An EF-hands protein, centrin-1, is an EGTA-sensitive SUMO-interacting protein inmouse testis
Niina Tanaka1, Miki Goto1, Azusa Kawasaki1, Takashi Sasano1, Ko Eto1, Ryotaro Nishi2,
Kaoru Sugasawa2, Shinichi Abe1 and Hisato Saitoh1, 3, 4, 5

1

Department of Biological Sciences, Graduate School of Science and Technology, Kumamoto University,

Kumamoto, Japan
2

Biosignal Research Center, Organization of Advanced Science and Technology, Kobe University, Japan3

Global COE (Centers of Excellence) Program, Global Initiative Center for Pulsed Power Engineering,

Kumamoto University, Kumamoto, Japan
4

Department of New Frontier Sciences, Graduate School of Science and Technology, Kumamoto University,

Kumamoto, Japan5

To whom correspondence should be addressed: Department of New Frontier Sciences, Graduate School of

Science and Technology, Kumamoto University, Kumamoto, 2-39-1 Kurokami, Kumamoto 860-8555,
Japan
Email: hisa@kumamoto-u.ac.jp, Phone: +81-96-342-3450, Fax: +81-96-342-3450

Keywords: SUMO-2/3, centrin-1, calcium signaling, spermatogenesis

1

ABSTRACTA multifunctional calcium-binding protein, centrin-1, is specifically expressed in male germ cells,
certain neurons and ciliated cells. We identified centrin-1 as a protein interacting with SUMO-2/3 using
yeast two-hybrid screening of a mouse testicular cDNA library. In bead halo assays, the interaction
between centrin-1 and SUMO-2/3 was reduced in the presence of EGTA and facilitated by the addition of
CaCl2. Immunostaining of seminiferous tubules in 35-day-old mouse testes revealed that cells in the layer
containing spermatogonia showed colocalization of SUMO-2/3 with centrin-1 in cytoplasmic spots.
Identification of centrin-1 as the EGTA-sensitive SUMO-2/3-interacting protein indicates the possible
role of calcium in modulating the centrin-1–SUMO-2/3 interaction and suggests the importance of thisinteraction in mouse testis.

2

INTRODUCTION

Spermatogenesis is essential for sexual reproduction in animals. It is triggered by sequential mitotic
divisions of spermatogonia in the testis, followed by their differentiation into spermatocytes. After two
meiotic divisions, spermatocytes develop into haploid spermatids possessing half the normal complementof genetic material and then into spermatozoa, the mature male gametes in many sexually reproducing
organisms. This complex process appears to be controlled by cooperation between several hormones andtesticular somatic cells that leads to subsequent changes in a wide variety of cell morphology and cellularsignaling regulated, in part, by post-translational protein modifications.1
The small ubiquitin-related modifier (SUMO) is a post-translational protein modifier, similar toubiquitin in terms of its structure and its mechanism of conjugation to target proteins. SUMO is involvedin many diverse cellular functions, including DNA replication, repair and recombination, RNAtranscription, nucleocytoplasmic trafficking, and protein stability.2,3 In higher eukaryotic cells, such as in
the human and mouse, at least two paralogs, SUMO-1 (SMT3C) and SUMO-2/3 (SMT3A/B), have beenreported.4 Although several groups have tried to elucidate the role of the SUMO modification pathways inthe differentiation of male germ cells and to seek key signaling molecule(s) that might link the SUMO
pathways with the onset and/or progression of meiosis during spermatogenesis, most of them remain
largely uncharacterized.5–7
Centrins are relatively small acidic proteins that contain four helix–loop–helix motifs, the so-called
EF-hands, which represent potential calcium binding sites.8–10 In mammals, at least three centrin proteins
have been identified so far,10-13 centrin-1 and centrin-2 share approximately 80–90% identity invertebrates, and sequences of the yeast centrin and the related mammalian centrin-3 isoform have aminoacid identities of only about 55%, compared with the other isoforms. Human centrin-1 and centrin-2 havebeen reported to bind preferentially to SUMO-2/3, unlike SUMO-1.14 Nucleocytoplasmic shuttling ofcentrin-2 is controlled by the SUMO modification system and the xeroderma pigmentosum group C(XPC) protein, suggesting the importance of a dynamic interplay among centrin-2, SUMO-2/3 andnuclear/chromatin protein(s). In line with studies on the subcellular localization of centrin-2 and its
3

