Molecular Determinants of Binding between Gly-Leu-Phe-Gly Nucleoporins and the Nuclear Pore Complex
2007; Elsevier BV; Volume: 282; Issue: 47 Linguagem: Inglês
10.1074/jbc.m707911200
ISSN1083-351X
AutoresGary A. Ratner, A.E. Hodel, Maureen A. Powers,
Tópico(s)RNA regulation and disease
ResumoThe vertebrate nucleoporin Nup98 can be expressed in two distinct forms from differentially spliced mRNAs, either as a 98-kDa protein or as the 195-kDa Nup98/Nup96 polyprotein. Both forms undergo autoproteolytic processing to generate the 90-kDa Nup98 and either an 8-kDa tail or the nucleoporin Nup96. An equivalent cleavage event occurs in one yeast ortholog, Nup145, to produce Nup145N and Nup145C. We previously proposed that Nup145N, and possibly the other orthologs Nup116 and Nup100, might bind to Nup145C as demonstrated for Nup98 and Nup96. Here we have further investigated the interaction of both yeast and vertebrate Gly-Leu-Phe-Gly nucleoporins with the nuclear pore. We find that dynamic Nup98 binding can be recapitulated in vitro and that simultaneous translation and folding as a polyprotein are not required to allow subsequent binding between Nup98 and Nup96. We show that Nup145N and Nup145C do indeed bind to each other, and we have determined the dissociation constants for these interactions in vitro. Additionally, we characterize two sites of molecular interaction for each binding pair. Of the yeast orthologs, Nup116 binds far less robustly to Nup145C than does Nup145N, and Nup100 binding is barely detectable. Thus, we conclude that Nup116 and Nup100 likely use means of incorporation into the nuclear pore complex that are distinct from those used by Nup145N. The vertebrate nucleoporin Nup98 can be expressed in two distinct forms from differentially spliced mRNAs, either as a 98-kDa protein or as the 195-kDa Nup98/Nup96 polyprotein. Both forms undergo autoproteolytic processing to generate the 90-kDa Nup98 and either an 8-kDa tail or the nucleoporin Nup96. An equivalent cleavage event occurs in one yeast ortholog, Nup145, to produce Nup145N and Nup145C. We previously proposed that Nup145N, and possibly the other orthologs Nup116 and Nup100, might bind to Nup145C as demonstrated for Nup98 and Nup96. Here we have further investigated the interaction of both yeast and vertebrate Gly-Leu-Phe-Gly nucleoporins with the nuclear pore. We find that dynamic Nup98 binding can be recapitulated in vitro and that simultaneous translation and folding as a polyprotein are not required to allow subsequent binding between Nup98 and Nup96. We show that Nup145N and Nup145C do indeed bind to each other, and we have determined the dissociation constants for these interactions in vitro. Additionally, we characterize two sites of molecular interaction for each binding pair. Of the yeast orthologs, Nup116 binds far less robustly to Nup145C than does Nup145N, and Nup100 binding is barely detectable. Thus, we conclude that Nup116 and Nup100 likely use means of incorporation into the nuclear pore complex that are distinct from those used by Nup145N. Transport between the nucleus and cytoplasm of a eukaryotic cell is mediated by nuclear pore complexes (NPCs), 2The abbreviations used are: NPC, nuclear pore complex; GFP, green fluorescent protein; GST, glutathione S-transferase. 2The abbreviations used are: NPC, nuclear pore complex; GFP, green fluorescent protein; GST, glutathione S-transferase. highly selective gateways embedded in the nuclear envelope. NPCs are composed of about 30 different protein species, known as nucleoporins or Nups. Each NPC has numerous copies of each nucleoporin and an approximate total mass of 40 or 60 MDa in yeast or metazoans, respectively. Structural studies, most recently by cryoelectron microscopy, have revealed with considerable detail an NPC with 8-fold symmetry about an axis perpendicular to the plane of the nuclear envelope (1Beck M. Forster F. Ecke M. Plitzko J.M. Melchior F. Gerisch G. Baumeister W. Medalia O. Science. 