In this study, we have examined the function of the Tpr protein through a molecular dissection of its various domains. In particular, we have identified distinct regions of the molecule that can confer localization either to the nuclear face of NPCs or to the nuclear interior. Since Tpr was first identified in NH2-terminal oncogenic fusions with certain protein kinases, we were especially interested in the role that the Tpr NH2-terminal domain might play in intracellular targeting. Finally, we have identified regions of the Tpr protein that when expressed in mammalian cells cause an accumulation of nuclear poly(A)+ RNA. Through this analysis, we have been able to implicate Tpr in the process of mRNA export from the nucleus.
Previous studies have shown that Tpr is often fused with the protein kinase domains of several protooncogenes (met, raf, and trk) in human tumors (Park et al., 1986
; Soman et al., 1991
; Greco et al., 1992
), and that the Tpr-Met chimera dimerizes in vivo inducing autophosphorylation and constitutive activation of the protein kinase domain (Rodrigues and Park, 1993
). Although it was possible that Tpr also directed localization to the NPC, thereby tethering an active protein kinase at the nuclear periphery, we found that the NH2
-terminal region of Tpr implicated in oncogenesis does not in fact contain such a targeting function. While this NH2
-terminal domain does occasionally form striking filamentous arrays extending throughout the cytoplasm, this only occurs at extremely high expression levels and it is doubtful that this phenomenon contributes to the transforming activity of Tpr-Met chimeras. Based on these observations, it appears that that the role of Tpr in the activation of protooncogenes is unrelated to NPC localization but instead is most likely the promotion of dimerization or higher-order oligomerization (Rodrigues and Park, 1993
A minimal sequence lying between residues 435 and 649 of the Tpr protein can confer localization to the nucleoplasmic region of NPCs in transient transfection assays. This domain appears to be essential for NPC association of the Tpr molecule since both NH2- and COOH-terminal deletions that extend into the sequence abolish NPC localization. Domains containing NH2-terminal truncations do, however, accumulate within the nucleoplasm. At present, we cannot rule out the possibility that, in the context of the full-length molecule, other regions of Tpr may be involved in interactions with the NPC. Furthermore, the minimal 225-residue NPC targeting or association region that we have identified could actually represent a domain within the molecule that functions in self association. In this way, NPC targeting by this minimal sequence could be by virtue of interaction with endogenous Tpr. This is not an issue that can be immediately resolved in normal cells that constitutively express the full-length protein. However, if this segment of Tpr does have a direct function in NPC-association it must logically interact with another component of the NPC or the nuclear envelope. Identification of such protein(s), by two hybrid analysis or more conventional in vitro binding assays for instance, would lend support for a direct role in NPC targeting. Such findings would also provide further insight into the role of Tpr in the maintenance of the normal architectures of both the NPC and nucleus.
A COOH-terminal domain of Tpr (amino acids 1,626– 1,989) localizes to the nucleus and also imparts nuclear localization to GFP suggesting that this domain contains a nuclear localization signal. A sequence closely matching the consensus NLS for import via the importin α/β pathway (Dingwall, 1991
) is contained within this domain, but as we have not shown that this sequence is responsible for nuclear localization, it is also possible that the nuclear localization domain contains a nuclear import signal unrelated to the classical basic domain NLS, (as recently described by Pollard et al., 1996
; Pemberton et al., 1997
; Rosenblum et al., 1997
; Rout et al., 1997
). A signal-mediated transport mechanism could explain how a protein as large as Tpr becomes associated with the nucleoplasmic face of the NPC, since it is far too large to diffuse through the NPC on its own. Alternatively, Tpr may “piggy-back” its way into the nucleus via binding to another protein that is itself transported into the nucleus. This possibility has been suggested for the nucleoporins Nup153 and Nup98, which also associate with the nucleoplasmic face of the NPC but at the same time do not appear to use a recognizable NLS (Bastos et al., 1996
). This scheme, however, seems unlikely for Tpr since it is almost entirely localized to the nucleus even at expression levels that might be expected to saturate endogenous levels of a non-shuttling carrier protein, another NPC protein for example.
While it is clear that the COOH-terminal portion of Tpr contains a nuclear localization domain, it is also possible that an additional nuclear localization signal exists within the NH2-terminal coiled-coil domain. The HA-Tpr1-734 domain localizes to the NPC (Fig. ) as does a larger construct HA-Tpr1-1387 (not shown), although both of these constructs are too large to diffuse through the NPC. As suggested above, it is possible that these molecules enter the nucleus by association with another nuclear protein, but a second nuclear localization sequence between the amino acids 304 and 734 cannot be ruled out.