associated factors, it is interesting to note that centrin-2 might function in mRNA export and this function
seems to be conserved. 15,16 Thus, it is reasonable to speculate that centrin’s subcellular localization can
influence its associated molecules/factors in the nucleus. In contrast to centrin-2, little is known
concerning centrin-1. In part, this is because of its restricted expression in the testis and retina, which haveflagella and cilia, respectively,11–13 whereas centrin-2 is expressed in most cell types except for the testis.
Here we describe the isolation of centrin-1 cDNA in the yeast two-hybrid screening of a mouse
testicular cDNA library using the GAL4–SUMO-3–SUMO-3 fusion protein as a bait. This confirmed
previous data indicating that centrin-1 and centrin-2 bind SUMO-2/3 in the yeast two-hybrid system.14 In
addition, we show here for the first time that the in vitro interaction of centrin-1 with SUMO-3 is reducedin the presence of EGTA and influenced by calcium, suggesting a possible role of metal ions flux incontrolling the centrin-1–SUMO-2/3 interaction. Moreover, biochemical and immunostaining
experiments using the adult mouse testis suggested that there is spatiotemporal interaction of the two
proteins in the cells localized at the layer containing spermatogonia in seminiferous tubules. Therefore,
we propose centrin-1 as a previously unappreciated form of calcium-modulated EGTA-sensitive
SUMO-interacting protein that might play a role in the differentiation of male germ cells.

MATERIALS AND METHODS

Plasmids
To generate enhanced green fluorescent protein (EGFP)–centrin-1 and glutathione S-transferase(GST)–centrin-1 expression vectors, mouse centrin-1 was amplified by polymerase chain reaction (PCR)
and cloned into the pEGFP-C2 (Clontech) and pGEX (GE Healthcare) vectors, respectively. Centrin-2,
SUMO-1/2/3 and MCAF1-SIM expression plasmids were as described previously.17,18,19

Antibodies
A rabbit polyclonal antibody against SUMO-2/3 was used as described.17,18 Anti-centrin-1 andanti--tubulin antibodies were obtained from Santa Cruz Biotechnology and Sigma-Aldrich, respectively.
4

The secondary antibodies used in this study were as described.17,18

Bead halo assay
This method was based on a modification of a low-affinity protein interaction procedure, known asbead halo assay, described previously.20 In brief, a 50 ml culture of Escherichia coli carrying
pET28-EGFP-SUMO-3 was used to express recombinant SUMO-3 fused to EGFP. The bacterial pellet
was dissolved in 5 ml of phosphate-buffered saline (PBS, pH 7.0), followed by sonication. The sonicatedbacterial lysate was centrifuged and the supernatant was stored at –20oC until use (approximately 0.01 mg
EGFP-SUMO-3/ml of bacterial lysate). In the experiments described in Figs 2C and 2D, expressed
EGFP-SUMO-3 was purified using Ni-beads according to the manufacture’s protocol and added to thereaction mixture in the presence or absence of appropriate amount of CaCl2 as indicated in the figures.
The GST-centrin-1 and -centrin-2 were expressed in E. coli and were then incubated withglutathione-Sepharose 4B beads (GE Healthcare). We used 10 µl of packed beads for bacterial lysate
prepared from 10 ml of bacterial culture. GST-fusion proteins were immobilized at high concentrations on
the surface of the Sepharose beads. Approximately 5-10 µg of GST-fusion proteins were bound to 10 µlof packed beads. To elucidate the effect of calcium described in Figs 2C and 2D, the beads were preparedwith PBS containing 2 mM EGTA followed by extensive washing with PBS without EGTA. The beads
were resuspended as a 50% slurry in PBS and a 2 µl portion of the slurry, in which approximately 30
particles of beads were included, was mixed, on a 76  26 mm Micro Slide Glass with 2.0 µl of either
bacterial lysate or purified EGFP-SUMO-3 in the presence or absence of EGTA/CaCl2. As soon as the
incubation started, the beads were viewed in real-time under equilibrium conditions and photographedusing a BIOREVO BZ-9000 fluorescence microscope (Keyence). When EGFP-SUMO-3 binds to an
immobilized GST-centrin protein, the interaction is visible through GFP filters as a halo of fluorescencearound the beads.