2004; 306: 1387-1390Crossref PubMed Scopus (400) Google Scholar, 2Tran E.J. Wente S.R. Cell. 2006; 125: 1041-1053Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 3Peters R. Methods Mol. Biol. 2006; 322: 235-258Crossref PubMed Scopus (27) Google Scholar). The central core region containing most of the mass of the complex consists of rings on each face of the nuclear envelope with spokes in between. Fibrils extend from the rings into either the cytoplasm or nucleoplasm and on the nucleoplasmic face join to form a basket-like structure. Nups of the core region are distributed symmetrically with respect to the plane of the envelope, whereas the cytoplasmic and nuclear fibrils are each composed of a distinct set of nucleoporins. In yeast, a few Nups have been shown to have a biased distribution relative to the nuclear envelope, being enriched on but not exclusive to one face or the other (4Rout M.P. Aitchison J.D. Suprapto A. Hjertaas K. Zhao Y. Chait B.T. J. Cell Biol. 2000; 148: 635-651Crossref PubMed Scopus (1149) Google Scholar). Many Nups associate into subcomplexes that act as building blocks in NPC assembly, underscoring the modular nature of this symmetrical structure (2Tran E.J. Wente S.R. Cell. 2006; 125: 1041-1053Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar, 5Schwartz T.U. Curr. Opin. Struct. Biol. 2005; 15: 221-226Crossref PubMed Scopus (137) Google Scholar, 6Devos D. Dokudovskaya S. Williams R. Alber F. Eswar N. Chait B.T. Rout M.P. Sali A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 2172-2177Crossref PubMed Scopus (225) Google Scholar). Additionally there are interspersed throughout the NPC a class of Nups containing repeat domains, each with many copies of the sequence Phe-Gly (FG) or variants of this sequence such as FXFG (were X is any amino acid) or Gly-Leu-Phe-Gly (GLFG). These domains function as natively unstructured regions responsible for interaction with transport receptors (7Denning D.P. Patel S.S. Uversky V. Fink A.L. Rexach M. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 2450-2455Crossref PubMed Scopus (375) Google Scholar, 8Bayliss R. Littlewood T. Stewart M. Cell. 2000; 102: 99-108Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar), for the permeability barrier of the NPC (9Frey S. Richter R.P. Gorlich D. Science. 2006; 314: 815-817Crossref PubMed Scopus (434) Google Scholar, 10Patel S.S. Belmont B.J. Sante J.M. Rexach M.F. Cell. 2007; 129: 83-96Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar), and for the association of some nucleoporins with the pore (11Griffis E.R. Craige B. Dimaano C. Ullman K.S. Powers M.A. Mol. Biol. Cell. 2004; 15: 1991-2002Crossref PubMed Scopus (89) Google Scholar, 12Stochaj U. Banski P. Kodiha M. Matusiewicz N. Exp. Cell Res. 2006; 312: 2490-2499Crossref PubMed Scopus (10) Google Scholar). The preceding findings present a static, architectural picture of the NPC. However, more recent studies in vertebrate cells indicate that NPC structure is, at least in part, dynamic. Although many Nups reside stably in the pore, others associate more transiently, and, in some cases, shuttle between nuclear and cytoplasmic compartments (13Griffis E.R. Altan N. Lippincott-Schwartz J. Powers M.A. Mol. Biol. Cell. 2002; 13: 1282-1297Crossref PubMed Scopus (204) Google Scholar, 14Rabut G. Doye V. Ellenberg J. Nat. Cell Biol. 2004; 6: 1114-1121Crossref PubMed Scopus (355) Google Scholar). To fully understand the function of the NPC, it will be necessary to characterize the molecular structures of the individual nucleoporins and the nature of the interactions between them, both stable and dynamic. One case in which interaction between two nucleoporins has been described at the molecular level is the pore-targeting interaction of the Nup98 C-terminal domain with Nup96. Nup98 is the sole GLFG repeat nucleoporin in metazoans, although there is a family of orthologs in Saccharomyces cerevisiae, Nup145, Nup116, and Nup100. Nup98 is found on both cytoplasmic and nucleoplasmic faces of the NPC and is a relatively dynamic Nup (13Griffis E.R. Altan N. Lippincott-Schwartz J. Powers M.A. Mol. Biol. Cell. 2002; 13: 1282-1297Crossref PubMed Scopus (204) Google Scholar, 14Rabut G. Doye V. Ellenberg J. Nat. Cell Biol. 2004; 6: 1114-1121Crossref PubMed Scopus (355) Google Scholar). Nup98 can be translated from two major alternatively spliced mRNA transcripts. The shorter transcript encodes a 920-amino acid, 98-kDa protein. The larger transcript encodes a 195-kDa Nup98/Nup96 polyprotein (Fig. 1). Post-translationally, both proteins are cleaved by cis-autoproteolysis