Our data indicate that Tpr has a potential role in the export of mRNA. Moderate overexpression of Tpr domains that localize to the NPC or to the nuclear interior cause the dramatic accumulation of poly(A)+
RNA within the nucleus. In contrast, NH2
-terminal domains that do not associate with the NPC have no such effect, even at extremely high expression levels. A recent publication by Shah et al. (Shah et al., 1998
) has provided compelling evidence that Tpr also interacts with components of the nuclear import machinery, in particular importin α/β heterodimers. Whether this reflects a role in import or a function in the recycling of the NLS receptor subunits to the cytoplasm has yet to be satisfactorily resolved. However, the observation that interaction of importin α/β with Tpr occurs only in the absence of binding of an NLS-bearing transport substrate would perhaps be more consistent with the latter interpretation. We have not been able to detect any inhibition of nuclear protein import at Tpr expression levels that have a clear effect on poly(A)+
RNA distribution. Nevertheless, at very high Tpr expression levels we observe a partial redistribution of importin α. However, the abundance of this and other soluble transport factors is sufficiently great that the overall impact of excess Tpr on their availability may be only marginally detectable in terms of distribution of nuclear import substrates. Thus, the results of Tpr overexpression are not inconsistent with the results of Shah et al. (Shah et al., 1998
). In any case, the fact that the nuclear import of the glucocorticoid receptor-β-galactosidase fusion protein (which probably exists as a ~750 kD tetramer) is largely unaffected by Tpr overexpression, suggests that the effect on RNA distribution is not due simply to a loss or occlusion of NPCs.
The mechanism by which Tpr over expression inhibits poly(A)+ RNA export is still a matter of speculation, particularly since two separate domains that localize to either the NPC or the nuclear interior both disturb the normal distribution of poly(A)+ RNA. Although it is possible that the NPC-binding region domains might displace the full-length Tpr from NPCs, there are likely additional reasons since immunofluorescence observations on cells overexpressing such domains reveal no major alterations in the localization of the endogenous molecule (Bangs, P., unpublished results). As discussed above, it is possible that this NPC targeting region could actually function at least in part by virtue of association with the endogenous protein as its coiled coil structure predicts. Such an interaction would most likely be formed in the cytoplasm soon after synthesis. The hybrid full-length/mutant Tpr dimer would then be imported into the nucleus (full-length provides the NLS) followed by localization to the NPC. The prediction of course is that this heterodimer would be nonfunctional (or would have reduced function) in terms of mediating RNA export. This model would explain why we do not see any major redistribution of endogenous Tpr from the NPC, since a 50% reduction (the maximum that could be achieved in this dimerization scheme) might not be immediately apparent by immunofluorescence microscopy. An alternative to the self association that would explain the poly(A)+ RNA effect is that a large excess of the NPC association region of Tpr might potentially block assembly of one or more additional nucleoporins that are essential for normal RNA trafficking. The merits of either model will be better evaluated once we have more information on the identities of proteins with which the Tpr NPC targeting region (435–649) interacts.
A somewhat different scenario is likely to account for the block in poly(A)+
RNA export caused by overexpression of the Tpr COOH-terminal domain. Since this truncated protein exhibits no preferential association with the NPC and instead localizes uniformly throughout the nucleus, the resulting accumulation of poly(A)+
RNA is probably not due to a disruption of activity at the site of the NPC. It is more likely the result of titration of factors necessary for efficient RNA export. These could include any of the recently identified carriers that mediate export from the nucleus (Fornerod et al., 1997
; Kutay et al., 1997
; Stade et al., 1997
), as well as RNA-binding proteins necessary for the assembly of transportable RNA-protein complexes, a possibility suggested previously (Cordes et al., 1997
Based on the identification of distinct Tpr domains that localize to the NPC and the nuclear interior and the ability of these domains to inhibit mRNA export but not nuclear import, we conclude that Tpr is involved in the export of macromolecules from the nucleus. Our data are consistent with a previously suggested model in which Tpr, by virtue of its extensive coiled-coil structure, is an integral component of the intranuclear filaments that extend from the NPC and that the acidic COOH terminus of this protein interacts with soluble transport factors to facilitate the passage of RNA-protein complexes to the transport machinery of the NPC (Cordes et al., 1997
). Discovery of the NPC association domain of Tpr will facilitate the identification of proteins that localize Tpr to the NPC and subsequently provide a better understanding of the role of Tpr in nuclear export.