Cell culture and transfection
HeLa cells were cultured in Dulbecco’s modified Eagle’s medium with Ham’s F-12 nutrient mixture5

(Sigma-Aldrich) with 5% fetal calf serum and antibiotics, in 5% CO2. Transfection was carried out asdescribed.17 Indirect immunofluorescence was carried out as described.18

Immunoblotting and immunoprecipitation analyses
Testes (0.01–0.07 g) obtained from different ages of mice were homogenized in 3xSDS-sample buffer
with a sonicator (Tomy-UD201). The supernatant (2 mg/ml protein) was used in immunoblotting andimmunoprecipitation analyses.18

Immunohistology
C57BL/6 mice (purchased from Kyudo Co., Ltd., Japan) were used and were maintained, operated on,
euthanized and dissected according to the guidelines of Kumamoto University. A small piece of testicular
tissue was subjected to 6 m cryosections (MICROM HM505N, Uniscience). The sections were fixed byimmersion in 4% paraformaldehyde in PBS and were immunostained using anti-centrin-1 (dilution
1/200) and anti-SUMO-2/3 antibody (dilution 1/100). Nonspecific antibody-binding sites were blocked
using Roche blocking reagent (Roche). To visualize the nucleus, the sections were incubated in DAPI for
10 min. The sections were observed using BIOREVO BZ-9000.

RESULTS

Identification of centrin-1 as a protein interacting with SUMO-2/3 and characterization of its subcellular
localization in cultured cells
To identify a protein(s) that interacts with SUMO, we performed a GAL4-based yeast two-hybrid
screening using the SUMO-3–SUMO-3 fusion protein as bait.17,18 One of the interesting clones encoded amouse homologue of centrin-1, a relatively small acidic protein that is expressed specifically in the testis
and retina and contains EF-hands, which represent potential calcium-binding sites. As shown in Figs 1A
and 1B, a bead halo assay—a simple and rapid procedure that was developed to detect low affinity
interactions in real time under equilibrium binding conditions demonstrated that centrin-1 interacts
6

directly with SUMO in vitro in preference to SUMO-3, consistent with a previous observation.14
Despite the known localization of centrin-2 at the centrosome as well as in the nucleus and at thenuclear rim of mammalian cultured cells, the subcellular localization of centrin-1 in mammalian cultured
cells remained unclear.14,15,21 Therefore, to clarify its subcellular localization and to test on any functional
overlap between centrin-1 and centrin-2 at the centrosome, we transiently expressed EGFP–centrin-1 in
HeLa cells. As shown in Fig. 1C, green fluorescence was detected in both the cytoplasm and thenucleoplasm, with prominent accumulation as spots in the cytoplasm and the nuclear rim. Because thecytoplasmic spots were co-stained with anti--tubulin antibody, this transient expression of
EGFP-centrin-1 revealed that centrin-1 can be targeted to the centrosome in Hela cells. It should be notedthat similar patterns were observed when EGFP-centrin-1 was expressed in a mouse germ seminoma cellline, GC1 (data not shown), suggesting that centrin-1 accumulates in both the nucleoplasm and cytoplasm
with a potential to localize to the centrosome in mammalian cells. Taken together, these data imply that
centrin-1 is a protein that interacts with SUMO-2/3 and has ability to localize at multiple subcellularcompartments, including the centrosome.