to yield an N-terminal cleavage product of 90 kDa (generally referred to as Nup98) and a C-terminal cleavage product of either a 57 amino acid peptide (referred to here as the Nup98 "tail") or the nucleoporin Nup96, which is identical in its first 51 amino acid residues to the Nup98 tail (15Fontoura B.M. Blobel G. Matunis M.J. J. Cell Biol. 1999; 144: 1097-1112Crossref PubMed Scopus (195) Google Scholar). An equivalent cleavage event takes place in the yeast ortholog, Nup145, yielding the nucleoporins Nup145N and Nup145C (16Teixeira M.T. Siniossoglou S. Podtelejnikov S. Benichou J.C. Mann M. Dujon B. Hurt E. Fabre E. EMBO J. 1997; 16: 5086-5097Crossref PubMed Scopus (88) Google Scholar, 17Teixeira M.T. Fabre E. Dujon B. J. Biol. Chem. 1999; 274: 32439-32444Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Two other yeast orthologs, Nup100 and Nup116, are homologous only to the N-terminal cleavage product, Nup98, and do not carry out autoproteolysis. The targeting of Nup98 to the NPC is, in part, through binding between its C-terminal domain and Nup96 (18Griffis E.R. Xu S. Powers M.A. Mol. Biol. Cell. 2003; 14: 600-610Crossref PubMed Scopus (131) Google Scholar, 19Hodel A.E. Hodel M.R. Griffis E.R. Hennig K.A. Ratner G.A. Xu S. Powers M.A. Mol. Cell. 2002; 10: 347-358Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 20Vasu S. Shah S. Orjalo A. Park M. Fischer W.H. Forbes D.J. J. Cell Biol. 2001; 155: 339-354Crossref PubMed Scopus (132) Google Scholar), and in keeping with this, the products of Nup98 autoproteolysis can bind each other noncovalently in vitro. Because of the sequence identity between the Nup98 tail and Nup96, both bind identically to Nup98 and can compete with each other for binding (18Griffis E.R. Xu S. Powers M.A. Mol. Biol. Cell. 2003; 14: 600-610Crossref PubMed Scopus (131) Google Scholar). Therefore in vivo, if Nup98 is expressed from the smaller transcript, the tail peptide must be released to allow Nup98 to associate with Nup96 at the NPC (18Griffis E.R. Xu S. Powers M.A. Mol. Biol. Cell. 2003; 14: 600-610Crossref PubMed Scopus (131) Google Scholar, 19Hodel A.E. Hodel M.R. Griffis E.R. Hennig K.A. Ratner G.A. Xu S. Powers M.A. Mol. Cell. 2002; 10: 347-358Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Furthermore, although Nup96 incorporates into a large and highly stable subcomplex of the NPC, Nup96 expressed in vivo independently of Nup98 did not enter the nucleus and did not incorporate into NPCs with normal efficiency (15Fontoura B.M. Blobel G. Matunis M.J. J. Cell Biol. 1999; 144: 1097-1112Crossref PubMed Scopus (195) Google Scholar). Thus, proper assembly of Nup98 and Nup96 into the NPC requires a complex series of interactions and processing. The crystal structure of the Nup98 C-terminal domain revealed molecular details of the binding interaction between Nup98 and Nup96. Binding is mediated in large part by interactions between the first seven residues of the tail/Nup96 and a binding groove in Nup98 (19Hodel A.E. Hodel M.R. Griffis E.R. Hennig K.A. Ratner G.A. Xu S. Powers M.A. Mol. Cell. 2002; 10: 347-358Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). In addition, a run of acidic residues in the tail/Nup96, although not directly visible in the structure, were constrained to lie in proximity to a basic patch in Nup98; electrostatic interactions in this region would further contribute to binding between Nup98 and Nup96. Amino acids involved in both autocatalysis and binding are generally conserved between yeast Nup145 and human Nup98. We, therefore, postulated that Nup145N and Nup145C were likely to interact analogously at the yeast NPC (19Hodel A.E. Hodel M.R. Griffis E.R. Hennig K.A. Ratner G.A. Xu S. Powers M.A. Mol. Cell. 2002; 10: 347-358Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). Although Nup116 and Nup100 lack residues required for autocatalytic cleavage, molecular modeling predicts some structural similarity to both Nup98 and Nup145N, including conservation of the patch of basic amino acids. Thus, Nup145C might act also as a binding site for Nup116 and Nup100 in addition to Nup145N. Here we have further investigated the nature of the interactions between these GLFG nucleoporins and the NPC. We find that the dynamics of the Nup98/Nup96 interaction