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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >NUCLEAR LOCALIZATION SIGNALS OF HUMAN AND THERMOPLASMA PROTEASOMAL ALPHA SUBUNITS ARE FUNCTIONAL IN VITRO
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NUCLEAR LOCALIZATION SIGNALS OF HUMAN AND THERMOPLASMA PROTEASOMAL ALPHA SUBUNITS ARE FUNCTIONAL IN VITRO

机译：人和热血浆蛋白体α亚基的核定位信号在体外具有功能

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摘要

Proteasomes are located both in the nuclei and in the cytoplasm of eukaryotic cells. Active transport of these complexes through the nuclear pores has been proposed to be mediated by nuclear localization signals (NLS), which have been found in several of the alpha-type proteasomal subunits, We have tested three different putative NLS sequences from human alpha-type proteasomal subunits (HscL, Hsc9, and Hsc3), as well as a putative NLS-type sequence from the archaeon Thermoplasma acidophilum, for their ability to direct nonnuclear proteins to the nucleus. Synthetic peptides containing these putative NLS sequences were generated and conjugated to large fluorescent reporter molecules: allophycocyanin or fluorescein-labeled bovine serum albumin. The conjugates were introduced into digitonin-permeabilized HeLa and 3T3 cells in the presence of cell lysate and ATP, and nuclear import was monitored by fluorescence microscopy. All three putative NLS sequences from human proteasomal subunits were able to direct the reporter molecules to the nucleus in both fell types, although differences in efficiency were observed. Substitution of threonine for the first lysine residue of the eukaryotic NLS motifs inhibited nuclear import completely. Interestingly, the putative NLS sequence found in T. acidophilum was also functional as a nuclear targeting sequence.
机译：蛋白酶体位于真核细胞的细胞核和细胞质中。已经提出这些复合物通过核孔的主动转运是由核定位信号（NLS）介导的，NLS已在几种α型蛋白酶体亚基中发现。我们已经从人α型中检测了三种不同的推定NLS序列蛋白酶体亚基（HscL，Hsc9和Hsc3），以及来自古细菌嗜酸嗜热菌的推定NLS型序列，因为它们能够将非核蛋白引导至细胞核。生成了包含这些假定的NLS序列的合成肽，并将其与大型荧光报告分子：别藻蓝蛋白或荧光素标记的牛血清白蛋白偶联。在细胞裂解液和ATP存在的情况下，将缀合物引入到经过洋地黄酮渗透的HeLa和3T3细胞中，并通过荧光显微镜监测核的进口。尽管观察到效率差异，但来自人类蛋白酶体亚基的所有三个推定的NLS序列均能够将报告分子导向两种核酸类型的核。用苏氨酸替代真核NLS基序的第一个赖氨酸残基完全抑制了核输入。有趣的是，在嗜酸乳杆菌中发现的推定的NLS序列也可作为核靶向序列。

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