Aitchison J D, Blobel G, Rout M P
Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York 10021-6399, USA.
J Cell Biol. 1995 Dec;131(6 Pt 2):1659-75. doi: 10.1083/jcb.131.6.1659.
To extend our understanding of the mechanism by which the nuclear pore complex (NPC) mediates macromolecular transport across the nuclear envelope we have focused on defining the composition and molecular organization of the yeast NPC. Peptide sequence analysis of a polypeptide with a M(r) of approximately 100,000 present in a highly enriched yeast NPC fraction identified a novel yeast nucleoporin we term Nup120p. Nup120p corresponds to the open reading frame (ORF) YKL057c identified by the yeast genome sequencing project. The ORF predicts a protein with a calculated molecular mass of 120.5 kD containing two leucine zipper motifs, a short coiled-coil region and limited primary sequence similarity to Nup133p. Nup120p was localized to the NPC using a protein A-tagged chimera in situ by indirect immunofluorescence microscopy. Deletion of the NUP120 gene caused clustering of NPCs at one side of the nuclear envelope, moderate nucleolar fragmentation and slower cell growth. Transfer of nup120 delta cells to 37 degrees C resulted in the nuclear accumulation of poly(A)+ mRNA, extensive fragmentation of the nucleolus, spindle defects, and cell death.
为了拓展我们对核孔复合体(NPC)介导大分子穿过核膜机制的理解,我们着重于确定酵母NPC的组成和分子组织。对高度富集的酵母NPC组分中一种分子量约为100,000的多肽进行肽序列分析,鉴定出一种新的酵母核孔蛋白,我们将其命名为Nup120p。Nup120p对应于酵母基因组测序项目鉴定的开放阅读框(ORF)YKL057c。该ORF预测一种计算分子量为120.5 kD的蛋白质,含有两个亮氨酸拉链基序、一个短的卷曲螺旋区域,且与Nup133p的一级序列相似性有限。通过间接免疫荧光显微镜,利用蛋白A标记的嵌合体将Nup120p定位到NPC。NUP120基因的缺失导致NPC在核膜一侧聚集、核仁中度碎片化以及细胞生长缓慢。将nup120Δ细胞转移至37℃会导致多聚腺苷酸(poly(A)+)mRNA在细胞核内积累、核仁广泛碎片化、纺锤体缺陷以及细胞死亡。