We are using a novel nuclear envelope fractionation procedure to characterize more completely the protein components making up the mammalian NPC. Based on this fractionation procedure and separation of the isolated proteins by reverse phase column chromatography, we estimate that less than one-third of the mammalian nucleoporins have been characterized at the molecular level. This factor contributes significantly to our lack of understanding about how the NPC is assembled, and how it functions to regulate nucleocytoplasmic transport. The fractionation procedure that we have developed yields nucleoporins and NPC-associated proteins in high purity and yield, and has allowed us to identify a significant number of novel proteins. The first NPC-associated protein that we characterized using this fractionation procedure was the SUMO-1 modified form of RanGAP1 (Matunis et al., 1996
). Here, we have reported the characterization of a second protein, a new nucleoporin of 96 kD termed Nup96.
The most intriguing aspect of Nup96 is its unusual pathway of biogenesis. Through analysis at a number of levels, we have demonstrated that Nup96 is synthesized as a precursor that is proteolytically cleaved in vivo. Cleavage of this precursor generates not only Nup96, but also the GLFG-containing nucleoporin, Nup98. Nup98 can also be produced independently of Nup96, by what appears to be an alternatively spliced mRNA that does not include the Nup96 open reading frame (Radu et al., 1995
; Borrow et al., 1996
; Nakamura et al., 1996
). Whereas it was not previously recognized that this predicted Nup98 protein is proteolytically processed, we have shown that it is processed like the Nup98-Nup96 precursor. Evidence for the synthesis and processing the Nup98-Nup96 precursor and the Nup98 precursor in vivo are: (a) Northern blot analysis that supports the existence of the precursor mRNAs, and isolation of cDNAs that predict the precursor proteins; (b) NH2
-terminal peptide sequence of Nup96 that supports the cleavage of the Nup98-Nup96 precursor between phenylalanine 863 and serine 864; (c) mutagenesis of the cleavage sites that yield the uncleaved precursors, both in vitro and in vivo; (d) homology with S. cerevisiae
Nup145p, which undergoes posttranslational in vivo cleavage at an identical site (Dockendorff, 1997; Emtage et al., 1997
; Teixeira et al., 1997
The Nup98-Nup96 precursor is the apparent vertebrate homologue of S. cerevisiae
Nup145p. The NH2
terminus of both proteins, Nup98 and N-Nup145p, are ~20% identical over their entire length and contain highly conserved GLFG repeats. N-Nup145p is not essential, but it may have RNA-binding activity and function in RNA transport in yeast (Fabre et al., 1994
; Emtage, 1997; Teixeira et al., 1997
). In vertebrates, Nup98 is located on the nucleoplasmic side of the NPC, at or near the basket, consistent with a possible role in RNA export (Radu et al., 1995
). Further supporting such a role, antibodies against Nup98 inhibit export of multiple classes of RNAs when injected into the nuclei of Xenopus
oocytes (Powers et al., 1997
). The COOH-terminal domains of both precursors, Nup96 and C-Nup145p, are also 20% identical over their entire length, but contain no obvious motifs or homologies to proteins of know function. Studies in yeast, nonetheless, indicate a role for C-Nup145p in mRNA export (Dockendorff, 1997; Emtage, 1997; Teixeira et al., 1997
). Like Nup98, Nup96 also localizes to the nucleoplasmic side of the NPC, at or near the basket, consistent with a potential role in mRNA export. Our data obtained from in situ labeling of intact cells indicate that Nup96 may be located near the distal end of the nucleoplasmic basket, in proximity to the site where Tpr associates with the NPC.
Of particular interest is the domain around the Nup98-Nup96 cleavage site, as it is especially conserved among protein homologues in yeast, plant, worm, and man. A similar domain is also present at the extreme COOH-terminus of two additional yeast GLFG repeat-containing nucleoporins, Nup100p and Nup116p (Wente et al., 1992; Radu et al., 1995
; Teixeira et al., 1997
). In particular, the ~50 amino acids preceding the cleavage site, and the ~12 amino acids after the cleavage site, are ~40% identical between yeast and human (relative to 20% identity between other regions of the proteins), suggesting that this region may be important for the cleavage process. Surprisingly, however, a large portion of this highly conserved domain is not required for proteolytic processing of Nup145p in vivo (Emtage et al., 1997
). This raises the possibility that the domain preceding the cleavage site may have another function, a function that could possibly be regulated by the cleavage process. This domain includes a conserved RNP1 consensus site that has been suggested to contribute to the RNA-binding properties of N-Nup145p (Fabre et al., 1994
). It will be interesting to determine whether the RNA-binding properties of either Nup98 or N-Nup145p are affected by proteolytic cleavage. The conserved residues surrounding the cleavage site itself, H-F-S, do not resemble the consensus site for any known protease, and the exact mechanism for the cleavage process remains to be elucidated. It is also unclear when, or where the cleavage event occurs, although pulse labeling experiments indicate that the Nup98-Nup96 precursor is cleaved within five minutes of being synthesized (data not shown).
