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

在本研究中，我们计划利用酿酒酵母中tRNA加工内切酶的一个亚基Sen 2 p作为标记物，来鉴定内核被膜蛋白定位所必需的因子。这种蛋白质被认为定位于内核被膜中，因为它应该在细胞核中发挥作用，并且具有适于穿过膜的疏水片段。令人惊讶的是，我们的新数据强烈表明，“森”复合物不在酵母细胞核中。免疫荧光显微镜分析显示，Sen 2 p是本地化的线粒体和未知的细胞质颗粒。Sen 2 p不是一种完整的膜蛋白，而是一种外周膜蛋白，其自身暴露于线粒体上的细胞质。其N端90个氨基酸的片段包含线粒体靶向信息。一个223-243个氨基酸的疏水性足以跨膜的片段作为跨膜结构域定位于内质网， ...更多信息 m，如果该区域单独在酵母细胞中表达。另一方面，在野生型蛋白中，更多的C-末端区域（第244- 297个氨基酸）掩盖了这种活性。含有该疏水区段的C-末端一半与作为可溶性蛋白质的古细菌核酸内切酶高度同源，使得该疏水区段将充当折叠在可溶性蛋白质内部的疏水核心。我们还发现，酵母tRAN核酸内切酶Sen 54的另一个亚基，它是直接与Sen 2 p，定位于线粒体的外表面。虽然Sen 54 p具有与SV 40大T抗原的核定位信号（NLS）相同的氨基酸序列，但其第213- 313位氨基酸序列中含有短的芳香族氨基酸序列，具有碱性，是Sen 54 p的线粒体定位所必需和充分的，在本研究中，我们没有预料到这些结果，在某种意义上，无法进行原计划的分析。但这些发现可能会改变我们以前对tRNA生物发生的看法，我们将继续分析内切酶亚基。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