A new role for vertebrate centrin at the nuclear pore has been identified in this study. Despite the observation in 2000 that centrin was a structural component of the yeast nuclear pore (103
) and the fact that its functional role was revealed later (31
), the intervening years have never shown a hint that centrin has any connection to the vertebrate nuclear pore, either structurally or functionally. We have now observed the interaction between vertebrate centrin 2 and the critical Nup107-160 complex of the nuclear pore from Xenopus
to human cells (Fig. and ). Indeed, although centrin 2 was previously visualized to be a centrosomal protein by immunofluorescence, we have found that centrin 2 colocalizes extensively with nuclear pores in human and Xenopus
cultured cells, as well as with the pores of nuclei reconstituted in vitro in Xenopus
egg extracts (Fig. and ). Although the association of centrin 2 with NPCs is readily apparent in Xenopus
cells and in vitro-reconstituted nuclei, the substitution of digitonin for Triton X-100 is required to observe the NPC association in HeLa cells. Disruption of the nuclear pore targeting/assembly protein ELYS/MEL-28 by RNAi, which is known to lead to a loss of nuclear pores, causes a dramatic decrease in centrin 2 at the nuclear pore under conditions where the nuclear lamina remains unaltered (Fig. ). Instead, in ELYS RNAi-treated cells, centrin 2 relocates to FG nucleoporin-containing cytoplasmic aggregates (annulate lamellae), demonstrating that centrin 2 interacts with both nuclear and cytoplasmic pore complexes (Fig. ). We additionally identified a functional role for centrin 2 at the nuclear pore. Overexpression of either the N-terminal or C-terminal Ca2+
binding EF-hand domain of human centrin 2 leads to the nuclear accumulation of poly(A)+
RNA, consistent with a disruption of mRNA export (Fig. ). This mRNA export defect mirrors the nuclear poly(A)+
RNA accumulation caused by overexpression of Nup160 fragments, which are either direct or indirect binding sites for centrin 2 (Fig. ), and also mirrors the Cdc31 defect observed with yeast (31
). In addition, overexpression of either half of human centrin 2 has a dominant-negative effect on RGG protein export but not its import (Fig. ). Depletion of centrin 2 by RNAi causes similar disruptions of both mRNA and protein export (see Fig. S4 in the supplemental material). We conclude that centrin 2 interacts with a major subunit of the nuclear pore, exhibits a nuclear pore localization, and has a functional role in mRNA and protein export.
Centrins have long been associated structurally with the centrosome (105
). Traditional immunofluorescence with human cells, using methanol or formaldehyde/Triton X-100, has previously revealed centrin 2 to be localized at the centrosome (29
). RNAi experiments demonstrated that centrin 2 is required for centriole duplication (108
). Moreover, a physical interaction between centrin 2 and the centrosomal protein Sfi-1, observed for both yeast and human cells, plays a key role in the regulation of centrosome structure and centriole duplication (59
). It is, thus, clear that centrins are an essential component of the vertebrate centrosome and yeast spindle pole body. A single higher eukaryotic, noncentrosomal nuclear function for centrin 2 was previously observed for the XPC nucleotide excision repair complex, as described in the introduction (4
Our identification of vertebrate centrin 2 as a novel binding partner for the Nup107-160 complex in immunoprecipitations originally suggested a possible mitotic, centrosome-related role for the interaction. Indeed, the Nup107-160 complex moves to the kinetochores and spindle poles at mitosis (11
), and we previously demonstrated that it is required for correct bipolar spindle assembly: immunodepletion of the Nup107-160 complex from Xenopus
mitotic extracts results in severely defective spindle assembly in vitro (93
Our finding that centrin 2 is present at the nuclear pore in interphase and is involved in mRNA and protein export now shows that centrin 2 has another important novel interphase function in vertebrates. This conclusion brings an unexpected but remarkable symmetry to the studies of yeast and vertebrate centrins. The single centrin of S. cerevisiae
, Cdc31, is an essential component of the yeast centrosome equivalent, the spindle pole body, and an established member of the yeast nuclear pore complex (31
). Indeed, a functional role for yeast Cdc31 in mRNA export was identified; the expression of a mutant Cdc31 allele (Cdc31-151) or the absence of Cdc31 expression leads to nuclear accumulation of poly(A)+
). Here, we find that the overexpression of either half of the human centrin 2 has a dominant-negative effect on mRNA export, in both human and Xenopus
cultured cells (Fig. ). Moreover, centrin 2 affects Crm-1 mediated protein export but not at least one major type of protein import (Fig. ). Thus, our data indicate for the first time that vertebrate centrin 2 is at the nuclear pore and plays a role in mRNA and nuclear protein export during interphase.
