hsHec1p plays an essential role in mammalian cell mitosis. Cells injected with antibodies specific for hsHec1p exhibit several features of abnormal mitotic phenotypes, including the formation of multiple spindle poles and disordered metaphase chromosome alignment. These cells complete all aspects of division, including anaphase, cytokinesis, and re-formation of the nuclear envelope, but nonetheless missegregate chromosomes to daughter cells (8
). To elucidate the molecular bases of hsHec1p in M-phase progression, an S. cerevisiae
homolog of the hsHEC1
gene was identified and characterized. Consistent with previous observations of Wigge et al. (61
), we found that scHEC1/NDC80
encodes an 80-kDa cellular protein that is 36% identical to the human hsHec1p protein in its N-terminal one-third. Although the overall homology between the two proteins is not high, both proteins contain a long stretch of leucine heptad repeats that constitute two coiled-coil enriched domains in their otherwise divergent C-terminal regions. Genes with similar structure were also identified in fission yeast, C. elegans
, and mouse genomes (data not shown). Moreover, the hsHEC1
gene can complement the function of its yeast counterpart in cells deleted for scHEC1
. The result suggests that the essential functions of Hec1p proteins have been conserved throughout eukaryotic evolution.
The conservation of Hec1p function provided a useful tool to explore in a simple unicellular organism the molecular mechanisms by which hsHec1p functions. Our results suggest that hsHec1p function is essential for cell viability and M-phase progression. Its function is required during M phase, since conditional hshec1 mutations led to the mitotic delay, accumulation of large-budded cells, and increased lethality during M-phase arrest. Given the structural divergence that exists between hsHec1p and scHec1p, it was unexpected that the phenotypes of the mutants of these two proteins would be almost identical. This observation, however, strongly suggests that Hec1 proteins are functionally very conserved from yeast to human, and it is therefore feasible to study the in vivo properties of a human protein in an organism as simple as the budding yeast.
The chromosome missegregation resulting from mutated hshec1
may stem from a defect in the interaction between hsHec1p and Smc1p. The interaction between hsHec1p and the second coiled-coil domain of the human SMC1 protein was originally indicated by yeast two-hybrid screening. Later, the interaction of hsHec1p with yeast Smc1p was also found to be mediated by the corresponding region in this yeast homolog (Fig. ). The coiled-coil domains of SMC proteins have been implicated in oligomerization or interaction with other proteins (40
). The SMC1-type proteins, yeast Smc1p and Xenopus
XSMC1, have been implicated in sister chromatid cohesion (21
). The association of hsHec1p or scHec1p with Smc1p might therefore link one of the functions of Hec1p to the regulatory machinery controlling the process of chromatin assembly. At nonpermissive temperatures, the mutant hshec1-113p fails to bind to Smc1p, and this may cause a defect in sister chromatid separation, leading to chromosome missegregation. Consistently, a defect in sister chromatid cohesion has been proposed as the cause leading to chromosome missegregation. Our observation that overexpression of both yeast and human Hec1p can suppress the temperature sensitivity of the smc1
mutant suggests that increasing amounts of Hec1 proteins may augment their affinity for the mutant smc1p and help maintain the proper function of sister chromatid cohesion. Thus, the interaction between Hec1p and SMC1 protein is essential for the physiological function of Hec1p in chromosome segregation and cell viability.
Hec1p is also associated with Smc2p in yeast. Several lines of evidence have revealed the connection between sister chromatid cohesion and chromosome condensation (21
). A subunit (Scc1p/Mcd1p) of the Smc1-containing cohesion complex is proposed to function as a linker molecule that connects these two different chromatin-structuring activities on yeast mitotic chromosomes (22
). S. cerevisiae
Trf4p, a protein required for rDNA condensation, interacts with both Smc1p and Smc2p (5
), and a trf4
mutant also exhibits a cohesion defect (24
). Like these proteins, Hec1p may modulate both chromatin-structuring activities through association with Smc1p or Smc2p.
