©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The C Terminus of Mitosin Is Essential for Its Nuclear Localization, Centromere/Kinetochore Targeting, and Dimerization (*)

(Received for publication, May 26, 1995; and in revised form, June 16, 1995)

Xueliang Zhu (§) Kai-Hsuan Chang (§) Dacheng He (1) Michael A. Mancini (¶) William R. Brinkley (1) Wen-Hwa Lee (**)

From the Center for Molecular Medicine/Institute of Biotechnology, the University of Texas Health Science Center at San Antonio, San Antonio, Texas 78245 Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Mitosin is a novel 350-kDa nuclear phosphoprotein that dramatically relocates from the evenly nuclear distribution in S phase to the centromere/kinetochore and mitotic apparatus in M phase. The dynamic relocalization of mitosin is accompanied by the phosphorylation of itself, suggesting that mitosin plays a role in mitotic progression. The molecular basis of nuclear localization and targeting of mitosin to the centromere/kinetochore were characterized using a set of epitope-tagged deletion mutants. The data indicate that the extreme C terminus (amino acids 2,487-3,113) of mitosin has both an independent centromere/kinetochore targeting domain and an unusually spaced bipartite nuclear localization signal. Moreover, the same centromere/kinetochore targeting domain was shown to be essential for the ability of mitosin to bind to itself or other putative mitosin-associated proteins through use of the yeast two-hybrid system. These results suggest that the C terminus of the mitosin is essential for its role in influencing cell cycle progression.


INTRODUCTION

The kinetochore is a multifunctional trilaminar structure on the centromere surface believed to be responsible for, at a minimum, microtubule attachment and chromosome segregation during mitosis and meiosis. The composition of this specialized nucleic acid and protein complex remains poorly identified(1, 2, 3, 4) . Proper chromosome segregation during mitosis depends on the coordinated bipolar assembly of spindle fibers emanating from microtubule organizing centers toward components of the kinetochore. Kinetochore dysfunction is thought to be involved in the genesis of aneuploidy, a form of genomic instability that can lead to tumorigenesis(5) . A detailed understanding of kinetochore structure and function is essential to elucidate the mechanism underlying the fidelity of chromosome movement.

A number of centromere/kinetochore proteins have been identified using antibodies obtained from the sera of patients with autoimmune diseases or derived by using chromosomal scaffolds as immunogens. These proteins can be broadly classified into two groups. In one group, including centromere protein (CENP)-A, -B, and -C, (^1)the association with the centromere/kinetochore is independent of cell cycle (constitutive)(3) ; in contrast, proteins such as the inner centromere proteins(6) , the chromatid-linking proteins(7) , CENP-E(8, 9) , and CENP-F (10, 11) are temporally associated with the centromere/kinetochore in a cell cycle-dependent fashion (facultative)(3) . Interestingly, sequence comparison between these proteins reveals no homology(12) . Accurate targeting to the centromere/kinetochore region and the exact function(s) of these proteins are still being investigated. To date, the N-terminal 28-amino acid region of CENP-B, which binds centromeric DNA, is the only identified centromeric targeting signal(13) .

A 350-kDa, facultative kinetochore protein named mitosin has recently been discovered by its association with retinoblastoma protein. (^2)Mitosin is a nuclear phosphoprotein of 3,113 amino acid residues and does not exhibit significant homology to any known proteins in GenBank. The expression of mitosin is cell cycle-dependent.^2 Mitosin is undetectable during most of G(1) and appears in a uniform, fine speckled pattern throughout the nucleus just prior to S but is absent in nucleoli. During progression into M phase, the nuclear localization of mitosin is dramatically reorganized into paired dots at the kinetochore and the spindle apparatus and to the midbody in telophase just prior to its complete disappearance at the end of telophase. The dynamic spatial reorganization of mitosin coincides with its temporal phosphorylation pattern; mitosin is underphosphorylated in interphase and maximally phosphorylated during mitosis. Identification of the role played by mitosin during cell division was explored through examination of potential dominant-negative effects on cell cycle progression by transiently over-expressing a series of deletion mutants.^2 Overexpression of N-terminal deletion mutants, which retain their ability to locate within the nucleus, result in accumulation of cells mainly in G(2)/M phase.^2

