Genetic search for factors enabling inner nuclear envelope localization of membrane protein

在本研究项目中，我们计划利用酿酒酵母中tRNA加工内切酶的亚基Sen2p作为标记物，确定内核包膜蛋白定位所必需的因素。这种蛋白被认为定位于核膜内，因为它应该在细胞核内起作用，并且具有适合穿过膜的疏水段。令人惊讶的是，我们的新数据强烈表明“Sen”复合物不在酵母的细胞核中。免疫荧光显微镜分析显示，Sen2p定位于线粒体和未知的细胞质颗粒。Sen2p不是一个完整的膜蛋白，而是一个暴露在线粒体细胞质上的外周膜蛋白。它的n端90个氨基酸片段包含线粒体靶向信息。一个223-243个氨基酸的片段是疏水的，足以跨越膜，作为跨膜结构域，定位于内质网…如果这个区域单独在酵母细胞中表达，则更多。另一方面，在野生型蛋白中，更多的c端区域（第244-297个氨基酸）掩盖了这种活性。含有这个疏水片段的c端一半与古细菌内切酶高度同源，这是一种可溶性蛋白质，因此，这个疏水片段将作为折叠在可溶性蛋白质内部的疏水核心。我们还发现酵母tRAN内切酶Sen54的另一个亚基，它与Sen2p直接相关，定位于线粒体的外表面。虽然Sen54p与SV40大T抗原的NLS（核定位信号）具有相同的氨基酸链，但其213-313个氨基酸片段含有短链芳香氨基酸，具有基本性质，是Sen54p线粒体定位的必要和充分条件。在这项研究中，我们没有预料到这些结果，从某种意义上说，无法执行原计划的分析。但是这些发现可能会改变我们以前对tRNA生物发生的看法，我们将继续对内切酶亚基进行分析。少

In this research project, we planned to identify factors that are essential for localization of inner nuclear envelope proteins, using Sen2p, a subunit of tRNA processing endonuclease in Saccharomyces cerevisiae as a mmarker. This protein has been thought to be localized in the inner nuclear envelope since it should have functioned in the nucleus and has a hydrophobic segment suitable for traversing membrane. Surprisingly, our new data strongly indicates that the "Sen" complex is not in the nucleus in the yeast. Immuno-fluorescent microscopic analysis revealed that Sen2p is localized to mitochondria and unknown cytoplasmic granules. Sen2p is not an integral membrane protein but a peripheral membrane protein exposing itself to the cytoplasm on the mitochondria. Its N-terminal 90-amino acids segment contains mitochondrial targeting information. A 223-243 amino acids segment that is hydrophobic enough to span membrane acts as a transmembrane domain and is localized to endoplasmic reticulu … More m if this region alone is expressed in the yeast cell. On the other hand, in the wild type protein, more C-terminal region (244-297th amino acids) masks this activity. The C-terminal half containing this hydrophobic segment is highly homologous to archaebacterial endonuclease that is a soluble protein so that, this hydrophobic segment will act as a hydrophobic core folded inside of soluble protein. We also found that another subunit of yeast tRAN endonuclease Sen54, which is directly associated with Sen2p, is localized to the outer surface of the mitochondria. Though Sen54p has the same amino acid stretch as NLS (nuclear localization signal) of SV40 large T antigen, its 213-313th amino acids segment that contains short stretches of aromatic amino acids and has basic nature was found to be necessary and sufficient for Sen54p's mitochondrial localization.In this research, we did not expect these results and, in a sense, could not perform analysis originally planned. But these findings may enforce to change our previous view of tRNA biogenesis and we will continue the analysis of the endonuclease subunits. Less