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

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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