In order to understand the mechanism by which mitosin influences mitotic progression, we have extended our characterization of its biochemical and biological properties. In this paper, we have further defined that mitosin specifically localizes heterogeneously on the coronal surface of the outer kinetochore plate by electron microscopy. Through analyses of a series of deletion forms of mitosin, essential motifs required for nuclear localization and centromere/kinetochore targeting have been mapped to its C terminus. This region, containing three leucine heptads, may be a required structural motif for homodimerization and heterodimerization with a candidate mitosin-associated protein.


MATERIALS AND METHODS

Plasmid Construction

For studies in mammalian cells, DNA fragments of mitosin were cloned into pCEP4FLAG, a mammalian expression plasmid derived from pCEP4 (Invitrogen) to express FLAG-tagged fusion proteins.^2 All subsequent constructs were confirmed by restriction mapping and sequencing analysis to ensure the correct reading frames and modification.

To identify the nuclear targeting signal in mitosin, a series of constructs were engineered (see Fig. 1). The construction of pCF-10 (construct A; aa 632-3,113) and pCF-10Xh (construct E; aa 1,582-3,113) was described previously.^2 Construct B (containing aa 632-2,513) was made by removing the AccI-HindIII fragment (5.7 kb) from construct A. Construct C (pCF-10RV; aa 632-1,767) was constructed by removing the EcoRV-HindIII fragment (4.3 kb) from pCF-10 (construct A). For the construction of construct EDeltaL, (aa 2,927-3,113; containing basic regions I and II; see Fig. 1), one of the two lysine residues at the end of the FLAG tag (which may itself resemble the bipartite nuclear targeting motif KKLARASGKRQR) (15) was deleted by treatment with mung bean nuclease after HindIII cleavage, and the subsequent vector was ligated with a 10-base BamHI linker (New England BioLabs) to maintain the correct reading frame. To construct EDeltaC1 (aa 1,582-2,947; containing basic regions I and II), the BspMI site at nucleotide 8,916 of mitosin cDNA in construct E was destroyed by filling in with Klenow enzyme to create a new stop codon after five additional residues, ARELF. Construct EDeltaC2 (aa 1,582-3,037; basic regions I, II, and III) was made by self-ligation after removal of the PflMI-HindIII fragment (0.48 kb) from construct E. Construct EDeltaRvB was made by deleting the EcoRV-BspMI fragment (3.5 kb; 1,767-2,945; with only basic region III) from construct E, followed by Klenow fill in and religation.


Figure 1: A diagram showing the constructs used to determine mitosin NLS. The two putative bipartite nuclear targeting signals are underlined; the basic amino acid regions are identified as I, II, and III. The subcellular localization of the constructs is also summarized.



To define the centromere/kinetochore targeting region of mitosin, the series of deletion mutants was expanded. Construct EDeltaN (aa 2,094-3,113) was made by deleting the XhoI-SfiI fragment (1.5 kb) from construct E and then treating the remaining fragment with mung bean nuclease before religation. Construct EDeltaR (aa 1,582-3,113, and deleted from aa 2,178 to 2,359) was made by self-ligation of the vector after removing the EcoRI fragment (0.5 kb) from construct E. EDeltaG (aa 2,488-3,113) was created by cloning the StuI-BglII fragment (2.0 kb) into pCEP4F cleaved by NheI (fill in with Klenow) and BamHI. Construct EDeltaZ (aa 1,582-3,113, and deleted from aa 2,489-2,901) was constructed by deleting the EcoNI fragment (1.2 kb) from construct E and filling in the resulting vector with Klenow before religation. EDeltaNB was made by self-ligation of the vector after removing the SstI fragment (aa 2,666-2,756).