Whereas the physiological relevance of producing both Nup98 and Nup96 through proteolytic cleavage of a precursor is not known, the conservation of this pathway from yeast to human suggests a potentially important function. Posttranslational proteolytic cleavage of precursor proteins is widely used to regulate many different cellular functions. Processing of many neuropeptides, hormones and certain plasma proteins is used to regulate the formation of mature, active factors without the need for de novo protein synthesis (Resnick and Zasloff, 1992
; Seidah and Chretien, 1997
). Protelolytic cleavage can also function to indirectly activate proteins by regulating their localization within the cell. Examples include the signal-mediated nuclear targeting of Notch-1 from the plasma membrane (Schroeter et al., 1998
), and relocalization of the sterol regulatory element binding proteins from the nuclear membrane to the nucleus in response to sterol levels (Brown and Goldstein, 1997
). Other possible functions of precursor synthesis and proteolytic cleavage include protein folding, as has been suggested for certain ribosomal protein-ubiquitin precursors (reviewed by Johnson and Hochstrasser, 1997
), and as a mechanism to strictly control protein stoichiometry. Another important role for regulated proteolytic cleavage is in the assembly and maturation of large molecular complexes, best exemplified by virus particle assembly (reviewed in Krausslich and Welker, 1996
). By this analogy, proteolytic cleavage of nucleoporins could be related to the orderly assembly of the NPC. As an example, the Nup98-Nup96 precursor could be inserted into a newly synthesized pore and cleaved only after the appropriate cleavage factor is assembled. Cleavage may then result in conformational changes, or exposure of binding sites on Nup98 and/or Nup96, that allow other sets of proteins to bind in an orderly sequence.
Our data demonstrate that proper processing of both the Nup98-Nup96 precursor and the Nup98 precursor is required for efficient NPC association, indicating that there is an ordered series of events leading to the association of these nucleoporins with the NPC. Mutations in the cleavage site that prevent processing lead to accumulation of both proteins in the nucleoplasm, but not at NPCs. In the case of Nup98, this result suggests that the COOH-terminal 58 amino acids (that would normally be removed) either tether Nup98 to intranuclear sites, or mask the NPC-targeting domain. Interestingly, in addition to not being targeted to NPCs, unprocessed Nup98 was also absent from nucleoli when over expressed. Although the significance of processed Nup98 appearing in nucleoli is not clear, it has previously been suggested that Nup98 may not be absolutely confined to NPCs (Powers et al., 1995
). These data raise the intriguing possibility that Nup98 could shuttle between nucleoli and NPCs.
In addition to cleavage being important, our studies also demonstrate that proper targeting of Nup98 and Nup96 to the NPC is influenced by the synthesis of the precursor protein. In particular, we found that targeting of Nup96 to the NPC is dependent on its being synthesized as a precursor. When expressed independently, Nup96 accumulates in the cytoplasm, suggesting that its nuclear import may be Nup98-mediated (Nup98 expressed alone is imported into the nucleus). Nup98-mediated import of Nup96 could occur in one of two ways. Either the Nup98-Nup96 precursor could be imported into the nucleus before proteolytic processing, or Nup98 and Nup96 could remain associated after proteolytic processing in the cytoplasm, and then be imported as a complex. At present, there is no data to suggest that Nup98 and Nup96 remain associated with each other once they are inserted into the NPC. The sub-complex of nucleoporins containing Nup96, Nup107, and mSec13 that we isolated does not contain Nup98, and experiments in yeast indicate that the analogous sub-complex also lacks N-Nup145p (Teixeira et al., 1997
To date, relatively few sub-complexes of the mammalian NPC have been identified. And although strikingly similar at a morphological level (Yang et al., 1998
), no homologous sub-complexes of mammalian and yeast NPCs have previously been identified. This has raised the notion that the molecular structure of the NPC may be less well conserved than its morphological structure. We have identified a complex of mammalian nucleoporins that contains Nup96, Nup107, mSec13, at least one Sec13-related protein, and at least two additional proteins of ~150 kD. This complex is highly similar to the Nup84p complex of yeast, and therefore represents one of the first homologous complexes of nucleoporins identified between yeast and mammals. There is also evidence for homology between a yeast nucleoporin complex containing Nup159p, Nup82p, and Nsp1p and a vertebrate complex containing Nup214/CAN, Nup88, and possibly p62 (Macaulay et al., 1995
; Bastos et al., 1997
; Fornerod et al., 1997
; Belgareh et al., 1998
). The identification of these complexes suggests that the molecular structure of the NPC may be more highly conserved than was anticipated. Similarly, homologous sub-complexes will likely be identified in the future, as more mammalian and yeast nucleoporins are identified and characterized. In the immediate future, further purification of this complex will allow us to identify several additional nucleoporins, and possibly obtain information about its structural features. Of particular interest is the presence of the Sec13-related proteins in this complex. Whereas the exact role that Sec13 may have at the NPC has been previously discussed (Siniossoglou et al., 1996
), our combined data suggest that this function is likely to be exerted on the nucleoplasmic side of the NPC, given the localization Nup96. It will be interesting to investigate the role that Sec13 may have in NPC biogenesis using Xenopus
nuclear envelope reconstitution systems.
What are the functions of Nup98 and Nup96 once assembled into the NPC? As indicated, both Nup98 and Nup96 localize to the nucleoplasmic side of the NPC, at or near the basket. The nucleoplasmic basket appears to have an active role in binding RNP complexes and mediating their export from the nucleus. Electron micrographs of Balbiani ring RNPs being transported through the NPC show intimate interactions between the basket and the RNP, implying that the proteins in this structure actively engage the particles during transport (Kiseleva et al., 1996
). It is not yet known whether the nucleoporins at the basket bind the RNA itself, and/or the hnRNP proteins that accompany the RNA during transport (Nakielny et al., 1997
). As implied from their localization, and the functional studies of Nup145p in yeast and Nup98 in Xenopus
, Nup98 and Nup96 are likely to be important factors mediating the docking and translocation of RNPs through the NPC. The challenge ahead will be to understand how these nucleoporins interact with RNPs as they are transported through the NPC, and how these interactions regulate the transport process.