Using both in vivo and in vitro approaches, we have shown that Nup53 plays an essential role in nuclear pore complex assembly and nuclear envelope formation. Through our analysis, we have identified the regions of Nup53 that are necessary for its interactions with its neighbors Nup93, Nup155, and the transmembrane protein NDC1, and we have defined the roles of these regions of Nup53 in mediating its targeting to the nuclear rim in vivo and its function in NPC assembly and NE formation in vitro. Each of the nup interacting regions of Nup53 can associate with the NPC in vivo. However, all three regions are required for the efficient targeting of Nup53 to the NPC. The involvement of NDC1 in Nup53 localization is consistent with our previous conclusion that the interaction of Nup53 subcomplex with the membrane is mediated by NDC1 via its association with the COOH-terminal region of Nup53 (Mansfeld et al., 2006
). In addition, we have shown that the region of Nup53 required for binding to Nup155 plays a critical role in NPC and NE assembly in the Xenopus
system, suggesting the formation of a Nup53/Nup155 complex is a key step in this process.
The NPC is a multisubunit complex composed of several structurally defined components positioned at specific locations within the NPC (Schwartz, 2005
; Fahrenkrog, 2006
). A major challenge in the field is to develop a model for how individual Nups within the subcomplexes are locally organized and how, in turn, these subcomplexes interact with one another and the pore membrane. This portrait of the NPC is, however, only the initial step in our understanding of the complex and dynamic steps that lead to its assembly. As a step toward addressing this process, we have focused on defining interactions between Nup53 and several of its evolutionarily conserved binding partners, including Nup93 and Nup155. This subcomplex is a component of the NPC core, and interactions of two members of this subcomplex, Nup53 and Nup155, with pore membrane proteins [(Mansfeld et al., 2006
); ; Mitchell and Wozniak, unpublished data] suggest it lies adjacent to the membrane.
In this study, we assessed the interactions between members of the Nup53-containing subcomplex using human Nup53 truncations. Minimal binding regions of Nup53 were defined that are required for maintaining interactions with its known partners. Interestingly, as shown in , these regions are separable and not entirely interdependent. Nup93 binds an NH2
-terminal segment (amino acids residues 1-143) of Nup53, likely together with its binding partner Nup205 as this nup was universally detected in fractions containing Nup93 (data not shown). Nup155 interacts specifically with a more central region (residues 167-300) of Nup53, whereas NDC1 binds a COOH-terminal segment that contains an amphipathic helix located in the last 26 amino acid residues of Nup53. It is noteworthy that the Nup531-300
fragment, lacking the NDC1 interaction domain, binds better to Nup155 than full-length Nup53. We interpret these data to suggest that although the Nup155 and NDC1 binding sites on Nup53 are distinct, the binding of Nup53 to the two partner Nups is not entirely independent. One possible explanation for the increased binding of the Nup531-300
fragment to Nup155 is that the COOH-terminal 26-amino acid residues of Nup53 inhibits binding to Nup155. In vivo this could function to inhibit their interaction until the COOH-terminal region of Nup53 engages the membrane, binds NDC1, or interacts with an as yet unidentified protein. In our in vitro binding assays these factors would be absent (in the case of membrane) or potentially present in substoichiometric amounts (i.e., free NDC1), thus leading to the reduced binding of Nup155 to full-length Nup53 compared with the Nup531-300
fragment. This model is attractive as it is consistent both with the observation that Nup155 is not removed from Nup53-depleted soluble extracts (C) and with the apparent interdependence of Nup53 and Nup155 for incorporation into the assembling NE and NPC (Franz et al., 2005
; Supplemental Figure S1).
In vivo analysis of the Nup53 truncation mutants showed that each of the interaction domains of Nup53 with its neighboring nups (Nup93, Nup155, and NDC1) play a role in the efficient incorporation of Nup53 into the NPC. Individually, these separate regions can bind the NPC to some extent but with reduced efficiency relative to the wild-type protein. This would suggest that the cumulative interactions of Nup53 with its neighbors, rather than one specific binding event, are required for the stable association of Nup53 with the NPC (). Studying the dynamics of GFP-Nup53 truncation mutants in more detail in live vertebrate cells and using molecular dynamics simulation approaches, as used to study the binding of importin-β to FG repeat nucleoporins (Isgro and Schulten, 2005
), could shed more light on the interactions between the individual members of the Nup53 subcomplex.
