部翻译途径是那些识别mRNAs中的短基序，通过主动运输将它们运送到蛋白质合成位点的途径。然而，可能存在其他不依赖于mRNAs中的位置信息的本地化翻译机制。要证明这些途径的存在是困难的，因为新合成的蛋白质会立即与已经存在的旧分子池混合。在这个方案中，我们计划开发三种基于分裂泛素(Ub)的策略来测量单个细胞中高空间和时间分辨率的翻译和易位。在一种方法中，蛋白质合成将通过标记翻译或易位机制的Ub的C端一半(Cub)和新生多肽链的N端Ub的一半(Nub)来测量。Cub会被自发荧光的mCherry和GFP(CCG)夹在中间，从而促使CCG融合在荧光显微镜下发出黄色的光。一旦Cub标记的机器参与了它的Nub标记底物的合成或移位，两个Ub-肽之间产生的接近将迫使它们折叠成像Ub一样的天然多肽。绿色荧光蛋白将瞬间从幼崽身上分离出来，并被降解，留下翻译或易位蛋白，将它们的颜色从黄色转换为红色。不同的红色和黄色标记蛋白质的细胞分布将是所研究的蛋白质所特有的，并反映其合成和/或转位的热点。在另一种方法中，我们将测量新生的链与其自己的mRNA的接近程度。CCG模块将与噬菌体外壳蛋白(MS2CCG)融合，MS2CCG与RNA环序列具有高亲和力，RNA环序列将整合到编码核融合蛋白的mRNA的3UTR中。一旦该核被翻译，它将诱导从MS2CCG上切割GFP，该MS2CCG与其自身的核糖体参与的mRNA结合。同样，mCherry/GFP比率的局部增加表明了NUB融合蛋白的合成位置。对该分析方法进行轻微的重新配置也将使我们能够测量mRNA和mRNA结合蛋白之间的相互作用，从而为mRNA的靶向和翻译打开一个互补的视角。通过综合所有三种分析的结果，我们的目标是建立一个翻译和易位图谱，突出蛋白质生物发生的基因特异性热点。我们将在酵母中建立这种方法，在那里删除基因的容易程度将极大地促进鉴定参与局部合成的决定因素。然而，这项技术应该很容易适用于高等真核生物的细胞。

Spatial distribution of protein translation and of protein translocation in the yeast Saccharomyces cerevisiaeThe concept that proteins are synthesized and possibly translocated at the places where they later act is still quite new but applies to a growing list of proteins. The best known and best understood pathways of local translation are those that recognize short motifs in mRNAs to deliver them by active transport to the sites of protein synthesis. However, there might exist other mechanisms of localized translation that do not depend on the positional information in mRNAs. The proof of the existence of these pathways is difficult as the newly made proteins will immediately mix with the already existing pool of old molecules. In this proposal we plan to develop three Split-Ubiquitin (Ub)-based strategies to measure translation and translocation in single cells with high spatial and temporal resolution. In one approach protein synthesis will be measured by tagging the machinery of translation or translocation with the C-terminal half of Ub (Cub) and the nascent polypeptide chain with the N-terminal half of Ub (Nub). Cub will be sandwiched by the autofluorescent mCherry and GFP (CCG) thus prompting the CCG fusion to emit yellow light under the fluorescence microscope. Once the Cub-labelled machine is engaged in the synthesis or translocation of its Nub-labelled substrate the generated proximity between the two Ub-peptides will force them to fold into the native like Ub. The GFP will be instantaneously cleaved off from the Cub and degraded leaving behind translation or translocation proteins that switch their colour from yellow to red. A distinct cellular distribution of red- and yellow-labelled proteins will be specific for the investigated protein and reflect hotspots of its synthesis and/or translocation.In an alternative approach we will measure the proximity of the nascent chain to its own mRNA. The CCG module will be fused to a phage coat protein (MS2CCG) that binds with high affinity to RNA loop sequences that will be integrated into the 3UTR of a mRNA encoding a Nub-fusion protein. Once the Nub is translated it will induce the cleavage of the GFP from the MS2CCG that is bound to its own ribosome-engaged mRNA. Again a local increase in the ratio of mCherry/GFP indicates the site of the synthesis of the Nub fusion protein. A slight reconfiguration of the assay will also enable us to measure the interactions between mRNA and mRNA-binding proteins thus opening a complementary perspective on the targeting and translation of mRNA. By including the results of all three assays we aim to establish a map of translation and translocation that highlights gene-specific hotspots of protein biogenesis.We will establish the method in yeast where the ease of deleting genes will greatly facilitate the identification the determinants involved in local synthesis. However, the technique should be readily applicable to cells of higher eukaryotes.