Immunofluorescence and Immunoblotting

Mouse kidney CV1 cells were transfected with 20 µg of the expression plasmid by the calcium phosphate method. Two days after transfection, cells were fixed with cold methanol and then immunostained by using anti-FLAG M2 antibody (Eastman Kodak Co.) and anti-mitosin antibodies (alpha10C), and then they were analyzed as described previously.^2 Nuclear DNA was stained with DAPI (Sigma). Cells of interest were photographed using Ektachrome P1600 film. Procedures for immunoprecipitation and immunoblotting were described previously.^2

Immunogold Electron Microscopy

Mitotic HeLa cells were shaken from the flask and cytocentrifuged onto plastic coverslips. After permeabilization with 0.2% Triton X-100 for 1 min, cells were then fixed with 3.7% paraformaldehyde plus 0.05% glutaraldehyde in phosphate-buffered saline for 30 min. The coverslips were treated with NaBH(4) (1 mg/ml) for 15 min and then incubated with 1% bovine serum albumin, 0.1% gelatin, and 5% normal goat serum in phosphate-buffered saline for 30 min to reduce the nonspecific background. Samples were incubated with antibody 14C10 (1:40) for 45 min at 30 °C, followed by a thorough wash, and then incubated with 1.4 nm of gold-conjugated goat-anti-mouse antibody (Nanoprobes, Inc.) (1:25) at 30 °C for 1 h. The samples were postfixed with 3% glutaraldehyde after washing and were then silver-enhanced for 3 min (Nanoprobes, Inc.). The samples were then embedded with Spurr (Polysciences Inc.) sectioned, and stained according to the standard electron microscopy procedure and observed with a Philips 410 electron microscope.

Yeast Two-hybrid System

The yeast two-hybrid system was used to search for proteins that potentially associate with mitosin (16) . Two sets of hybrid plasmids were constructed; one plasmid named pAS1-E/StuI-BglII contains the DNA sequence coding for the DNA-binding domain of the yeast transcriptional factor GAL4 (amino acid residues 1-147), fused in-frame with the sequence for the C-terminal region of mitosin containing centromere/kinetochore-targeting region, aa 2,487-3,113. A second plasmid, pSE1107L, contains the DNA sequence for GAL4 activation domain II (amino acid residues 768-881) fused to a cDNA library prepared from Epstein-Barr virus-transformed human peripheral lymphocytes. Both plasmids (pAS1-E/StuI-BglII and pSE1107L) were used to transform the yeast strain Y153, which has been constructed to provide a dual selection system (His His+ and the presence of beta-galactosidase activity) to efficiently screen cDNA expression library for clones interacting with a protein of interest. After selection, transformants were first screened for His+ prototrophy and then beta-galactosidase activity by using a filter lifting assay. Fifty His+ colonies turned out to be blue within 12 h. His+ blue colonies considered positive in the initial screen were rescreened for verification of binding. Colonies with the strongest qualitative binding were first analyzed through DNA sequencing. To test further whether the phenotype observed in the original screen was reproducible and dependent upon mitosin, library-derived plasmids were selectively recovered by virtue of the yeast LEU2 gene carried on those plasmids to complement a leuB6 mutation present in the E. coli strain JA226. Isolated plasmids were used to transform Y153 either alone or with the plasmid pAS1-E/StuI-BglII. The transformants were assayed for beta-galactosidase activity by using the filter lift method.

The yeast two-hybrid system was also used to define the regions of the C terminus of mitosin responsible for dimerization. Three deletion constructs were created: 1) pAS1-E/StuI-MscI (aa 2,487-2,925, which contains three leucine heptad repeats); 2) pAS1-EDeltaZ/StuI-BglII, in which these three leucine heptad repeats (aa 2,487-2,901) were deleted; and 3) pAS-1-EDeltaNB/StuI-BglII, where the space between aa 2,666 and 2,756 was deleted. Yeast strain Y153 was cotransformed with one of the above plasmids and pSE1107-E/BglII, which contains the C terminus of mitosin (aa 2,490-3,113). Transformants were assayed for beta-galactosidase activity, first qualitatively via the filter lift method and then quantitatively by chlorophenol red-beta-D-galactopyranoside (Boehringer Mannheim) measurement(16) .


