Schonberger O, Knox C, Bibi E, Pines O
Department of Molecular Biology, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
Proc Natl Acad Sci U S A. 1996 Sep 3;93(18):9612-7. doi: 10.1073/pnas.93.18.9612.
The assembly of functional proteins from fragments in vivo has been recently described for several proteins, including the secreted maltose binding protein in Escherichia coli. Here we demonstrate for the first time that split gene products can function within the eukaryotic secretory system. Saccharomyces cerevisiae strains able to use sucrose produce the enzyme invertase, which is targeted by a signal peptide to the central secretory pathway and the periplasmic space. Using this enzyme as a model we find the following: (i) Polypeptide fragments of invertase, each containing a signal peptide, are independently translocated into the endoplasmic reticulum (ER) are modified by glycosylation, and travel the entire secretory pathway reaching the yeast periplasm. (ii) Simultaneous expression of independently translated and translocated overlapping fragments of invertase leads to the formation of an enzymatically active complex, whereas individually expressed fragments exhibit no activity. (iii) An active invertase complex is assembled in the ER, is targeted to the yeast periplasm, and is biologically functional, as judged by its ability to facilitate growth on sucrose as a single carbon source. These observation are discussed in relation to protein folding and assembly in the ER and to the trafficking of proteins through the secretory pathway.
最近已经报道了几种蛋白质在体内从片段组装成功能性蛋白质的情况，包括大肠杆菌中分泌的麦芽糖结合蛋白。在这里，我们首次证明了分裂基因产物能够在真核生物分泌系统中发挥作用。能够利用蔗糖的酿酒酵母菌株会产生转化酶，该酶通过信号肽靶向中央分泌途径和周质空间。以这种酶为模型，我们发现以下几点：（i）转化酶的多肽片段，每个片段都含有一个信号肽，可独立转运到内质网（ER）中，经糖基化修饰后，沿着整个分泌途径到达酵母周质。（ii）同时表达独立翻译和转运的转化酶重叠片段会导致形成具有酶活性的复合物，而单独表达的片段则无活性。（iii）活性转化酶复合物在内质网中组装，靶向酵母周质，并且具有生物学功能，这可以通过其促进酵母利用蔗糖作为单一碳源生长的能力来判断。我们结合内质网中的蛋白质折叠和组装以及蛋白质通过分泌途径的运输来讨论这些观察结果。
