(Received for publication, May 26, 1995; and in revised form, June 16, 1995)
From the
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.
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, ()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. ()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.
Mitosin is undetectable during most of
G
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.
Overexpression of N-terminal deletion mutants, which retain their
ability to locate within the nucleus, result in accumulation of cells
mainly in G
/M phase.
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.
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. 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 E
L, (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 E
C1 (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 E
C2 (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 E
RvB 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 EN (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 E
R (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. E
G (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 E
Z (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. E
NB was made
by self-ligation of the vector after removing the SstI
fragment (aa 2,666-2,756).
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-EZ/StuI-BglII, in which these three leucine
heptad repeats (aa 2,487-2,901) were deleted; and 3)
pAS-1-E
NB/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
-galactosidase activity, first qualitatively via the
filter lift method and then quantitatively by chlorophenol
red-
-D-galactopyranoside (Boehringer Mannheim)
measurement(16) .
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
10C (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
E
L; 6-6`, construct E
C1; 7-7`, construct E
C2; 8-8`,
construct E
RvB. Bar, 20
µm.
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.
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 10C 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
10C does not recognize C, endogenous
mitosin labeling is shown for comparison. Panels7-9, representative immunolabeling showing the
centromeric staining of E
N. Panels10-12 are representative of patterns of E
Z, which does not show
centromere/kinetochore staining, and panels13-15 are E
NB 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 -galactosidase activity was quantitatively
measured by using the chlorophenol
red-
-D-galactopyranoside
method.
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-EZ/StuI-BglII
(in which the three leucine heptad repeats were deleted), and 3)
pAS1-E
NB/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.
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
-helical domains, multiple leucine heptad repeats, a large
inverted repeat, and a putative bipartite NLS.
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 EC1.
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.