Cell Division

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Meiosis specific coiled-coil proteins in Shizosaccharomyces pombe
Ayami Ohtaka1, Takamune T Saito1,2, Daisuke Okuzaki1 andHiroshi Nojima*1
Address: 1Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871,
Japan and 2Department of Genetics, Harvard Medical School 77 Avenue Louis Pasteur, New Research Building, Room 334, Boston, MA 02115,
USA
Email: Ayami Ohtaka - aohtaka@biken.osaka-u.ac.jp; Takamune T Saito - tsaito@genetics.med.harvard.edu;
Daisuke Okuzaki - dokuzaki@biken.osaka-u.ac.jp; Hiroshi Nojima* - snj-0212@biken.osaka-u.ac.jp* Corresponding author

Published: 18 May 2007
Cell Division 2007, 2:14

doi:10.1186/1747-1028-2-14

Received: 29 April 2007
Accepted: 18 May 2007

This article is available from: http://www.celldiv.com/content/2/1/14© 2007 Ohtaka et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Many meiosis-specific proteins in Schizosaccharomyces pombe contain coiled-coil motifs which playessential roles for meiotic progression. For example, the coiled-coil motifs present in Meu13 andMcp7 are required for their function as a putative recombinase cofactor complex during meiotic
recombination. Mcp6/Hrs1 and Mcp5/Num1 control horsetail chromosome movement by astral
microtubule organization and anchoring dynein respectively. Dhc1 and Ssm4 are also required forhorsetail chromosome movement. It is clear from these examples that the coiled-coil motif in these
proteins plays an important role during the progression of cells through meiosis. However, there
are still many unanswered questions on how these proteins operate. In this paper, we briefly review
recent studies on the meiotic coiled-coil proteins in Sz. pombe.

Background
Meiosis, a fundamental biological phenomenon conserved from yeast to mammals, is a specialized form ofcell division that generates haploid gametes from diploidparental cells and that presumably evolved from mitosis.
The fission yeast, Schizosaccharomyces pombe, is an ideal
model organism to study the molecular mechanism of
meiotic regulation because meiosis can be induced from amitotic cell simply by removing the nitrogen source fromthe growth medium. Sz. pombe is most stable in the haploid state and behaves asexually in rich nutritional environments. Upon nitrogen deprivation, two haploid cellswith opposite mating types conjugate without delay, after
which their nuclei fuse and undergo meiosis. After a single
round of DNA replication, the nucleus (or chromosome)begins to oscillate between the cell poles and acquires an
elongated shape called a horsetail. During this horsetailmovement, pairing of homologous chromosome andgenetic recombination occur, which generates new alleliccombinations in the resulting gametes, thereby increasing
the genetic diversity of the offspring. Subsequently, tworounds of successive chromosome segregation, calledmeiosis I (MI) and meiosis II (MII), occur, which results
in a 2-fold reduction in chromosome number. During aprocess called sporulation, intracellular formation of adouble layer forespore membrane (FSM) occurs, whichmatures into spore walls. Thus, one cell produces one
ascus containing four spores.
The expression of specific genes that regulate meiosis are
induced throughout the process. These meiotic genes areclassified as early, middle and late genes based on the timing of their expression during meiosis. These genes are further classified according to their structural motifs. One
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structural motif that is frequently observed is the coiledcoil motif, a ubiquitous protein folding structure containing seven-residue repeats which form alpha-helices thatwrap around each other [1-4]. Understanding the function of meiotic coiled-coil proteins is important not only
to understand their direct role in meiotic regulation but
also their indirect role through their interaction and regulation of other meiotic coiled-coil proteins. Indeed, someof the meiotic coiled-coil protein interactions in Sz. pombeare pivotal during meiosis. Here, we describe recent findings on the meiotic coiled-coil proteins of Sz. pombe.
Meiosis specific coiled-coil proteins of Sz. pombe
The genome sequence of Sz. pombe [5] has 4,824 open
reading frames (ORFs) (which can be viewed through theSz. pombe GeneDB [6]) which encode 955 proteins containing coiled-coil motifs (Fig. 1). The genome-wide transcriptome analysis [7] identified 180 genes which
encoded proteins with coiled-coil motifs whose expressions are upregulated during meiosis. Among those 180
genes, 25 (13.9%), 113 (62.8%) or 42 (23.3%) are early,
middle or late genes, respectively. Expression analysis foreach gene has shown that at least 21 of these genes are
expressed solely during meiosis and not during mitosis [819]. These 21 meiosis specific coiled-coil proteins include
8, 11 or 2 early, middle or late genes, respectively (Table1). In contrast, 159 out of 180 genes are also expressed
during mitosis although their expression levels are low
compared with their expression during meiosis. It remains
to be determined if there are other genes whose expression is specifically upregulated during meiosis.

