Abstract
The Ras-related nuclear protein, Ran, has been implicated in nuclear transport. By screening a HeLa cell lambda expression library with Ran-GTP and sequencing overlapping cDNA clones, we have obtained the derived primary structure of a protein with a calculated molecular mass of 358 kDa. Using antibodies raised against an expressed segment of this protein, we obtained punctate nuclear surface staining by immunofluorescence microscopy that is characteristic for nucleoporins. Electron microscopy of immunogold-decorated rat liver nuclear envelopes sublocalized the 358-kDa protein at (or near) the tip of the cytoplasmic fibers of the nuclear pore complex (NPC). In agreement with current convention, this protein was therefore termed Nup358 (for nucleoporin of 358 kDa). Nup358 contains a leucine-rich region, four potential Ran binding sites (i.e. Ran binding protein 1 homologous domains) flanked by nucleoporin-characteristic FXFG or FG repeats, eight zinc finger motifs, and a C-terminal cyclophilin A homologous domain. Consistent with the location of Nup358 at the cytoplasmic fibers of the NPC, we found decoration with Ran-gold at only the cytoplasmic side of the NPC. Thus, Nup358 is the first nucleoporin shown to contain binding sites for two of three soluble nuclear transport factors so far isolated, namely karyopherin and Ran-GTP.
Highlights
From the Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10021
Using antibodies raised against an expressed segment of this protein, we obtained punctate nuclear surface staining by immunofluorescence microscopy that is characteristic for nucleoporins
nuclear pore complex (NPC) consists of a central opening of -40 nm in diameter surrounded by a scaffold that is attached to the pore membrane
Summary
Molecular Cloning-Recombinant Ran was prepared and loaded with [0'_32PIGTP as described (13). Production of a Recombinant Protein and Antibodies and Immunological Analyses-A DNA fragment encoding amino acids 2550-2837 was obtained by PCR using clone 6-2 as a template and was inserted into Escherichia coli expression vector pQE-30 (Qiagen). Ran binding to isolated NEs was detected using recombinant Ran (13) conjugated, as described by the manufacturer (Amersham Inc.), directly to 10 nm gold. A standard binding reaction consisted of: 20 ILl of isolated NEs (derived from 3 X 106 nuclei) suspended in STM plus 0.5 mM phenylmethylsulfonyl fluoride, and 1 ILg/ml each of leupeptin, pepstatin A, and aprotinin, 14 ILl of buffer B (20 mM Hepes (pH 7.3), 110 mM potassium acetate, 2 mMMg(OAc)2' 1 mM EGTA), 2 ILl of mM GTP in buffer B, 2 ILl of 20 mg/ml bovine serum albumin in buffer B, and 2 ILl of Ran-gold in buffer B. Samples were viewed by EM after staining with 2% uranyl acetate
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