The interaction between centrin-1 and SUMO-3 is modulated by calcium
Given that the major domains of centrin-1 comprise calcium-binding EF-hand motifs,8-10 weinvestigated whether the affinity of centrin-1 to SUMO-2/3 might be affected by calcium. To do this, wefirst wished to determine whether centrin-1 binding to SUMO-3 could be reduced by the addition of
EGTA, a calcium chelating agent, in the bead halo assay. In Figs 2A and 2B, we prepared the beads andincubated them with the lysate in a buffer containing 0 mM or 2 mM EGTA, respectively. Compared with
the incubation conditions of 0 mM EGTA, addition of EGTA produced significant reduction of green
fluorescence around the centrin-1 beads. In contrast, EGTA had little effect on the green fluorescentsignals around the beads containing GST-MCAF1-SIM. Given that in vitro binding and nuclear magnetic
resonance (NMR) spectroscopy analyses concerning the SUMO-2/3–MCAF1-SIM interaction revealed
that there is no requirement of calcium and/or other metal ions for interaction, we chosen MCAF1-SIM asa suitable control protein that binds to SUMO in a manner independent of EGTA.17,22 Thus our data7

indicate that EGTA effectively reduces the association of centrin-1 with SUMO-3, and that the effect of
EGTA was specific to the interaction between centrin-1 and SUMO-3. Moreover, a bead halo assay
revealed that GST-centrin-2, another centrin family protein which has been reported to bind SUMO-2/3, 21
reduced its ability to bind with EGFP-SUMO-3 in the presence of EGTA (Fig. 2E), implying that the
EGTA-sensitive binding to SUMO-2/3 might be a common feature among centrin-1 and -2 family
proteins.
Given that EGTA is known as a calcium chelating agent, we then tested whether an increased
concentration of calcium in the reaction buffer would facilitate the binding of SUMO-3 to centrin-1.
When conducting this experiment, we expected that centrin-1 prepared by treatment with EGTA might bea calcium-free form that would incorporate calcium rapidly when CaCl2 was added to the reaction buffer(see Materials and Methods section in detail). As shown in Figs 2C and 2D, green fluorescence was notdetected when the bead halo assay was performed in the absence of CaCl2. In contrast, green fluorescenceappeared around the beads when the assay was carried out in the presence of 0.1 mM, 1 mM or 2 mM
CaCl2. Since no significant effect of addition of CaCl2 on the GST–MCAF1-SIM interaction (Figs 2C andD), our results suggest that the interaction between centrin-1 and SUMO-2/3 can be facilitated bycalcium.

Mapping of SUMO-interacting domain/motif in centrin-1
Next, to determine the region responsible for binding to SUMO-2/3, we generated a series of deletionmutants of recombinant centrin-1 and performed a bead halo assay. Because many SUMO-binding
proteins reported so far contain a short polypeptide sequence which has key hydrophobic residues often
flanked by acidic residues, referred to as SUMO interacting motif (SIM),17,18,22-26 we expected that
centrin-1 was a canonical SIM-containing protein. As shown in Figs 3A and 4B, GST protein fused to a
C-terminal fragment of centrin-1, residues 94–172 bound strongly with EGFP-SUMO-3; conversely thebinding of GST proteins fused to the N-terminal fragment 1–98 or to each of the EF-domain (residues
1-64 for EF-1, residues 65-98 for EF-2, residues 94-145 for EF-3 and residues 139-145 for EF-4,
respectively) was notably weaker than that for the residues 94–172. These results indicated that the region
8