can be recapitulated in vitro using purified protein domains, indicating that additional factors are not required to facilitate such exchange. Binding can occur between the independently expressed Nup98 pore-targeting domain and the Nup98 tail/Nup96 fragment, suggesting that simultaneous translation and folding are not required to produce the appropriate structural folds. Using fluorescence polarization, we have determined the binding constant for this interaction. Furthermore, we extended our analysis to the yeast GLFG orthologs and find that, as expected, Nup145N utilizes the same mechanism for interaction with Nup145C. However, despite sharing considerable sequence homology, Nup116 binds far less robustly to Nup145C, and significant binding between Nup100 and Nup145C was not detected, suggesting that these nucleoporins use, at least in part, distinct means of incorporation into the NPC. Production of Recombinant Proteins—For expression of GST fusions, fragments of Nup98, Nup145, Nup100, or Nup116 genes were cloned into pGEX-4T vectors and expressed in BL21(DE3) cells induced at either 37 °C for 3 h or 17°C overnight. Frozen cell pellets from 1 liter of culture were resuspended in binding buffer 1 (BB1; 10 mm Tris, pH 8.0, 100 mm NaCl, 1 mm β-mercaptoethanol, 1 mm EDTA) with 0.1% Triton X-100 and COMPLETE protease inhibitors (Roche Applied Science) to a final volume of 15 ml/liter of culture and lysed in a French press. Insoluble material was removed by centrifugation, and clarified lysates were either frozen for later use or incubated with glutathione-Sepharose (GE Healthcare) at 700 μl of beads/liter of culture for 1 h at 4°C. The beads were washed extensively, first in BB1 with 0.1% Triton-X and then in BB1 alone. Bound fusion proteins were eluted with 100 mm reduced glutathione in 100 mm Tris, pH 8.0, 120 mm NaCl, and dialyzed against BB1. Products were analyzed by SDS-PAGE and Coomassie Blue staining and judged to have purity in excess of 90%. Purified proteins or clarified lysates were stored in aliquots at –80 °C after addition of glycerol to 7% final concentration. To produce C-terminal GFP fusions, fragments of Nup96, Nup98/96, Nup145, or Nup145C were cloned upstream of a modified GFP2 gene (21Prasher D.C. Eckenrode V.K. Ward W.W. Prendergast F.G. Cormier M.J. Gene (Amst.). 1992; 111: 229-233Crossref PubMed Scopus (1763) Google Scholar) in pET-28a and expressed in BL21(DE3) cells induced at 37° for 3 h. For purification of GFP-hexahistidine-tagged proteins, frozen cell pellets were resuspended in nickel binding buffer consisting of 50 mm sodium phosphate, pH 7.4, 250 mm NaCl, 0.1% Triton X, 1 mm phenylmethylsulfonyl fluoride and lysed in a French press. After the pelleting of insoluble material, the protein was bound to a HiTrap chelating column (GE Healthcare) and eluted with a gradient of 0–500 mm imidazole in nickel binding buffer. Fractions containing the protein were pooled and dialyzed against BB1 containing 10% glycerol, separated into aliquots, and stored at –80 °C. Protein concentrations were determined using the Bio-Rad Protein Assay with bovine serum albumin as a protein standard. Mutagenesis—Site-directed mutagenesis was performed using QuikChange mutagenesis (Stratagene; Austin, TX), and mutations were verified by sequencing. Mutant proteins were expressed and purified as described above. Bead Binding Assays—For trans binding assays, 75 μgof purified GST-fusion protein and 37.5 μl of washed glutathione-Sepharose beads in a total volume of 250 μl of BB1 were incubated for 75 min at 4 °C. After incubation, beads were pelleted and washed 3 times in cold BB1 with 0.1% Triton X and once with BB1 alone. 1.5 mg of purified tail-GFP-His-tagged protein in 500 μl of BB1 was then added to the washed beads and incubated for 3 h at 4°C to allow binding. After incubation, beads were pelleted and washed once in BB1 with 0.1% Triton-X and 3 times with BB1 alone. Bound proteins were then eluted by boiling in Laemmli gel sample buffer (22Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207012) Google Scholar) and analyzed on 14% acrylamide gels followed by Coomassie Blue R250 staining. Final loads were adjusted to give approximately equal amounts of GST head protein. For exchange binding assays, 37.5 μl of washed beads were added to 500 μl of clarified lysate containing GST fusion protein, corresponding to 80 ml of expression culture. These samples were incubated to allow binding and washed in the same manner as the trans binding assays. 