Fontoura B M, Dales S, Blobel G, Zhong H
Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021, USA.
Proc Natl Acad Sci U S A. 2001 Mar 13;98(6):3208-13. doi: 10.1073/pnas.061014698.
The Nup98 gene codes for several alternatively spliced protein precursors. Two in vitro translated and autoproteolytically cleaved precursors yielded heterodimers of Nup98-6kDa peptide and Nup98-Nup96. TPR (translocated promoter region) is a protein that forms filamentous structures extending from nuclear pore complexes (NPCs) to intranuclear sites. We found that in vitro translated TPR bound to in vitro translated Nup98 and, via Nup98, to Nup96. Double-immunofluorescence microscopy with antibodies to TPR and Nup98 showed colocalization. In confocal sections the nucleolus itself was only weakly stained but there was intensive perinucleolar staining. Striking spike-like structures emanated from this perinucleolar ring and attenuated into thinner structures as they extended to the nuclear periphery. This characteristic staining pattern of the TPR network was considerably enhanced when a myc-tagged pyruvate kinase-6kDa fusion protein was overexpressed in HeLa cells. Double-immunoelectron microscopy of these cells using anti-myc and anti-TPR antibodies and secondary gold-coupled antibodies yielded row-like arrangements of gold particles. Taken together, the immunolocalization data support previous electron microscopical data, suggesting that TPR forms filaments that extend from the NPC to the nucleolus. We discuss the possible implications of the association of Nup98 with this intranuclear TPR network for an intranuclear phase of transport.
Nup98基因编码几种可变剪接的蛋白质前体。两种体外翻译并经自身蛋白酶切割的前体产生了Nup98-6kDa肽和Nup98-Nup96的异二聚体。TPR（易位启动子区域）是一种蛋白质，它形成从核孔复合体（NPC）延伸至核内位点的丝状结构。我们发现体外翻译的TPR与体外翻译的Nup98结合，并通过Nup98与Nup96结合。用针对TPR和Nup98的抗体进行的双重免疫荧光显微镜检查显示了共定位。在共聚焦切片中，核仁本身仅被弱染色，但核仁周围有强烈染色。从这个核仁周围环发出明显的穗状结构，当它们延伸到核周边时逐渐变细成更细的结构。当一种带有myc标签的丙酮酸激酶-6kDa融合蛋白在HeLa细胞中过表达时，TPR网络的这种特征性染色模式显著增强。使用抗myc和抗TPR抗体以及二级金偶联抗体对这些细胞进行双重免疫电子显微镜检查，产生了金颗粒的行状排列。综上所述，免疫定位数据支持先前的电子显微镜数据，表明TPR形成从NPC延伸至核仁的细丝。我们讨论了Nup98与这种核内TPR网络的关联对于核内运输阶段的可能影响。