between amino acids 94-172 of centrin-1, which includes both EF-3 and EF-4 domains, is important for
efficient binding to SUMO-3, although other region of centrin-1, including EF-1 and EF-2, might also
contribute to the interaction with SUMO-3 to some extent.
To further localize SIM in the region between amino acids 94-172 of centrin-1, we looked for thecanonical SIM in amino acids 94-172. Although a sequence perfectly matched to the canonical SIM was
not detected in this region, we found that the amino acids 133-142 (LGESLTDEEL) were reasonably
similar to the canonical SIM. To elucidate whether this polypetide functions in SUMO binding, we madea GST protein fused to amino acids 133–142 (GST-133-142) and performed a bead halo assay. As shownin Figs 3A and 3B, GST-133-142 fusion protein failed to bind EGFP-SUMO-3, suggesting that, although
the sequence showed similarity to the canonical SIM, this polypeptide was not sufficient for
SUMO-binding. Thus we failed to map a SUMO interacting motif to a small region in centrin-1,
suggesting that centrin-1 contains an unusual SIM or binds to SUMO in a manner different from theinteraction with the canonical SIM.

Immunohistology of the centrin-1–SUMO-2/3 interaction in mouse seminiferous tubules
Because centrin-1 is expressed exclusively in male germ cells, certain neurons and ciliated cells, wechose the mouse testis to investigate the possible in vivo interaction between centrin-1 and SUMO-2/3.11–13
For this, we first determined the expressions of centrin-1 and SUMO-2/3 at the protein level. We
performed immunoblot analyses using lysates from mice testes and detected centrin-1 in 21-, 28- and30-day old mouse testes (Fig. 4A). In these extract, SUMO-2/3 was also detected, suggesting that both
centrin-1 and SUMO-2/3 were expressed in adult mouse testes.
Next, to investigate whether centrin-1 and SUMO-2/3 interacted in vivo and to elucidate any
physiological significance, we performed immunostaining on the seminiferous tubules to test whether we
could visualize a subset of testicular cells that showed colocalization of these proteins. Sections ofindividual tubules obtained from the adult mouse testis contain the full range of spermatogenic stages,
which can be divided spatially into three cell layers. The outermost layer of each tubule contains
spermatogonia and interspersed Sertoli cells; the middle zone contains primary and secondary9spermatocytes and the innermost zone contains spermatids and spermatozoa. Thus, immunostaining of theadult seminiferous tubules allowed investigation of the centrin-1–SUMO-2/3-interaction among multiple
cell types during the course of spermatogenesis. No signal was observed in control experiments, either
omitting the incubation with anti-centrin-1 or anti-SUMO-2/3 antibodies (data not shown). However, as
shown in Fig. 4B, a section of individual tubules obtained from a 35-day-old mouse testis showed positive
signals, suggesting that the immunofluorescent signals in the sections were specific to the antibodies.
When the sections were observed, it was obvious that the cells present in the middle zone enriched withprimary and secondary spermatocytes (region #3 in Fig. 4B) showed significant labeling with theanti-SUMO-2/3 antibody. Because previous reports demonstrated that the components in the SUMO
pathway, including SUMO-2/3, SUMO-1, Ubc9 (SUMO-E2), Zimp7 (PIAS family of SUMO-E3) andPML (SUMO-substrate), were localized to the XY body in pachytene and diplotene spermatocytes, we
speculate that the prominent signals detected by anti-SUMO-2/3 antibody in our specimen represent the
XY-body.5-7,27 In contrast, centrin-1 signals at the XY-body were barely detected, indicating that centrin-1
might not be a major component of the XY body in spermatocytes. On the other hand, the cytoplasmic
spots detected by the anti-centrin-1 antibody were apparent in many cells not only in the mid zone but
also the outer (region #1 and #2) and inner (region #4) layers, suggesting that centrin-1 is a component of
the centrosome during spermatogenesis, consistent with a previous observation.28 Intriguingly, whenobserving the immunostaining pattern of anti-centrin-1 antibody in consecutive sections, there were
multiple cells containing cytoplasmic spots that costained with anti-centrin-1 and anti-SUMO-2/3
antibodies in cells in the outer zone, but not in the middle and inner layers. Thus these observations
suggest that there might be a temporal difference in antigen availability of SUMO-2/3 in the cytoplasm,
possibly at the centrosome region. Alternatively, the localization of SUMO-2/3 in the cytoplasm, inparticular at around the centrosomal region, might be regulated temporally during spermatogenesis. Taken
together, these immunostaining results on the mouse testis suggest that there is interaction between
centrin-1 and SUMO-2/3 in the cytoplasmic spots in cells present in the outer zone of seminiferous
tubules, containing mostly spermatogonia.