1.5 mg of purified GFP-His-tagged protein in 1 ml of BB1 was then added to the washed beads, and the samples were incubated, washed, and processed in the same manner as trans binding assays. For immunoblots of bead binding assays, proteins were transferred from polyacrylamide gels to Immobilon membranes (Millipore; Billirica, MA), and tail fragments were detected using a mouse antibody against the hexahistidine tag (H-15; Santa Cruz Biotechnology; Santa Cruz, CA) and goat anti-mouse horseradish peroxidase conjugate (Zymed Laboratories Inc.; South San Francisco, CA). Fluorescence Polarization Assays—For polarization assays, three fluorescently labeled peptides (sequences given in Table 1) were prepared by solid phase peptide synthesis at the Keck Biotechnology Resource Center at Yale University, New Haven, CT. After the actual Nup96 or Nup145C sequence, each peptide terminates with a diglycine linker followed by an ϵ-(5/6-carboxyfluorescein)-l-lysine residue, referred to as K(fam), and a final glycine required C-terminal to K(fam) in the synthetic process. High performance liquid chromatography analysis revealed no significant peaks other than those corresponding to the intended products.TABLE 1Binding constants for analogs of autoproteolytic nucleoporin cleavage productsN-terminal fragmentC-terminal peptideC-terminal peptide sequenceKdμmNup98Nup98/96 β-strand plus acidic residuesSKYGLQDSDEEEEEGGK*G0.11 ± 0.01Nup98Nup98/96 β-strand onlySKYGLQDSGGK*G2.6 ± 0.1Nup98Nup145 β-strand plus acidic residuesSIWGLVNEEDAEIDEDDGGK*G0.25 ± 0.01Nup145Nup145 β-strand plus acidic residuesSIWGLVNEEDAEIDEDDGGK*G0.12 ± 0.01Nup145Nup98/96 β-strand plus acidic residuesSKYGLQDSDEEEEEGGK*G0.16 ± 0.01Nup145Nup98/96 β-strand onlySKYGLQDSGGK*G1.6 ± 0.1Nup116Nup145 β-strand plus acidic residuesSIWGLVNEEDAEIDEDDGGK*G1.3 ± 0.1Nup100Nup145 β-strand plus acidic residuesSIWGLVNEEDAEIDEDDGGK*G36 ± 1 Open table in a new tab To obtain binding curves, purified GST head fragments in concentrations ranging from 1 nm to greater than 50 μm were incubated at room temperature with 1.0 × 10–8 m fluoresceinated peptide in BB1 for at least 2.75 h to reach full equilibrium. Fluorescence polarization was measured at 25 °C over 15 read cycles on a Beacon 2000 Fluorescence Polarization System (Invitrogen). For each combination of head fragment and tail peptide, measurements were made at 20 or more different concentrations of head protein resulting from serial dilutions of 1:1.6. Fluorescent peptide was kept at 1.0 × 10–8 m in all incubations. Four independent dilution series were prepared for each combination of head fragment and tail peptide unless otherwise indicated. A binding constant, KD, was determined for each binding curve by nonlinear least squares fitting using ProFit software (QuantumSoft; Uetikon am See, Switzerland) and Equation 1, θ=(HT+KD+TT)-(-HT-KD-TT)2-4HTTT2TT(Eq. 1) where θ is the fraction of tail protein bound given as the fraction of the polarization maximum observed at saturating concentrations of head protein, HT is the total concentration of head protein, bound and unbound, in each reaction, and TT equals 10.0 nm, i.e. the total concentration of tail peptide, both bound and unbound, in each reaction. Nup98 is one of a subset of nucleoporins that undergo dynamic interactions with the nuclear pore. Nup98 is unique among nucleoporins in that the precise site of its interaction with the pore has been determined at the molecular level (18Griffis E.R. Xu S. Powers M.A. Mol. Biol. Cell. 2003; 14: 600-610Crossref PubMed Scopus (131) Google Scholar, 19Hodel A.E. Hodel M.R. Griffis E.R. Hennig K.A. Ratner G.A. Xu S. Powers M.A. Mol. Cell. 2002; 10: 347-358Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The Nup98 C-terminal domain undergoes an autocatalytic cleavage that releases either an 8-kDa protein tail or the nucleoporin Nup96 depending upon whether Nup98 was expressed as a single protein or as the polyprotein precursor, Nup98/Nup96 (Fig. 1 and Fontoura et al. (15Fontoura B.M. Blobel G. Matunis M.J. J. Cell Biol. 