Hicks G R, Smith H M, Lobreaux S, Raikhel N V
Michigan State University-Department of Energy, East Lansing 48824-1312, USA.
Plant Cell. 1996 Aug;8(8):1337-52. doi: 10.1105/tpc.8.8.1337.
The import of proteins into the nucleus is a poorly understood process that is thought to require soluble cytosolic factors in vertebrates and yeast. To test this model in plants and to identify components of the import apparatus, we developed a direct in vitro nuclear import assay by using tobacco protoplasts that were permeabilized without detergents such as digitonin or Triton X-100. Substrates were imported specifically by a mechanism that required only guanine nucleotides. Moreover, in vitro import did not require exogenous cytosol. To investigate this novel finding, we isolated a full-length cDNA encoding an Arabidopsis homolog of vertebrate and yeast nuclear localization signal receptors and produced an affinity-purified antibody. The plant receptor was tightly associated with cellular components in permeabilized protoplasts, even in the presence of 0.1% Triton X-100, indicating that this factor and probably others were retained to an extent sufficient to support import. The lectin wheat germ agglutinin bound to the nucleus; however, it did not block translocation in our system, indicating that direct interaction with polysaccharide modifications at the nuclear pore complex was probably not essential for import in plants. Other features of in vitro import included reduced but significant import at low temperature.
蛋白质进入细胞核的过程目前还知之甚少，一般认为在脊椎动物和酵母中这一过程需要可溶性胞质因子。为了在植物中验证这一模型并鉴定输入装置的组成成分，我们利用烟草原生质体开发了一种直接的体外核输入测定法，这些原生质体在不使用诸如洋地黄皂苷或 Triton X - 100 等去污剂的情况下进行透化处理。底物通过一种仅需要鸟嘌呤核苷酸的机制特异性地导入。此外，体外输入不需要外源性胞质溶胶。为了研究这一新颖的发现，我们分离出了一个全长 cDNA，它编码脊椎动物和酵母核定位信号受体的拟南芥同源物，并制备了一种亲和纯化抗体。即使在存在 0.1% Triton X - 100 的情况下，植物受体仍与透化原生质体中的细胞成分紧密结合，这表明该因子以及可能的其他因子保留的程度足以支持输入。凝集素麦胚凝集素与细胞核结合；然而，它在我们的系统中并不阻止转运，这表明与核孔复合体处多糖修饰的直接相互作用可能对植物中的输入并非必不可少。体外输入的其他特征包括在低温下输入减少但仍很显著。