Our data suggest that Nup53 plays an important role in nuclear assembly. Previously, depletion of the protein from mammalian cultured cells by RNAi (Hawryluk-Gara et al., 2005
) was shown to cause a severe defect in nuclear morphology and reduction of accumulation of Nup93, Nup155, and Nup205 at the nuclear rim suggestive of defects in NPC assembly. Moreover, depletion of the C. elegans
counterpart of Nup53 caused an embryonic lethal phenotype along with a severe block to nuclear formation after mitosis (Galy et al., 2003
). As in the C. elegans
case, upon depletion of Nup53 from Xenopus
extracts, we observed a strong inhibition of NE formation. Membrane vesicles were bound to the chromatin surface, but they did not fuse to form a closed NE and no NPC assembly was detected either by immunofluorescence or electron microscopy. Of the nucleoporins thus far tested, depletion of only three have produced a similar defect in NE formation (Finlay et al., 1991
; Powers et al., 1995
; Grandi et al., 1997
; Walther et al., 2001
). Two nucleoporins are the integral pore membrane proteins NDC1 and POM121 (Antonin et al., 2005
; Mansfeld et al., 2006
) that reside, together with lamin B receptor, in a membrane vesicle population, which has a high avidity for the chromatin surface (Antonin et al., 2005
; Ulbert et al., 2006
). The third nucleoporin is Nup155 (Franz et al., 2005
). The depletion of Nup155 from C. elegans
embryos also prevents NE formation (Franz et al., 2005
). Nup155, although recruited late during NE formation, therefore defines an essential step in NPC and NE assembly.
The similarities in phenotype observed upon depletion of POM121, NDC1, Nup53, and Nup155 might suggest that these proteins function on a similar branch of the assembly pathway. In support of this idea, our analysis of the Nup53 truncation mutants revealed that the region that bound Nup155 (Nup53167-300; ) was sufficient to restore both NE and NPC formation and Nup155 recruitment in assembly assays depleted of Nup53. These results suggest that complex formation with Nup155 is critical to the function of Nup53 in NE and NPC assembly. Interestingly, the Nup531-300 fragment, which, as discussed above, binds Nup155 in vitro at a higher efficiency than full-length Nup53, but it does not interact with NDC1, rescues the depletion phenotype to almost the same extent as full-length Nup53. This implies that although NDC1 interacts with Nup53, and this association is likely to play a role in linking Nup53 to the pore membrane, this specific interaction is not essential for the assembly of the Nup53/Nup155 complex within the NPC. It is possible that functionally redundant mechanisms exist that mediate the association of the Nup53/Nup155 complex with the pore membrane including, for example, the presence of additional interactions interfaces between this complex and NDC1 or other membrane proteins. Recent observations suggest the latter may exist as we have detected a direct interaction between Nup155 and POM121 (Mitchell and Wozniak, unpublished data).
Interestingly, within the Nup53167-300
truncation is an evolutionarily conserved RNA recognition motif (RRM) that is positioned within amino acid residues 167–252 of human Nup53 (Devos et al., 2006
). The crystal structure of this RRM has recently been solved for M. musculus
Nup53 (Handa et al., 2006
). These authors proposed that the Nup53 RRM, rather than acting as a nucleic acid binding domain, might be a homodimerization domain or contribute to protein–protein interactions. However, we could not detect any interactions between the RRM (in a Nup53144-265
truncation) and either endogenous Nup53 or other nups, including Nup155, by using our pulldown assay (data not shown). Furthermore, the Nup53144-265
fragment did not rescue the NE and NPC assembly activity of the Nup53 depleted Xenopus
extracts (data not shown). There was also no detectable effect of adding the RRM fragment in excess to nuclear assembly reactions (data not shown).
This work is the first extensive in vitro and in vivo analysis of nucleoporin truncation fragments. It clearly highlights the importance of defining specific protein interaction domains within a known nucleoporin subcomplex, and it has allowed the analysis of key interactions in the process of NE and NPC assembly (). Moreover, we provide further evidence for the proposed checkpoint that links NE and NPC assembly in Xenopus
egg extracts (Antonin et al., 2005
) by demonstrating that the soluble nucleoporin Nup53 must be present and able to interact with Nup155 for NE membrane and NPC assembly to occur. Our data suggest that Nup53, although recruited to chromatin later than the Nup107-160 complex or POM121 (data not shown) plays a key role in the events that bring together the chromatin-associated Nup107-160 complex, vesicle bound transmembrane nucleoporins and soluble Nup155.