10

DISCUSSION

Our studies revealed that centrin-1 preferentially binds to SUMO-2/3 rather than SUMO-1 (Fig.1), andthat their interaction appears to be reduced in the presence of EGTA (Figs 2A and 2B). The
EGTA-sensitive SUMO-binding was also observed when centrin-2, another centrin family protein in
mammals, was subjected to a bead halo assay, suggesting that EGTA-sensitive SUMO-binding property
might be conserved among centrin-1 and -2 family proteins (Fig. 2E).
Protein structures at high resolution based on X-ray crystallographic data have been determined foryeast and human centrin.29,30 The molecular structure of centrin includes four EF-hands, which comprisethe prototypical bivalent metal ion binding helix–loop–helix motif. Chlamydomonas centrin binds four
calcium ions with dissociation constants ranging from 1 nM to 0.1 mM, while only the carboxy-terminal
EF-hands of human centrin-2 bind calcium ion at physiological concentrations.30–33 Therefore it isreasonable to speculate that depletion and addition of calcium may influence centrin-1-SUMO-2/3
interaction. Indeed the addition of EGTA, which may cause the reduction of calcium ion in the reaction
buffer via its preferential chelating activity toward calcium, resulted in reduction of centrin-1’s binding to
SUMO-3 (Figs 2A and 2B). Furthermore, as shown in Figs 2C and 2D, we detected that nearly fullrecovery of EGFP-SUMO-3 binding by addition of CaCl2 to the reaction buffer containing nearly
negligible calcium ion. As such, these results encourage us to propose calcium is an important metal ion
to regulate the interaction between centrin-1 and SUMO-2/3. However, it remains to be elucidated indetail which metal ion(s) besides calcium binds most efficiently to the EF-hands in centrin-1 or centrin-2
in our experimental conditions. We cannot rule out the possibility of substitution of other metal(s) for
calcium in the context of regulating the centrin-1-SUMO interaction in vitro as well as in vivo.
Attempts to map the SUMO interacting motif (SIM) in centrin-1 led to the realization of its difficulty(Fig. 3). One feasible explanation for our failure to map a SIM to a small region in centrin-1 is that theinteraction of centrin-1 to SUMO-2/3 might be distinct from the interaction of the canonical SIMs
Although the structural basis of EGTA-sensitive centrin-1–SUMO-2/3 interaction should be elucidated,
our in vitro data clearly indicate that the calcium-binding form of centrin-1 shows much higher affinity to
11

SUMO-2/3 than does the calcium-free form, suggesting that a metal-dependent conformational change isimportant for binding with SUMO-2/3. It would be intriguing to speculate that the interaction might beimportant for the conformational change if centrin-1 is modified by SUMO-2/3.
Immunostaining revealed colocalization of centrin-1 and SUMO-2/3 in the area containing a significant
population of spermatogonia (Fig.4). We believe that this is the first approach for characterizing
centrin-1–SUMO-2/3 interaction in the testis. Centrin-1 and SUMO-2/3 colocalized at cytoplasmic spots
in cells present in the outer zone of the seminiferous tubule, containing mostly spermatogonia. Based on
the nature of centrin-1 as a centrosomal component, this suggests that the cytoplasmic interaction between
centrin-1 and SUMO-2/3 might represent a spatiotemporal regulation of centrosomal proteins during
spermatogenesis. Klein and Nigg reported that human centrin-2 could be modified by either SUMO-1 or
SUMO-2/3, but indicated that localization of centrin-2 to the centrosome was independent of its
SUMOylation.14 Although their results argue that centrin-1 itself could be an efficient SUMOylation
substrate in the testis, we could not detect a SUMOylated form of endogenous centrin-1 using the lysatesof 35-day-old mice testes, suggesting that most of the endogenous centrin-1 in testis was modified very
poorly by SUMO (if at all), or that any SUMOylated centrin-1 in the testis was unstable (unpublisheddata). Thus, we assume that colocalization of centrin-1 and SUMO-2/3 at the cytoplasmic spots might
represent an interaction of centrin-1 with SUMOylated proteins that is distinct from SUMOylated
centrin-1. In future experiments, proteomic analysis of SUMOylated proteins in the mouse testis should
be explored to identify a centrosomal protein(s) that can be SUMOylated and interact with centrin-1.
In conclusion, the testis-specific and conspicuous spatiotemporal expression, together with thecalcium-binding property of centrin-1, suggest that this EGTA-sensitive interaction with SUMO-2/3
might be very important for the differentiation of male germ cells. Although further studies will be
required to provide insights into the physiological role of the interaction, this study improves
understanding of how protein SUMOylation is involved in centrin-1/2- or centrosome-function during
spermatogenesis as well as in the context of calcium signaling.