The biochemical basis of how Hec1p binds to Smc1p or Smc2p and participates in the activities of SMC proteins remains to be elucidated. It has been suggested by the in vitro activity of Xenopus
condensins that SMC proteins may act as motors facilitating formation of chromosome loops in an energy-dependent fashion (22
). The ATP-binding ability and ATPase activities of SMC protein complexes are proposed to provide them with motor energy and modulate their functions. Interestingly, other hsHec1p-associated proteins such as MSS1 and p45 of the 26S proteasome were also suggested to have ATPase activities (4
), and hsHec1p is able to down-regulate the in vitro ATPase activity of MSS1, the human Cim5p homolog (9
). Whether Hec1p can modulate ATPase activities of SMC proteins remains to be tested.
Besides a potential role in the modulation of sister chromatid cohesion or chromosome condensation, a kinetochore function can not be excluded for Hec1p in chromosome segregation since hsHec1p has been shown to localize to centromere regions in mammalian cells during M phase (8
). The phenotypes of the mutant forms of both human Hec1p (Fig. ) and the yeast Hec1p homolog (61
) are similar to those of ndc10-1, a mutant form of a centromere-binding protein (18
). The staining pattern observed for Hec1p/Ndc80p in this study and that previously reported by Wigge et al. (61
) is reminiscent of the staining of the spindle pole body and is consistent with centromere localization. Moreover, the staining of Ndc80p on microtubules adjacent to the spindle pole body and along the short spindle, an important characteristic observed with centromere proteins including Ndc10p (18
) and Cse4p (44
), was also demonstrated in a previous report (50
). A recent study suggested that Hec1p/Ndc80p interacts genetically with Ctf19p, which is a centromere protein (32
). The yeast Hec1p/Ndc80p, however, was initially purified from the spindle pole (61
), suggesting that Hec1p/Ndc80p may also localize to the spindle pole region. In addition, Hec1p may be distributed among other chromatin regions since cohesion and condensation occur at multiple places along the entire chromosome other than the centromere.
The interaction between Hec1p and SMC proteins may reflect a novel function for the SMC proteins. It is evident that SMC proteins are involved not only in sister chromatid cohesion or chromosome condensation but also in other activities such as DNA replication, recombination, and repair (24
). The chromatin assembly activities involved in the cohesion and condensation processes that occur at the centromere have the potential to function in the structural remodeling of centromeric chromatin during mitosis. In higher eukaryotes, the assembly of highly ordered centromeric chromatin structure is suggested to be essential for kinetochore functions. In S. cerevisiae
, specialized chromatin structures of the centromeres also appear to be important for kinetochore assembly (60
). Therefore, it is, possible that the interaction between Hec1p and SMC proteins is involved in the process of centromeric chromatin assembly and modulation of kinetochore function.
Hec1p may play multiple roles in M-phase progression since it has been shown to associate with other proteins such as the human homologs of Sug1p/Cim3p, Cim5p, and NIMA. It has been shown that Sug1p/Cim3p, Cim5p, and NIMA are important M-phase players because mutants of these proteins lead to G2
/M arrest (16
). Interestingly, the interactions between Hec1 proteins and their associated proteins appear to have been conserved (Table ). These interactions are likely to be common modes for regulation of M-phase progression in all eukaryotes.
Although hsHec1p may serve as a regulator of multiple mitotic pathways, it is itself regulated by higher-level modulators. The retinoblastoma protein Rb, as an hsHec1p-associated protein, is likely to be one of these modulators. hsHec1p is not the first protein linking Rb to M-phase progression. The association of Rb with mitosin/CENP-F (62
), H-nuc/CDC27 (7
), and protein phosphatase 1α (13
) has provided circumstantial evidence that Rb has an important role in M-phase progression. The higher-level regulatory function that we propose for Rb is less conserved in lower eukaryotes. First, no gene with sequence similarity to Rb exists in the entire S. cerevisiae
genome. Second, the specific I-C-E motifs found in hsHec1p sequences could well serve as Rb-binding domains (6
). The protein sequence of either the budding or fission yeast homolog, however, contains no I-C-E motif, consistent with the lack of interaction between budding yeast Hec1p and human Rb in the yeast two-hybrid system (Table ). The lack of Rb in yeast will allow us to address Rb function by using yeast machinery as a powerful assay tool, without interference from endogenous Rb. The strain in which the scHEC1
gene has been replaced by the hsHEC1
gene will be available for future studies of the in vivo interaction between Rb and hsHec1p and the biological consequences of such an interaction.