The size, number and position of the coiled-coil motifs
from meiosis specific coiled-coil proteins so far reported
are schematically presented in Figure 2. Since coiled-coilmotifs consist of two or more alpha-helices that wind
together in a supercoil, the motif can vary in size depending on the number of alpha-helices that form versatile
folds in each protein. Moreover, the locations andnumber of coiled-coil motifs in a protein impacts otherinteracting proteins. Indeed, 9 out of 21 meiosis specificcoiled-coil proteins (Mcp1, Mcp3, Mcp5, Mcp6, Meu1,
Meu23, Mde4, Ssm4, Dhc1) have more than two coiledcoil motifs. In particular, the coiled-coil motif region
occupies a large portion of Mcp3, Mcp6, Meu1 andMeu23 suggesting the indispensable roles of coiled-coilmotifs for the function of these proteins. The other proteins carry only a single and comparatively short stretch ofcoiled-coil sequence. However, this may not necessarily
suggest a less important role for these coiled-coil folds inprotein function.
Putative functions of early coiled-coil genes
The physiological role of the eight early meiosis specificgenes that encode coiled-coil proteins have been analyzed

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S. pombe genome

4,824̙ORFs
Peaks of expression
during meiosis

Coiled-coil proteins

Early
MiddleLate

955
Meiotically upregulated
coiled-coil proteins

180

42

25
113

Meiosis specificcoiled-coil proteins

21

FigureThe
teins,
sis
Sz.
specificpombe
numbers
meiotically
1 genome
coiled-coilof openupregulated
proteins
reading coiled-coilframesthat are(ORFs),
predicted
proteins,
coiled-coilfromand meiothe
proThe numbers of open reading frames (ORFs), coiled-coil proteins, meiotically upregulated coiled-coil proteins, and meiosis specific coiled-coil proteins that are predicted from theSz. pombe genome. The 180 genes that encode meioticallyupregulated coiled-coil proteins are divided into three categories according to their peak in expression levels (early,
middle and late).

in detail. Subcellular localizations and functions of these
proteins during meiosis are diverse. For example, Tht1, a
type I membrane glycoprotein with a single coiled-coilmotif in the middle of the molecule, plays a key role inkaryogamy, the nuclear fusion process occurring at thestart of meiosis in which two haploid nuclei fuse to produce a diploid nucleus [9]. Tht1 is present in the nuclear
envelope and endoplasmic reticulum (ER) during karyogamy (Fig. 3A), but is not observed during later stages ofmeiosis. In the tht1 mutant, nuclear congression and spindle pole body association occurs but fusion of nuclear
envelopes is blocked. Nonetheless, meiosis continues
which results in the generation of aberrant asci. The roleof the coiled-coil domain in Tht1 during nuclear envelope
fusion remains elusive.
Meu13, another meiosis specific coiled-coil protein, is
required for proper homologous pairing and recombination [13]. It is present on meiotic chromatin and plays apivotal role in chromosome pairing through a recombination-independent mechanism, promoting proper homologous pairing and recombination. Mcp7, another meiosis
specific coiled-coil protein, forms a complex with Meu13
during meiosis [16]. Since a single coiled-coil motif occupies the central part of each protein, it is likely that theirinteraction is mediated by coiled-coil motif. Both meu13Δ
cells and mcp7Δ cells have reduced recombination rates
and spore viability and generate spores with abnormal
morphology. The coiled-coil motifs in the mouse

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Table 1: List of meiosis-specific coiled-coil proteins.