ACKNOWLEDGMENTS
12

This work was supported by grants to H.S. from The Japanese Government Ministry of Education,
Culture, Sports, Science and Technology.

13

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FIGURE LEGENDS

Figure 1. Centrin-1 is an EF-hands protein localized at the centrosome.
A: Preferential binding of centrin-1 with SUMO-3 in the bead halo assay. A bacterial lysate containing
0.01 mg/ml of EGFP–SUMO-1 (left) or EGFP–SUMO-3 (right) was mixed with the bead-immobilized
GST–centrin-1 (upper) and GST alone (bottom), respectively. Aliquots of the recombinant
EGFP–SUMO-1, EGFP–SUMO-3, GST–centrin-1 or GST alone were separated by SDS–PAGE andstained with Coomassie blue. The mixtures were treated as described in the Materials and Methods andimaged using a fluorescence microscope with an appropriate green fluorescence filter.
B: The amount of green fluorescence on the GST-centrin-1-beads was measured (n = 50 beads). a.u.,
arbitrary fluorescence unit.
C: Subcellular localization of EGFP-tagged centrin-1. HeLa cells were transfected with an expression
vector for EGFP–centrin-1. After 24 h, the cells were fixed and green fluorescence was detected byfluorescence microscopy. To visualize the centrosome, indirect immunofluorescence was also performed
using an anti--tubulin antibody. Enlarged image of the boxed region is shown (inset). Merged images areon the right (Bar = 50 m).

Figure 2. In vitro interaction of centrin-1 with SUMO-2/3 is facilitated by Ca2+.
A: Interaction of centrin-1 with SUMO-3 was reduced in the presence of EGTA. Beads immobilized to
GST–centrin-1 (upper) or GST–MCAF1-SIM (lower) were prepared and treated with a bacterial lysate
containing 0.01 mg/ml of EGFP–SUMO-3 containing 0 mM or 2 mM EGTA, followed by washing thebeads with PBS. Aliquots of the recombinant EGFP–SUMO-3, GST–centrin-1 or GST-MCAF1-SIM
were resolved by SDS–PAGE and stained with Coomassie blue.
B: Quantification of centrin-1–SUMO-3 interaction observed in (A). The amounts of green fluorescenceon the beads bound to GST–centrin-1 (left axis) or GST–MCAF1-SIM (right axis) were measured (n =60 beads). a.u., arbitrary fluorescence unit.
C: Interaction of centrin-1 with SUMO-3 was enhanced by the addition of CaCl2. The GST-centrin-1 or
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GST-MCAF1-SIM expressed in E. coli was incubated with glutathione-Sepharose beads in PBS
containing 2 mM EGTA, expecting the production of the beads associated with calcium-free form of
GST-centrin-1 and –MCAF1-SIM. .Approximately 0.01 mg/ml purified EGFP-SUMO-3 in PBS
containing 0.1% Triton-X100 was mixed with the bead-immobilized GST–centrin-1 (upper) or
GST–MCAF1-SIM (lower) followed by incubation in buffer containing 0, 0.1, 1 or 2 mM CaCl2.
Aliquots of the recombinant EGFP–SUMO-3, GST–centrin-1 or GST–MCAF1-SIM were resolved by
SDS–PAGE and stained with Coomassie blue.
D: Quantification of centrin-1–SUMO-3 interaction in (C). The amounts of green fluorescence on thebeads bound to GST–centrin-1 (left axis) or GST–MCAF1-SIM (right axis) were measured (n = 60
beads). a.u., arbitrary fluorescence unit.
E: Centrin-2 is an EGTA-sensitive SUMO-interacting protein. Beads immobilized to GST–centrin-2 was
prepared and treated with a bacterial lysate containing 0.01 mg/ml of EGFP–SUMO-3 in the presence of
0 mM (left) or 2 mM EGTA (right), followed by washing the beads with PBS. Aliquots of therecombinant EGFP–SUMO-3 or GST-centrin-2 were resolved by SDS–PAGE and stained with
Coomassie blue. The amounts of green fluorescence on the beads bound to GST–centrin-2 were
measured (n = 60 beads). a.u., arbitrary fluorescence unit.