RESULTS

The Mitosin C Terminus Contains the Nuclear Localization Signal

Mitosin is a cell cycle-dependent nuclear protein.^2 From the deduced primary structure, a series of basic amino acid clusters in the C terminus of mitosin similar to the bipartite nuclear targeting sequence in Xenopus proteins was found(15) . In order to analyze whether the nuclear targeting signal of mitosin is carried by these clusters, a series of epitope-tagged deletion mutants were constructed (summarized in Fig. 1), and their corresponding nuclear location in transiently transfected CV1 cells was monitored by immunofluorescence using an anti-FLAG antibody (Fig. 2). As with the positive control (a near full-length construct A) shown in Fig. 2, panels1-1`, the N-terminal truncated mitosin (construct E) also localized to the nucleus (Fig. 2, panels4-4`), whereas the two C-terminal truncated proteins (constructs B and C) remained in the cytoplasm (Fig. 2, panels2-2` and 3-3`, respectively). These results are consistent with the putative bipartite nuclear localization signal (NLS) (15) found at the C-terminal region of mitosin. To further delineate the mitosin NLS, several additional deletion mutants were examined. In the mutant EDeltaL, the first basic KR (basic region I) important for a bipartite motif was deleted. As shown in Fig. 2, panels5-5`, the nuclear localization remained unchanged. EDeltaC1, which contains basic regions I and II, was constructed to test if the putative bipartite motif KRLSSGQNKASFKRQRS (15) can function as a minimal NLS for mitosin. When transfected into CV1 cells, the expressed EDeltaC1 protein was predominantly cytoplasmic (Fig. 2, panels6-6`). To test if basic region III (KKSKK), which is 20 amino acid residues away from the bipartite motif, contributed to nuclear localization, EDeltaC2, containing basic regions I, II, and III was created, and the expressed protein was predominantly nuclear (Fig. 2, panels7-7`). Basic region III alone, however, was not sufficient for nuclear targeting since EDeltaRvB, in which basic regions I and II were removed, localized only in the cytoplasm (Fig. 2, panels8-8`). These data suggest that basic regions II and III work collectively as an NLS, not the commonly found bipartite sequence of regions I and II.


Figure 2: Determination of the mitosin nuclear localization sequence. Immunofluorescent staining of unsynchronized CV1 cells transiently transfected with FLAG epitope-tagged constructs is shown. Approximately 20 µg of each DNA construct, described schematically in Fig. 1, was used for transfection. After 2 days, cells were fixed using cold methanol and then immunolabeled using alpha10C (green; panels1-8) and anti-FLAG (red; panels1`-8`) antibodies. 1-1`, construct A; 2-2`, construct B; 3-3`, construct C; 4-4`, construct E; 5-5`, construct EDeltaL; 6-6`, construct EDeltaC1; 7-7`, construct EDeltaC2; 8-8`, construct EDeltaRvB. Bar, 20 µm.



Localization of Mitosin to Subdomains of the Kinetochore

To determine the fine distribution of mitosin at the centromere/kinetochore structure, a higher resolution image of this region was analyzed by mitosin-labeled centromeres using immunogold microscopy. Mitosin is found in a unique, heterogeneous distribution on the coronal surface of the outer kinetochore plate (Fig. 3). Although consideration for the preservation of mitosin antigenicity precludes optimal ultrastructure, the organization of gold label indicates that mitosin is not present at the kinetochore center, even though microtubules attach throughout the kinetochore. This result further demonstrates that mitosin is a kinetochore protein and is consistent with the previous observations derived from the immunostaining method.^2


Figure 3: Mitosin localizes to the coronal surface of the outer kinetochore plate. Mitotic HeLa cells (obtained from a shake off of unsynchronized cultures) were pre-extracted with 0.2% Triton X-100 prior to immunolabeling (see ``Materials and Methods''). Silver-enhanced gold label is seen primarily on the outer surface of the kinetochore in all kinetochores, independent of the plane of section. When sectioned directly through the middle, mitosin label appears to be found specifically at the outer edges of the coronal surface (arrowheads). IP, inner kinetochore plate; OP, outer kinetochore plate. Bar, 0.2 µm.