Gene Name

ORF NameAmino acids

Category

Protein Features

tht1
meu13
mcp7
mcp6/hrs1
mcp5/num1
dhc1
ssm4
eta2
sgo1
meu14
mcp4
mcp3
meu1
meu18
meu23
spn5/mde9/meu28
mde4
mcp2
mcp1
meu6
mpf1

SPAC13C5.03
SPAC222.15
SPAC13A11.03
SPBC582.06C
SPBC216.02
SPAC1093.06C
SPAC27D7.13C
SPAC31G5.10
SPBP35G2.03C
SPBC1347.03
SPBC16E9.08
SPAC1006.04C
SPAC1556.06
SPBC409.11
SPCC613.11C
SPAC24C9.15C
SPBC6B1.04
SPCC1682.08C
SPAC1687.10
SPBC428.07
SPAC4G9.05

543
216
217
327
968
4196
670
569
319
335
355
952
776
553
254
464
421
703
661
651
581

early
early
early
early
early
early
early
early
middlemiddlemiddlemiddlemiddlemiddlemiddlemiddlemiddle
late
late
late
late

nuclear membrane protein involved in karyogamy
meiotic expression upregulated
meiosis specific coiled-coil protein
meiosis specific coiled-coil protein
meiosis specific coiled-coil protein
dynein heavy chain
p150-Glued
Myb family
shugoshinmeiotic expression upregulated
meiosis specific coiled-coil protein
meiosis specific coiled-coil protein
meiotic expression upregulated
NLS
B13958 domain
septinmei4 dependent protein
RNA-binding protein
meiosis specific coiled-coil protein
lysine-rich protein
meiotic PUF family protein 1

homologs Hop2 (Meu13) and Mnd1 (Mcp7) are essential
for their interaction [20]. The Hop2-Mnd1 complex
ensures that homologous chromosomes properly synapse
during meiosis by acting in concert with Rad51 and Dmc1
to promote the strand invasion step (D-loop formation)of homologous recombination [20].
Mcp6/Hrs1 is required for proper astral microtubule positioning to maintain the horsetail movement of chromosomes [17,21]. Mcp6/Hrs1 is present at the start ofkaryogamy where it localizes to the spindle-polebody(SPB) before it is degraded prior to chromosome segregation at meiosis I. Although the allelic recombination ratesof the mcp6Δ cells are 10–40% lower than of the wildtype, the ectopic recombination rates are doubled. This is
likely due to abnormal homologous pairing resulting
from aberrant horsetail movement in this mutant [17].
Hrs1 directly interact with Alp4, a component of gamma
tubulin ring complex (γ-TuRC), and Mto1, a gamma tubulin recruiting protein [21]. In hrs1Δ cells, Alp4 becomesdispersed around nucleus from its original localization at
SPB in early meiosis I. On the other hand, overexpressionof Hrs1 at mitotic interphase causes the homogeneously
distributed Alp4 to accumulate to SPB and induces movement of chromosome and formation of aster. From theseresults, it is proposed that Hrs1 recruits Alp4 to SPB, promotes the formation of horse tail astral array (HAA) after
formation of pre-HAA by Mto1, attracts minus-end microtubule to SPB via γ-TuRC and stabilizes it [21]. It is notable that all of the interacting proteins with Hrs1 (fulllength) so far identified, such as Alp4, Mto1 and Kms1,

posses coiled-coil motif, although their physiological significance remains elusive.
Mcp5/Num1 is also required for horsetail chromosome
movement [18,22]. Although in budding yeast the dynein
anchor Num1, a homolog of Mcp5, is expressed and functions during both meiosis and mitosis, Mcp5 in Sz. pombeis expressed and functions only during meiosis. Mcp5/
Num1 is located at the cell cortex and plays a role inanchoring the cortical dynein in meiotic cells [18,22]. The
repeat unit just after the coiled-coil motifs is essential forthe complete function of Mcp5/Num1 because mutants
which lack the repeat unit in Num1/Mcp5 display aberrant chromosome oscillation probably due to failure ininteraction with dynein [22]. This defect results in a
decline in recombination rate and in abnormal spore formation [18]. However, physiological roles of coiled-coilmotifs remain elusive. Function of Mcp5 during meiosis issummarized as follows. Namely, when the SPB (Fig. 3B,
blue circle) and the horsetail chromosome reach the cellcortex, the microtubules carrying the SPB and chromosome extend toward the opposite end. The dynein complex (Fig. 3B, green triangle) on the end of thesemicrotubules is then trapped and anchored by Mcp5 (redshapes). This may stimulate minus-end motor activity
which leads to the cortical sliding of the microtubules.
Subsequently, the nucleus turns and is pulled towards the
opposite side of the cell. When SPB arrives at the cell cortex where dynein is anchored by Mcp5, the pulling force
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Tht1(577)

100 a.a.