Figure 3. The interaction of each centrin-1-deletion mutant with SUMO-3 was detected by the beadhalo assay.
A: Schematic representation of centrin-1 and four individual EF-hands (EF-1, -2, -3, and 4), and asummary of the bead halo assay. Centrin-1 deletion constructs are represented as thin black lines.
Numbers indicated on the sides of the lines correspond to amino acid residues in centrin-1. The Table on
the right summarizes the interactions of each deletion mutant with EGFP-SUMO-3 in the bead halo assay
shown in B. The symbols +++ represents strong binding; +, sufficient binding; -/+, weak binding; and -,negligible binding.
B: Interaction of each GST–centrin-1-deletion mutant with SUMO-3. Beads immobilized to each
GST–centrin-1-deletion mutant as indicated were prepared and treated with a bacterial lysate containing
190.01 mg/ml of EGFP–SUMO-3 in PBS containing 1 mM CaCl2, followed by washing the beads with
PBS. Aliquots of the recombinant EGFP–SUMO-3 or GST–centrin-1 mutants were resolved by
SDS–PAGE and stained with Coomassie blue.

Figure 4. Centrin-1–SUMO-2/3 interaction is detectable in the adult mouse testis.
A: Expression of centrin-1 protein in mouse testis. Testicular lysates from 21-, 28- and 30-day-old mice
were used for immunoblotting analysis with anti-centrin-1 (upper) and anti-SUMO-2/3 (middle)
antibodies. The Coomassie blue stained SDS–PAGE result is shown at the bottom.
B: Immunofluorescence detection of centrin-1 and SUMO-2/3 in sections of seminiferous tubules.
Seminiferous tubules of 35-day-old mouse testis were sectioned and stained with DAPI, anti-centrin-1
and anti-SUMO-2/3 antibodies. DAPI stains DNA and visualizes the nucleus. The area circled by a white
dotted line indicates an individual tubule. Sections of individual tubules obtained from the adult mouse
testis contain the full range of spermatogenic stages, which can be divided into three cell layers. The
outermost layer of each tubule contains spermatogonia and interspersed Sertoli cells (box #1 and #2); themiddle zone contains primary and secondary spermatocytes (box #3) and the innermost zone contains
spermatids and spermatozoa (box #4). The left panel shows the merged image of triple-stained
immunofluorescence of a section. The right panel shows immunofluorescence images of each boxedregion stained with anti-SUMO-2/3 (left) and anti-centrin-1 (middle) antibodies. DAPI-stained andmerged images are on the right (Bar = 50 m). The arrowheads indicate the positions of cytoplasmic
spots detected by the anti-centrin-antibody. These cytoplasmic spots might represent the centrosome (andsee Fig. 1C).28

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