Centromere/Kinetochore Targeting of Mitosin Is Confined to the C Terminus

In order to identify the centromere/kinetochore targeting region of mitosin, a series of FLAG-tagged deletion mutants was constructed (Fig. 4). All these constructs synthesized their corresponding proteins after transfection into CV1 cells by immunoblotting analysis (data not shown). Their corresponding localizations were then examined by immunofluorescence as described previously.^2 We first tested the specificity of both antibodies (anti-FLAG and anti-mitosin alpha10C) used for indirect immunofluorescence staining. In untransfected CV1 nuclei of mitotic cells (Fig. 5, panels1 and 2), alpha10C labeled endogenous mitosin at the centromere/kinetochore, whereas anti-FLAG antibody did not, indicating that labeling with anti-FLAG was due to the cellular production of FLAG fusion proteins. The antigenic epitope for alpha10C has been previously mapped to the C terminus of mitosin.^2 In cells expressing the C-terminal truncated fusion protein C, the endogenous protein was shown to be associated with the centromere/kinetochore (Fig. 5, panels4 and 5); in contrast, the FLAG label was only in the cytoplasm. The N-terminal truncated mutant E localized to the centromere/kinetochore, suggesting the centromeric targeting domain of mitosin was located within the C terminus. Deletion of either the N terminus (EDeltaN; Fig. 5, panels7-8) or one inverted repeat (EDeltaR; data not shown), did not significantly affect centromere/kinetochore localization of the FLAG fusion proteins. Further deletion of the N terminus (EDeltaG) did not abolish centromeric targeting (data not shown). When the region including amino acid residues 2,488-2,902 was deleted from E to create EDeltaZ, the centromere/kinetochore targeting of the fusion protein was completely abolished ( Fig. 6and (11) ); however, the location of endogenous mitosin on the centromere/kinetochore was not affected ( Fig. 5and (11) ). Deletion of the space region between aa 2,666 and 2,756 creating a mutant EDeltaNB did not affect the centromere localization (Fig. 5). As summarized in Fig. 4, the region of mitosin important for centromere/kinetochore targeting is confined to a region of 414 amino acid residues (aa 2,487-2,901) containing three leucine heptad repeats located at amino acids 2,557-2,592, 2,797-2,818, and 2,866-2,887, respectively.


Figure 4: A diagram showing the constructs used to determine essential domains for centromere/kinetochore targeting. Summary of the localization data is also presented.




Figure 5: Determination of the centromere/kinetochore targeting domain of mitosin. Immunofluorescent staining of unsynchronized CV1 cells transiently transfected with FLAG epitope-tagged constructs is shown. Following transfection of CV1 cells with the DNA constructs of interest for 2 days, cells were fixed and immunofluorescently labeled. Mitotic cells expressing FLAG mitosin were recorded on Ektachrome P1600 film. The centromere/kinetochore localization was judged by the presence of discrete, paired dot staining on the chromosomes. The C-terminal region of mitosin was labeled by using alpha10C and fluorescein isothiocyanate-conjugated anti-rabbit IgG (Fisher). The FLAG epitope was identified by using anti-FLAG M2 antibody and Texas Red-conjugated anti-mouse IgG (Fisher). Nuclear DNA was identified with DAPI. A representative image of immunolabeled CV1 cells is shown. Centromeric staining is indicated by arrows. Panel1, typical centromeric and cytoplasmic labeling of endogenous mitosin in whole cells. Panel2, immunolabeling of untransfected cells with anti-FLAG M2 antibody. The film was overexposed to show the minimal background staining. Panel3, DAPI-stained DNA. Panels4-6, deletion mutant C did not localize to the centromere/kinetochore. Since alpha10C does not recognize C, endogenous mitosin labeling is shown for comparison. Panels7-9, representative immunolabeling showing the centromeric staining of EDeltaN. Panels10-12 are representative of patterns of EDeltaZ, which does not show centromere/kinetochore staining, and panels13-15 are EDeltaNB to show the localization of kinetochore.