A

Nuclear fusion in karyogamy

Meu13(216)

Tht1

Mcp7(210)
Mcp6/Hrs1(327)
Mcp5/Num1(968)
Dhc1* (4196 )B

Ssm4(670)

Meiotic recombination in horsetail phase

Eta2(569)
Sgo1(319): Coiled-coil: Nuclear localization signal: CAP-Gly domain: Pumilio-family RNA binding repeat

Meu14(335)

Meu13-Mcp7

Mcp4(355)

Mcp5/Num1

Recombinase cofactor

Mcp3(952)
Meu1(776)
Meu18(553)
Meu23(254)

Mcp6/Hrs1
Astral microtubule organizer

Sgo1

Mde4(421)
Mcp2(703)
Mcp1(661)

Regulator of microtubule dynamics

C Reductional division in meiosis I

Meu28/Mde9/Spn5 (464)

Meu6(651)

Dynein anchor

Dhc1-Ssm4

D: Cell cortex: NE and ER: Nucleus: SPB: Microtubule: Dynein/Dynactin: Maternal Chromosome: Paternal chromosome: Centromere: Forespore membrane

Mpf1(581)
Meu14

FigureThe
eachposition,
protein2
arenumberschematicallyand size depictedof the coiled-coilby grey boxes
motifs in
The position, number and size of the coiled-coil motifs ineach protein are schematically depicted by grey boxes. Additional motifs including the NLS (white box), microtubulebinding CAP-Gly domain (shaded box) and Pumilio-family
RNA binding repeat (black box) are also shown. The number
following each protein name represents the size of aminoacids of each protein. The size of Dhc1 alone is reduced to
30% of its original size. Bars, 100 amino acids.

horsetail chromosome will extend to the opposite end of
the cell.
Cytoplasmic dynein is a large multisubunit complex composed of two motor proteins, the dynein heavy chains(DHC), and several associated subunits that have been
named light, light intermediate, and intermediate chains[23]. The dynein motor complex transports various cellular cargos by walking along microtubules from its plusend to minus-end. Unlike mammalian cells, the DHC
subunit of Sz. pombe, Dhc1, that carries an ATP-dependent
minus-end directed motor activity, is expressed only during meiosis and localizes at astral microtubules and the
SPB at meiotic prophase I [10,24]. Dhc1 plays a pivotal
role in horsetail chromosome movement and homologous chromosome pairing by walking to the minus-end of
microtubules when it is anchored to the cell cortex bydynein anchor protein [10,25]. Recent observation indi-

Mcp4

Forespore formation and meiosis II

LEP protein

FigureSchematiction
of meiosis
3 representation
specific coiled-coilof the typical
proteins
subcellular localizaSchematic representation of the typical subcellular localization of meiosis specific coiled-coil proteins. (A) Tht1 is
detected at the nuclear envelope (NE) and endoplasmic
reticulum (ER) during karyogamy. (B) Mcp6/Hrs1, Meu13Mcp7 and Mcp5/Num1 localize to SPB, chromatin and cellcortex, respectively. Dhc1 and Ssm4 localize to SPB microtubule and cell cortex. (C) Sgo1 localizes to centromeres andprotects the centromeric cohesin from degradation during
anaphase I. (D) Meu14 forms a ring-shaped structure at theaperture of fore spore membrane (FSM) during the sporulation process. Mcp4 also forms a ring and is localized between
Meu14 and nucleus.

