Large subunit of translation initiation factor--3 p170 contains potentially functional nuclear localization signals

Eukaryotic translation factors and their subunits can have independent cellular functions, including regulation of nuclear events. We analyzed primary structure of p170 large subunit of human translation initiation factor eIF3 and found four potential bipartite nuclear localization signals (NLS). Then we studied whether these NLS were functional, that is were able to direct protein to cell nucleus. Complementary DNA of p170 fragments were expressed in cultured CV-1 and Cos-1 green monkey cells, and localization of fused with GFP proteins was determined by fluorescent microscopy. We established that p170 molecule possessed at least two functional NLS which determined nuclear localization of p170 fragments. At the same time more long p170 fragments containing the same functional NLS could be retained in cytoplasm. We speculate that either using specific factors or after limited proteolysis p170 can enter cell nucleus and participate in genome expression regulation. Also we do not exclude the possibility that functioning of p170 in cytoplasm can be regulated by reversible binding of importins to its NLS.     

Evidence for Gal3p's cytoplasmic location and Gal80p's dual cytoplasmic-nuclear location implicates new mechanisms for controlling Gal4p activity in Saccharomyces cerevisiae

Genetics and in vitro studies have shown that the direct interaction between Gal3p and Gal80p plays a central role in galactose-dependent Gal4p-mediated GAL gene expression in the yeast Saccharomyces cerevisiae. Precisely how Gal3p-Gal80p interaction effects induction is not clear. It has been assumed that Gal3p interacts with Gal80p in the nucleus upon galactose addition to release Gal80p inhibition of Gal4p. Although Gal80p has been shown to possess nuclear localization signal (NLS) peptides, the subcellular distribution of neither Gal80p nor Gal3p was previously determined. Here we report that Gal3p is located in the cytoplasm and apparently excluded from the nucleus. We show that Gal80p is located in both the cytoplasm and the nucleus. Converting Gal80p into a nucleus-localized protein (NLS-Gal80p) by exogenous NLS addition impairs GAL gene induction. The impaired induction can be partially suppressed by targeting Gal3p to the nucleus (NLS-Gal3p). We document a very rapid association between NLS-Gal3p and Gal80p in vivo in response to galactose, illustrating that the nuclear import of Gal80p is very rapid and efficient. We also demonstrate that nucleus-localized NLS-Gal80p can move out of the nucleus and shuttle between nuclei in yeast heterokaryons. These results are the first indication that the subcellular distribution dynamics of the Gal3 and Gal80 proteins play a role in regulating Gal4p-mediated GAL gene expression in vivo.     

核糖体蛋白L6/Taxreb107的核定位信号的分析

核糖体蛋白L6（RpL6,Taxreb107）含有三个具有核定位信号特征的基序.用作者构建的核定位信号捕获系统分析了这些核定位信号是否具有介导蛋白质进行核转位的功能.将RpL6/Taxreb107分段插入核定位信号捕获载体的克隆位点后转化宿主酵母,发现其前两个核定位信号可以介导融合蛋白进入细胞核,而第三个核定位信号无此作用.将RpL6/Taxreb107分段与绿色荧光蛋白融合后转染培养的哺乳类细胞,证实了以上在酵母中所得的结果.进一步发现RpL6/Taxreb107的前两个核定位信号同时具有核仁定位的功能.当在细胞中表达的早期,进入核内的融合蛋白优先定位于核仁.这些结果一方面有助于理解RpL6/Taxreb107核转位的机理,同时说明作者构建的核定位信号捕获系统也可用在蛋白质中寻找核定位信号.