Figure 6: Diagram describing the regions of mitosin responsible for homodimerization with itself (pSE1107-2) and for interaction with a mitosin-associated candidate protein (pSE1107-5). The beta-galactosidase activity was quantitatively measured by using the chlorophenol red-beta-D-galactopyranoside method.



The Mitosin C Terminus Is Also Essential for Homo- and Heterodimerization

The C terminus of mitosin is required for nuclear localization and centromere/kinetochore targeting. To determine whether this region is mechanistically required for association with other proteins that could influence its cellular targeting, interacting proteins were isolated by screening a ACT human lymphocyte cDNA library with the yeast two-hybrid method(16) . Fifty clones were obtained from the initial screen (listed in Table 1). The eight clones with the highest qualitative binding activity were characterized by DNA sequencing. Three clones were found to be identical with the C terminus (amino acid residues 2,490-3,113) of mitosin. Also, four clones were identified as being related to immunoglobulin genes. The remaining clone contains a novel gene sequence. The results indicate that mitosin can interact with itself and other proteins through its C terminus. This observation is consistent with the idea that different fusion protein constructs of the mitosin C terminus can interact with themselves in in vitro binding assays (data not shown).



Leucine heptad repeats are frequently involved in protein-protein interactions and are found three times in the mitosin C terminus(17) . In order to determine whether these leucine heptads play a role in homodimerization of mitosin or heterodimerization with the mitosin-associated candidate gene product (pSE1107-5), three deletion constructs were created: 1) pAS1-E/StuI-MscI (which contained all three leucine heptad repeats), 2) pAS1-EDeltaZ/StuI-BglII (in which the three leucine heptad repeats were deleted), and 3) pAS1-EDeltaNB/StuI-BglII (in which a space between the first and second leucine repeats is deleted). These constructs were then used to measure the contribution of the leucine heptads for dimerization of mitosin. As shown in Fig. 6, removal of the region containing the leucine heptad repeats abolishes the homodimerization and heterodimerization capacity of the C terminus.


DISCUSSION

In order to understand the cell cycle dynamics of mitosin localization, we have used deletion mutagenesis and subsequent transient expression of epitope-tagged proteins to identify functional subdomains responsible for nuclear and centromere/kinetochore targeting. From the deduced amino acid sequence of mitosin, this method reveals a highly complex molecular structure that includes predominant alpha-helical domains, multiple leucine heptad repeats, a large inverted repeat, and a putative bipartite NLS.^2 Our data show that the NLS of mitosin is composed of two interdependent basic clusters (aa 2,916-2,958; basic regions II and III) separated by an intervening 20 amino acids. The sequence is a variant of the bipartite nuclear targeting signal proposed by Dingwall(15) . The interdependence of the two clusters in the mitosin NLS is demonstrated by the finding that deletion of either cluster abolishes nuclear targeting of mitosin.

Immunoelectron microscopy performed upon detergent-extracted cells provides clear evidence that mitosin is differentially located on the coronal surface of the outer plate of kinetochores. The primary functions currently ascribed to the kinetochore are microtubule attachment and motor activity. The outer plate of the kinetochore is believed to provide binding sites for microtubules(3) . A facultative kinetochore protein, CENP-E, is a putative motor protein that interacts with microtubules presuming either through its N-terminal kinasin domain (18) or the C-terminal domain (100 residues), which is a basic (pI 9) and proline-rich (13%) region similar to those of and MAP2(19) . Interestingly, the extreme C terminus of mitosin is also basic (pI 10) and proline-rich (10%); however, the amino acid sequences of these structurally similar regions are not significantly homologous. Inasmuch as electron microscopic examination of the outer kinetochore surface reveals a homogeneous array of microtubules, an electron dense outer plate, and an amorphous corona, our finding of a discrete labeling pattern of mitosin in this structure suggest a possible role in guiding or stabilizing microtubules to the kinetochore. As mitosin does not contain known tubulin binding domains (such as kinesin family homology), demonstration of a mitosin-tubulin interaction, perhaps through its proline-rich and basic C terminus, will be required to support this hypothesis.

