In fission yeast and metazoa, the conserved MCM proteins are found in the nucleus throughout the cell cycle. We have examined the localization of MCM proteins in fission yeast and found that there is a role for the nuclear envelope in regulating these proteins even though their localization is not cell cycle dependent: it serves to maintain intact hexameric MCM complexes inside the nucleus. We propose that the nuclear targeting of Mcm2p and associated proteins requires assembly of the MCM proteins to activate the Mcm2p NLS. In contrast, when Mcm2p is not associated with other MCM proteins in the nucleus, the NLS is inaccessible and the protein is exported. Thus, assembly of an intact hexameric MCM complex is linked to the nuclear localization of individual MCM subunits.
An attractive model is that the association of at least the core MCM proteins (MCM4/6/7) and MCM2 in the cytoplasm is required for their targeting of the subcomplex to the nucleus (Figure ). Previously, we and others have shown that the MCM heterohexamer contains subcomplexes: a core of MCM4/6/7 bound by MCM2 and a peripheral dimer of MCM3/5 (Ishimi et al., 1996
; Kimura et al., 1996
; Adachi et al., 1997
; Kubota et al., 1997
; Thommes et al., 1997
; Sherman and Forsburg, 1998
; Sherman et al., 1998
). That coassembly of subcomplexes of MCM proteins occurs in the cytoplasm is demonstrated by the ability of the NLS1 mutant, Mcm2p-M9, to trap wild-type MCM proteins in the cytoplasm. In fission yeast, as in budding yeast, only Mcm2p and Mcm3p homologues have consensus nuclear localization sequences. Thus, Mcm3p may similarly target Mcm5p to the nucleus. In this model, retention of all six MCM proteins in the nucleus would require binding of the peripheral dimer MCM3/5 to the subcomplex MCM2/4/6/7 to inactivate NESs. The disruption of Mcm3p localization in the cdc19
mutant and the disruption of MCM core complex localization in the mcm3
mutant may indicate that assembly of the full hexameric complex is required for nuclear retention. Alternatively, the intact complex might assemble in the cytoplasm and require both sets of NLSs to target the hexamer to the nucleus.
Figure 7 MCM complex assembly and nuclear localization. A speculative model coupling MCM complex assembly and nuclear localization. The six MCM proteins depend on NLS sequences supplied by MCM2 and MCM3 for nuclear import. The NLS sequences on MCM2 and MCM3 are (more ...)
This model linking hexameric MCM complex assembly and nuclear localization in fission yeast is based on three major observations. First, MCM protein localization is interdependent: in mcm
temperature-sensitive strains, the wild-type MCM proteins are lost from the nucleus at the restrictive temperature. MCM protein associations are also disrupted under these conditions (Sherman et al., 1998
). In either the mcm3-HA
cold-sensitive or the cdc19
temperature-sensitive mutant, this redistribution of MCM proteins requires an active nuclear export system. It remains to be determined if each MCM protein contains an NES, whether MCM subcomplexes or individual subunits are targets for export, or if export depends on interaction with an NES-containing protein. There are potential NESs in Mcm2p at residues 627–638 and 771–780, based on comparison with consensus NESs (Kim et al., 1996
; Nakielny and Dreyfuss, 1997
; Nigg, 1997
; Mattaj and Englmeier, 1998
). Notably, Mcm2p-D10 retains the putative NES at residues 771–780 (Table ) and can be trapped in the nucleus in the nuclear export–defective strain. Masking of the NES has been proposed as a mechanism for regulating p53 subcellular localization (Stommel et al., 1999
). Tetramerization of p53 monomers blocks the NES elements, resulting in nuclear retention of the p53 complex.
Second, using a panel of mutations in Mcm2p, we show that mutants that disrupt complementation and MCM protein interactions (Forsburg et al., 1997
; Sherman et al., 1998
) also abolish nuclear localization, even in the presence of an intact NLS. Trivially, this could suggest that all mutant proteins fail to fold properly, even though they are all produced and stable (Forsburg et al., 1997
; Sherman et al., 1998
). However, several observations argue against this. (1) The NLS2 mutant Mcm2p-M10 also contains an intact NLS1, yet it fails to accumulate in the nucleus. In this case, however, the protein is still able to bind other MCM proteins (Sherman et al., 1998
) and can be rescued by adding a heterologous NLS, which suggests that its structure is still intact. (2) The protein tolerates deletions in the amino terminus (D1, D2, D3) that do not affect binding, localization, or complementation. (3) A mutation in the putative nucleotide-binding site, M7 (K540R), also produces a functional protein; an alanine mutation of the same lysine residue disrupts activity. Both mutant proteins exhibit reduced MCM protein binding and nuclear localization. These observations demonstrate that the protein can accommodate both point and deletion mutations. (4) Among the nearly 20 other mutant proteins with point and deletion mutations throughout the protein, all are produced and stable; none shows nuclear localization, despite the presence of NLS1 and NLS2. Although one can never prove that these proteins are folded normally, indirect evidence suggests that at least some of them are intact. We suggest that they are specifically deficient in targeting. An intriguing reason is that the NLS remains masked because the mutant proteins fail to interact with other MCM proteins. We also observed that overproduced Mcm2p does not accumulate in the nucleus in wild-type or crm1
mutant cells. This result suggests that some factor is limiting for either its import or its retention. This is unlikely to be attributable to an effect on general import machinery because overexpression of wild-type Mcm2p has no deleterious phenotype. Because we never observed nuclear localization without MCM binding, we deduce that binding is required for localization.