1999; 144: 1097-1112Crossref PubMed Scopus (195) Google Scholar)). After cleavage, Nup98 remains bound to amino acid residues immediately after the cleavage site. These residues are identical in either the tail peptide or the N terminus of Nup96, and this binding to Nup96 is thought to be the major site of Nup98 interaction with the NPC. To advance our understanding of the dynamic nature of the NPC, we set out to both further characterize the binding interaction between Nup98 and Nup96 and to extend our analysis to the orthologous yeast GLFG nucleoporins. In Vitro Binding Is Dynamic and Does Not Require Simultaneous Protein Folding—Initially it was uncertain whether dynamics of the interaction between Nup98 and the tail peptide/Nup96 would be recapitulated in vitro or whether some additional factor might be required to release the tail peptide from Nup98. To test this we developed an exchange binding assay in which a GST-tagged Nup98 NPC-targeting domain including the cleavage site and tail peptide (amino acids 712–920; "GST-head-tail") was bound to glutathione beads and then incubated with a second form of this same Nup98 domain in which the tail peptide was fused to GFP (Fig. 2A; "head-tail-GFP"). After incubation, the beads with bound GST-Nup98 head were retrieved, and the presence of the tail-GFP fragment was assessed by gel electrophoresis as an indication of dynamic exchange between the head and tail fragments. When the GST was fused to a wild type Nup98 head-tail construct, which could undergo autocatalytic cleavage to separate head and tail fragments, there was clearly dynamic exchange in which some of the original tail peptide was replaced by tail-GFP (Fig. 2A, lanes 3 and 4). When GST was fused to a mutant form of Nup98 (S864A), which we had previously shown to be inactive for autocatalytic cleavage and release of the tail (19Hodel A.E. Hodel M.R. Griffis E.R. Hennig K.A. Ratner G.A. Xu S. Powers M.A. Mol. Cell. 2002; 10: 347-358Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar), there was no exchange, and no tail-GFP was recovered on the beads (Fig. 2A, lanes 5 and 6). We concluded, therefore, that dynamic interaction between Nup98 and the tail/Nup96 residues downstream of the cleavage site did not require additional factors from the NPC; the interaction remained dynamic in vitro. The autocatalytic activity of Nup98 identifies it as a member of a family of self-processing proteases that includes both intein and NTN proteins (for review, see Ref. 23Perler F.B. Xu M.Q. Paulus H. Curr. Opin. Chem. Biol. 1997; 1: 292-299Crossref PubMed Scopus (138) Google Scholar). It has been suggested for some members of this family that expression as a polyprotein followed by autocatalytic cleavage to separate the components is necessary to obtain protein conformations that can only be achieved by simultaneous folding as a single polypeptide (24Xu Q. Buckley D. Guan C. Guo H.C. Cell. 1999; 98: 651-661Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Such conformations would not be obtainable from components expressed independently and then combined, as was shown in the case of glycosyl asparaginase (25Riikonen A. Tikkanen R. Jalanko A. Peltonen L. J. Biol. Chem. 1995; 270: 4903-4907Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Similarly, simultaneous folding in the form of Nup98 with its tail peptide or the Nup98/Nup96 polyprotein might be essential for Nup98 and Nup96 to achieve conformations that allow correct recognition of each other as binding partners. To test this possibility, we used a trans binding assay (Fig. 2B) in which GST-Nup98 head truncated at the site of cleavage (amino acids 712–863) was bound to glutathione beads and incubated together with independently expressed tail-GFP (amino acids 864–920). The ability of the proteins to bind each other was queried by retrieval of the beads and detection of bound tail-GFP by PAGE and Coomassie staining. Expression in bacteria leads to removal of the initiator methionine, thus leaving the authentic serine as the first residue of the tail-GFP construct. When these separately produced partners were mixed, we found that the two components had each independently folded into a conformation that was compatible with binding; tail-GFP was retrieved on glutathione