In yeast, the function of Cdc31 in mRNA export appears to be mediated through the interaction with an mRNA export complex, the yeast Sac3-Thp1-Sus1 complex (31
). Specifically, Cdc31 interacts with yeast Sac3p, a 150-kDa protein (9
). Vertebrate centrin 2 might also possibly bind and/or function through an as yet undiscovered vertebrate Sac3-Thp1-Sus1 complex.
Three potential vertebrate homologues of Sac3 have been identified, GANP, MCM3AP, and Shd1 (1
). B-cell g
rotein (GANP) and MCM3 a
rotein (MCM3AP) are derived from the same gene by alternative splicing (1
). GANP is 210 kDa, 60 kDa of which has 23% homology with a region of yeast Sac3 (aa 129 to 803). This region of yeast Sac3 contains its centrin-interaction domain (1
). GANP also has several N-terminal degenerate FG motifs (32
), a motif found almost exclusively in nucleoporins. However, the large GANP protein and its smaller alternatively spliced C-terminal isoform, MCM3AP, contain other non-Sac3 domains, including a GCN5-related N-acetyltransferase domain and a DNA primase activity, both of which are required for their function in DNA replication (60
The third vertebrate relative of Sac3p, the S
omain protein 1
(SHD1) is one-third the size of yeast Sac3p and lacks the yeast-centrin interaction domain (58
). However, SHD1 localizes to centrosomes, and RNAi of SHD1 causes abnormalities in centrosome duplication and spindle formation (58
). Thus, while both Shd1 and GANP/MCM3AP contain similarities to yeast Sac3, they contain unrelated additional domains. We cannot rule in or out a role for these proteins at the nuclear pore in mRNA export. As yet they have no direct connection to vertebrate centrins.
Our work identifies interactions between centrin 2 and several major nucleoporins involved in mRNA export. In addition to the Nup107-160 complex, we observed centrin 2 interacting with the FG nucleoporin Nup153 (Fig. ). Interestingly, yeast Sac3 is known to bind to the yeast FG nucleoporin Nup1, a distant homologue of Nup153 (31
). Our observed interaction of vertebrate centrin 2 with Nup153 may be direct or indirect and involve a vertebrate Sac3 equivalent, such as Shd1. Alternatively, the interaction between vertebrate centrin 2 and Nup153 may use the Nup107-Nup160 complex as an intermediary, since we have shown this complex binds both centrin 2 and Nup153 (Fig. ) (128
Structurally, the major domains of centrins are comprised of Ca2+
binding EF-hand motifs. It has been observed that mutations that specifically affect the calcium binding of yeast Cdc31 cause nuclear accumulation of poly(A)+
). Other actions of centrins are also altered by calcium. The affinity of Cdc31 for its spindle pole body partner, Kar1, increases 10-fold with calcium and affects its role in spindle pole body duplication (37
). The affinity of human centrin 2 for XPC similarly increases 28-fold in the presence of calcium, although how this affects XPC activity has not yet been determined (92
Calcium has been implicated separately as a possible regulator of the nuclear pore in a handful of studies (see references 26
and references therein). For example, atomic force microscopy observations showed structural changes in the pore with calcium addition, described as a calcium-induced, iris-like opening of the nuclear basket ring, the first pore structure encountered by proteins and mRNAs before export (116
). Specific changes in nucleoporin localization include the FG domain of Nup153, which changes its position within the pore with the addition of 2 mM Ca2+
), a Ca2+
change comparable to that of typical calcium flux from the endoplasmic reticulum (ER).
Calcium changes have been observed to influence not only nuclear pore structure but also pore function. Thapsigargin, which depletes the ER calcium stores, is seen to disrupt passive diffusion and nuclear transport through the vertebrate NPC in living cells (41
). Because the integral membrane pore protein, gp210, contains luminal calcium-binding motifs, and the expression of an Ab to this portion of gp210 within the ER lumen inhibits both passive diffusion and signal-mediated transport, one hypothesis has been that gp210 could be a calcium sensor that triggers such conformational NPC changes (26
). However, our identification of centrin 2 at the vertebrate nuclear pore introduces a potential more centrally located calcium sensor for the NPC.
In summary, we conclude that a population of vertebrate centrin 2 interacts with nuclear pore subcomplexes, localizes to the nuclear pore, and plays a role in both mRNA and protein export. Thus, we have identified a new and distinct interphase function for vertebrate centrin 2. Possible interaction of centrin 2 in an mRNA export complex, such as the yeast Sac3-Thp1-Sus1-Cdc31 complex, which is as yet undiscovered in vertebrates, may be of future interest. In addition, the ability of centrin 2 to bind calcium provides interesting potential roles for this protein as a regulatory or structural component of the nuclear pore.