Our third major observation is based on analysis of Mcm2p NLS sequences. Mcm2p contains at least one NLS (NLS1) that is sufficient to target a reporter protein to the nucleus, but that sequence is insufficient to target binding-defective Mcm2p mutants to the nucleus. Nor can NLS1 target an NLS2 mutant Mcm2p to the nucleus, even though its ability to assemble with other MCM proteins is normal. Mutations in either NLS have no effect on complex assembly (Sherman et al., 1998
) but abolish localization; this localization can be rescued by adding a heterologous NLS to the mutant protein. In addition, NLS mutants can be used to sequester wild-type MCM proteins in the cytoplasm. The NLS mutants allow us to uncouple complex assembly from localization and suggest that complex assembly precedes localization. Thus, we suggest that complex assembly is necessary but not sufficient for localization of Mcm2p.
We infer that the NLS sequences are not active or exposed unless Mcm2p is assembled with other MCM proteins. The Mcm2p-D10 mutant, which contains a large deletion but retains NLS1 and NLS2, shows weak nuclear localization in the crm1
mutant, suggesting that NLS masking is defective for this mutant protein. One possible explanation for this observation is that the large deletion in Mcm2p-D10 not only prevents association of the mutant protein with other MCM proteins but also prevents complete inactivation of NLS1 and NLS2. In the wild-type strain, Mcm2p-D10 with partially active NLS function is inefficiently imported, but the functional nuclear export mechanism removes the mutant protein from the nucleus to the cytoplasm. In the crm1
strain, Mcm2p-D10 is captured in the nucleus because of the failure of the nuclear export system, but its nuclear localization is substantially reduced compared with that of wild-type Mcm2p. There is precedent for intramolecular masking of NLS function. Recently, Humbert-Lan and Pieler (1999)
reported that a carboxy-terminal transport regulatory domain may mask two NLS sequences in the Xenopus
B-Myb transcription factor, thus restricting B-Myb to the cytoplasm. They propose that the domain acts via either intramolecular or intermolecular interactions to regulate NLS function.
Because the Mcm2/Cdc19 NLS mutants can sequester the other MCM proteins in the cytoplasm, and because localization of MCM subunits is interdependent, we propose that Mcm2p may be required for import of those MCM proteins that lack identifiable NLS elements. There have been several recent reports that complex assembly allows nuclear targeting of a protein that lacks a functional NLS. Piggyback mechanisms have been proposed for the Fanconi anemia protein complex (Naf et al., 1998
), mushroom homeodomain transcription factor complex (Spit et al., 1998
), cytomegalovirus capsid assembly (Plafker and Gibson, 1998
), mouse DNA primase (Mizuno et al., 1996
), mammalian DNA repair enzymes (Boulikas, 1997
), STAT proteins (Johnson et al., 1998a
), and IκB (Turpin et al., 1999
). Furthermore, two recent reports (Abu-Shaar et al., 1999
; Berthelsen et al., 1999
) have demonstrated that the subcellular localization of the Drosophila
homeodomain protein Extradenticle depends on regulating the accessibility of its NLS and NES elements. Heterodimerization of Extradenticle with Homothorax blocks NES accessibility and leads to nuclear accumulation of the Extradenticle/Homothorax complex. In each case, the cell assembles a protein complex in the cytoplasm and then imports the complex into the nucleus. The latter case provides an example of the role of nuclear export in maintaining appropriate stoichiometry of complex subunits. In this way, only active, intact complexes are present in the nucleus. Our data suggest that fission yeast MCM complexes may also have this spatial control of their assembly.
Such a mechanism may be conserved in other eukaryotes. Kimura and colleagues (1996)
showed that overexpression of murine MCM6 and MCM5 led to their accumulation in the cytoplasm unless MCM2 and MCM3, respectively, were coexpressed. In characterizing the NLS of budding yeast Mcm3p, Young and colleagues (1997)
speculated that it may provide nuclear access for other MCM proteins. Furthermore, Maiorano and colleagues (1996)
suggested that the cytoplasmic accumulation of overproduced fission yeast Mcm4p may indicate that Mcm4p depends on its association with an NLS-containing limiting factor for its nuclear import. Our interpretation is consistent with these data. It provides a mechanism for the cell to assemble intact hexameric MCM complexes in the nucleus and maintain the correct stoichiometry of the individual subunits. By requiring the subcomplexes to assemble in the cytoplasm before nuclear entry and actively exporting free MCM subunits or subcomplexes from the nucleus, the cell avoids having unassociated MCM proteins bind other factors or interfere with the function of the intact MCM complex. Interestingly, this active nuclear export may explain the shuttling behavior of MCM proteins reported in S. cerevisiae
(Hennessy et al., 1990
; Yan et al., 1993
; Dalton and Whitbread, 1995
; Young et al., 1997
; Young and Tye, 1997
); uniquely in this organism, MCM proteins leave the nucleus during S phase. This may indicate that the MCM complex is normally disrupted during the cell cycle in this species.
Nuclear localization of MCM proteins thus appears to reflect a balance of nuclear import and export. Access to the requisite targeting sequences may be mediated by the interaction of the complex components or associated factors. Even though this localization is not cell cycle dependent in normal growth, it clearly imposes spatial regulation on the assembly and activation of these conserved proteins. We expect that such regulation will be a common feature of the assembly of complex protein structures in the nucleus.