beads along with GST-head (Fig. 2B, lanes 2 and 3). As before, the non-cleavable mutant head, which cannot release the tail peptide, did not bind the tail-GFP protein (Fig. 2B, lanes 4 and 5). Nup98/Nup96 Binding Constant Determined by Fluorescence Polarization—Because we appeared to have recapitulated authentic dynamic binding in vitro, we next determined the binding constant for this interaction. For these measurements, we used fluorescence polarization assays in which GST-Nup98 head (amino acids 712–863) was incubated with a synthetic fluorescent peptide corresponding to the first 14 amino acids of the tail/Nup96 followed by a diglycine linker and a fluorescently labeled lysine residue (see the sequence in Table 1). This sequence includes the tail/Nup96 residues previously observed by crystallography to directly bind to Nup98 as well as the stretch of acidic residues we predicted to be positioned for interaction with a cluster of basic residues in Nup98 (Ref. 19Hodel A.E. Hodel M.R. Griffis E.R. Hennig K.A. Ratner G.A. Xu S. Powers M.A. Mol. Cell. 2002; 10: 347-358Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar and Table 1). The binding curve obtained from the polarization assays indicated a dissociation constant of 0.11 μm (Fig. 3 and Table 1). When the tail peptide was shortened by removal of the acidic residues, the binding constant increased ∼10-fold to 1.3 μm, indicating a significant contribution of the acidic residues to binding. S. cerevisiae Nup145N and Nup145C Bind Each Other in a Manner Equivalent to Nup98/Nup96—In the budding yeast S. cerevisiae, Nup98 has three orthologs Nup145, Nup116, and Nup100 (26Suntharalingam M. Wente S.R. Dev. Cell. 2003; 4: 775-789Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). Of these three proteins, only Nup145 undergoes autocatalytic cleavage to yield Nup145N and Nup145C. Nup145N resembles Nup98 in both the C-terminal and GLFG repeat domains, although it lacks a binding site for the mRNA export factor Rae1/Gle2 (Fig. 1). It is not established whether Nup145N interacts stably or dynamically with the yeast NPC; however, based upon our results with Nup98, we predicted that Nup145N would bind to the post-cleavage N terminus of Nup145C. To test this possibility, we expressed GST-Nup145N (amino acids 439–605; head) and Nup145C (amino acids 606–631; tail-GFP) constructs analogous to the GST-Nup98 head and Nup98 tail-GFP proteins and utilized these in both exchange (Fig. 4A, left panel) and trans (Fig. 4A, right panel) binding assays. Like Nup98, Nup145 fragments undergo dynamic interactions in vitro and are capable of binding each other efficiently when expressed in trans. As expected, the homologous uncleavable mutation in Nup145 (S606A) was completely unable to bind to the Nup145 tail-GFP in an exchange assay (Fig. 4A, lanes 4 and 5 and lanes 8 and 9). To further test our prediction that the post-cleavage interaction between Nup145N and Nup145C is equivalent to the interaction seen for Nup98 and Nup96, we synthesized a fluorescent Nup145C peptide analogous to the Nup98 tail peptide for fluorescence polarization binding analysis (27Checovich W.J. Bolger R.E. Burke T. Nature. 1995; 375: 254-256Crossref PubMed Scopus (164) Google Scholar). This peptide corresponded to the first 17 amino acids of Nup145C including the cluster of acidic residues (Table 1). Binding to the Nup145 head resulted in a binding curve with a dissociation constant of 0.12 μm (Fig. 4B and Table 1). This value is remarkably similar to that obtained from the Nup98 assays, suggesting an equivalent and conserved binding interaction. Indeed, we found that the Nup98 head could bind to the Nup145 tail with only slightly more than a 2-fold decrease in affinity compared with binding between the Nup98 head and Nup98 tail (Fig. 3 and Table 1). Conversely, the Nup145N head construct bound the Nup98 tail with virtually the same affinity as it bound its natural partner (Fig. 4B and Table 1). Again, there was a clear contribution of the acidic residues to both these heterologous interactions since, in their absence, the affinity of binding between the Nup145N head and the Nup98 tail was reduced 10-fold exactly as had been observed for binding between th
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