Kosova B, Panté N, Rollenhagen C, Hurt E
Biochemie-Zentrum Heidelberg, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany.
J Biol Chem. 1999 Aug 6;274(32):22646-51. doi: 10.1074/jbc.274.32.22646.
Human Nup93, the homologue of yeast Nic96p, is associated with a 205-kDa protein whose intracellular location and function is unknown. We show here that the yeast open reading frame YJL039c, which is homologous to this human p205, encodes the so far largest yeast nucleoporin. Accordingly, green fluorescent protein (GFP)-tagged YJL039c was localized to the nuclear pores and therefore named Nup192p. Affinity purification of ProtA-Nic96p from glutaraldehyde-fixed spheroplasts reveals association with Nup192p. NUP192 is essential for cell growth. A temperature-sensitive mutant nup192-15 is neither impaired in nuclear import of a SV40 nuclear localization sequence-containing reporter protein nor in mRNA export, but association of Nup49-GFP with nuclear pores is inhibited at the non-permissive temperature. By immunoelectron microscopy, Nup192p-ProtA is seen at the inner site of the nuclear pores, at a distance of 60 +/- 15 nm from the central plane of the pore. This suggests that Nup192p is an evolutionarily conserved structural component of the nuclear pore complex with a preferential location at the inner site of the nuclear membrane.
人类Nup93是酵母Nic96p的同源物,与一种205 kDa的蛋白质相关联,其细胞内定位和功能尚不清楚。我们在此表明,与这种人类p205同源的酵母开放阅读框YJL039c编码了迄今为止最大的酵母核孔蛋白。因此,绿色荧光蛋白(GFP)标记的YJL039c定位于核孔,因此被命名为Nup192p。从戊二醛固定的原生质球中亲和纯化ProtA-Nic96p显示其与Nup192p相关联。NUP192对细胞生长至关重要。温度敏感突变体nup192-15在含SV40核定位序列的报告蛋白的核输入或mRNA输出方面均未受损,但在非允许温度下,Nup49-GFP与核孔的关联受到抑制。通过免疫电子显微镜观察,Nup192p-ProtA出现在核孔的内部位置,距离孔的中心平面60±15 nm。这表明Nup192p是核孔复合体的一种进化保守的结构成分,优先位于核膜的内部位置。