Abstract
The nuclear pore complex is the gateway for selective traffic between the nucleus and cytoplasm. To learn how building blocks of the pore can create specific docking sites for transport receptors and regulatory factors, we have studied a zinc finger module present in multiple copies within the nuclear pores of higher eukaryotes. All four zinc fingers of human Nup153 were found to bind the small GTPase Ran with dissociation constants ranging between 5 and 40 mum. In addition a fragment of Nup153 encompassing the four tandem zinc fingers was found to bind Ran with similar affinity. NMR structural studies revealed that a representative Nup153 zinc finger adopts the same zinc ribbon structure as the previously characterized Npl4 NZF module. Ran binding was mediated by a three-amino acid motif (Leu(13)/Val(14)/Asn(25)) located within the two zinc coordination loops. Nup153 ZnFs bound GDP and GTP forms of Ran with similar affinities, indicating that this interaction is not influenced by a nucleotide-dependent conformational switch. Taken together, these studies elucidate the Ran-binding interface on Nup153 and, more broadly, provide insight into the versatility of this zinc finger binding module.
Highlights
Which includes integral membrane proteins and resides within the plane of the membrane; 2) a cytoplasmic ring and its extended filaments; and 3) a nuclear basket, which is composed of distal and proximal rings connected by eight fibers
One distinctive nucleoporin domain that has not been examined structurally is a zinc finger domain present in multiple copies in both Nup153 and Nup358/RanBP2. These two proteins localize to different sites on the pore, with Nup358 situated on the cytoplasmic filaments and Nup153 positioned on the nuclear basket
RanBP2-type zinc fingers conform to the consensus sequence pattern: W-XC-X(2,4)-C-X(3)-N-X(6)-C-X [2]-C
Summary
Nucleoporin; TFIIS, transcription factor S-II; COPI, coatomer protein; ZnF, zinc finger; GST, glutathione S-transferase; TEV, tobacco etch virus; DTT, dithiothreitol; TOCSY, total correlation spectroscopy; TROSY, transverse relaxation optimized spectroscopy; NOESY, nuclear Overhauser effect spectroscopy; HSQC, heteronuclear singlequantum coherence; NZF, Npl zinc finger; Ub, ubiquitin; -ME, -mercaptoethanol; Ni-NTA, nickel-nitrilotriacetic acid. Nup ZnF modules are present only in higher eukaryotes, suggesting a specialized role(s) in nuclear pore function. Consistent with this idea, the Nup ZnF regions of Nup153 and Nup358 are important in orchestrating mitotic nuclear envelope breakdown, a type of nuclear membrane remodeling unique to higher eukaryotes. In this context, the Nup ZnFs are proposed to serve as a scaffold for recruitment of the coatomer complex COPI [18, 19]. To begin to investigate how Nup ZnF modules function in nuclear pore biology, we have determined the three-dimensional structure of a nucleoporin zinc finger and characterized its Ran binding properties
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