The mechanism underlying the specific and novel distribution of mitosin to the coronal surface of the kinetochore outer plate is unknown. Mapping of deletion mutants of epitope-tagged mitosin suggested that the C-terminal 413 amino acids containing the three leucine heptad repeats is important for its centromere/kinetochore targeting. At present, the only other protein whose centromere targeting is well characterized is CENP-B, and it is defined as the first 28 amino acids of the N terminus(13) . This region displays the ``CENP-B box''-specific DNA binding activity, explaining (in part, if not entirely) how CENP-B may specifically target the interphase pre-kinetochore and the mature centromere. As might be expected, when amino acid sequence is compared with the inner centromere/kinetochore proteins CENP-B (20) or CENP-C(21) , the essential outer kinetochore-targeting regions of mitosin show no sequence homology. It is noteworthy, however, that targeting of the epitope-tagged mitosin mutants is observed only when the constructs contain a functional NLS; cytoplasmic contructs that contain the C-terminal leucine heptad repeats are not sufficient for centromere/kinetochore localization such as in the case of EDeltaC1. That mitosin is exclusively nuclear during interphase may suggest that proper centromere/kinetochore targeting requires nucleus-dependent processing, or the targeting domain can only function during a specific temporal period during genesis of the mature kinetochore.

It is probable that cellular proteins interacting with mitosin are involved in its ability to target kinetochore structures. Therefore, a C-terminal fragment of mitosin (2,488-3,113) was used to screen a ACT human lymphocyte cDNA library by the yeast two-hybrid method(16) . Three of the eight clones that survived the original screening were virtually identical to the mitosin C terminus itself (aa 2,490-3,113). This result strongly supports the idea that the C terminus can bind to itself. The functional significance of the self-association is unclear at present but does suggest that centromere/kinetochore targeting and homodimerization may possibly be coupled. Protein-protein interactions via leucine heptads (17) in the C terminus may regulate the signaling for kinetochore localization by conveying a conformational change within mitosin. Alternatively, homodimerization may create a new molecular surface that serves as the targeting signal, similar to dimerization domains between transcription factors creating DNA-binding surfaces(14) . Identification of mitosin-associated proteins that also localize to the centromere/kinetochore and bind mitosin homodimers would support this possibility.

Two hybrid screening also identified several other putative mitosin-binding proteins. One novel clone, pSE1107-5, was also shown to interact with the mitosin C terminus containing the three leucine heptad repeats that are also present in the region essential for centromere/kinetochore targeting. At present whether the protein encoded by pSE1107-5 is centromere/kinetochore-associated remains to be shown. However, its ability to interact with mitosin would be consistent with heterodimerization influencing centromere/kinetochore targeting.

The specific cellular targeting properties of mitosin thus far characterized can be assigned to the C-terminal fourth of the protein. Based on its deduced primary amino acid sequence, a number of leucine heptads are also found in the N terminus;however, functional properties that accompany these structural characteristics remain to be determined. Further characterization of mitosin will be necessary to fully assign function to specific amino acid residues and should provide a mechanistic insight into biological role of mitosin.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This work was supported by grants from the National Institutes of Health (to W. H. L. and B. R.), the Council for Tobacco Research (to W. H. L.), and the Texas Higher Education Board (to W. H. L.).

§
These two authors contributed equally to this work.

Supported by a postdoctoral fellowship from NCI, the National Institutes of Health.

**
To whom correspondence should be addressed: Center for Molecular Medicine/Institute of Biotechnology, the University of Texas Health Science Center at San Antonio, 15355 Lambda Dr., San Antonio, TX 78245. Tel.: 210-567-7353; Fax: 210-567-7377.

(^1)
The abbreviations used are: CENP, centromere protein; aa, amino acid(s); kb, kilobases; DAPI, 4,6-diamidino-2-phenylindole; NLS, nuclear localization signal.

(^2)
Zhu, X., Mancini, M. A., Chang, K.-H., Liu, C.-Y., Chen, C.-F., Shan, B., Jones, D., Yang-Feng, T. L., and Lee, W.-H. (1995) Mol. Cell. Biol.15, in press.


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