cates that the formation of bouquet arrangement of chromosomes at the telomeres, or telomere clustering, plays
an essential role in homologous chromosome pairing andthat Bqt1 and Bqt2 connect telomeres to the SPB by forming a bridge between Rap1 (a telomere protein) and Sad1
(an SPB protein) [26]. Considering that dynein light chain(dlc1), a component of dynein complex, interacts with
Kms1 (an SPB protein) [27] and that the Sad1 body (Sad1
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a kms1Δ mutant [28], it is interesting to explore if thedynein motor activity is required for telomere clustering
as a driving force [26].
The ssm4+ gene, a high copy-number suppressor gene ofmeiotic arrest encodes a meiosis specific coiled-coil protein carrying a microtubule-binding motif at its N-terminus [8]. Ssm4 (p150-Glued protein) is a component ofdynactin, a large multi-subunit complex implicated incytoplasmic dynein-driven motility, which interacts
directly with the cytoplasmic dynein complex and mediates the binding of dynein to cargo such as membranes[29]. Expression of Ssm4 is induced in response to mating
pheromone at the early stage of meiosis, when it isrequired for the horsetail movement of chromosome [24].
Like Dhc1, Ssm4 localizes at SPB and microtubule ofhorsetail phase. Loss of Ssm4 causes abnormal chromosome movement due to failure in dynein anchoring at thejunction of microtubule and cell cortex, which results infrequent generation of asci containing less than four
spores [24]. Although the direct evidence is lacking, thecoiled-coil motif in Ssm4 may be important for its fullfunction, because the coiled-coil motif of rat p150-Glued
protein plays a key role in its direct binding to the dyneinintermediate chain [30].
Eta2, also called Plx1 after multiplex transcript [31], is aputative transcriptional factor homologous to the mammalian oncogene Myb. This region of the genome, wherePlx1 mRNA is transcribed, generates variable sizes of transcripts in a meiosis specific manner [31]. Understanding
its physiological role requires further analysis.
Putative functions of middle coiled-coil genes
Studies of the physiological roles of the eleven middle
meiotic coiled-coil genes have revealed their pivotal roles
in proper meiotic progression, although the significanceof the coiled-coil motif for their functions remains elusive. For example, Sgo1 (shugoshin) cooperates with protein phosphatase 2A (Par1) to protect Rec8-containing
centromeric cohesin from degradation during meiosis I[15,32-35]. Sgo1 is also a component of the spindle
checkpoint which monitors chromosome alignment on
the mitotic spindle, and is required for sensing tension
between sister chromatids during mitosis [36]. Thus, Sgo1
provides a molecular link between sister chromatid cohesion and tension-sensing at the kinetochore-microtubuleinterface and activates the mitotic spindle checkpoint
when spindle tension is perturbed [37]. Both Asn-29 andIle-50 in the coiled-coil domain are required for full function of Sgo1 [15]. Fission yeast have a second Shugoshin
family member, Sgo2, which is not meiosis specific andcontrols docking of the passenger proteins on centromeres upon checkpoint activation in early mitotic cells[39,40]. Sgo2 promotes the localization of Aurora kinase

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complex to the pericentromeric region via interaction with
Bir1 (Survivin) component, and plays a role in correctionof erroneous attachment of kinetochores, thereby enabling tension-generating attachment.
Meu14 is dynamic during meiosis [14]. It is first observed
inside the nuclear region at prophase II, after which it
accumulates beside the two SPBs at metaphase II. Thereafter, it forms two ring-shaped structures that surrounded
the nucleus at early anaphase I. At this stage, Meu14
appears to localize at the border of the forespore membrane that later develops into spore walls at the end of
sporulation. It is then degraded at post-anaphase II. Meiotic meu14Δ cells are highly tetranucleate, form aberrant
forespore membranes, and fail to produce asci. Thus,
Meu14 participates in both nuclear division during meiosis II and in formation of the forespore membrane.
Mcp4 is required for the correct positioning of F-actin during meiosis [19]. Expression of Mcp4 is induced at thehorsetail phase and continues to be expressed throughsporulation. The subcellular localization of Mcp4 is
dynamic during meiosis, being located along side F-actin.
At metaphase II, Mcp4 assembles at the lagging face of thedividing nuclei, being sandwiched between F-actin andthe nucleus. The shape and viability of mcp4Δ spores arenormal, but they are sensitive to NaCl. F-actin, which isnormally dumbbell-shaped at anaphase II, adopts anabnormal balloon-shape in mcp4Δ cells. The coiled-coilmotif at the C-terminus of Mcp4 appears to be essential
for its full function because ectopically expressed Mcp4
lacking the C-terminal domain is aberrantly localized, losing its proper sandwiched distribution between F-actin
and the nucleus.
Mcp3 is also a meiosis-specific protein with three coiledcoil domains, whose expression profile and subcellular
localization during meiosis is similar to that of Mcp4 [19](Ohtaka et al., unpublished data; see Additional file 1).
Mcp3 is punctuate throughout the cytoplasm in Metaphase I and perinuclear during anaphase I. It thenclumps into four aggregates in anaphase II, before being
localized to the inner spore periphery at the end of meiosis (see Additional file 2). Subcellular distribution of
Mcp3-GFP is distinct from those of Golgi/endosome
structures. Mcp3 and Mcp4 only partially co-localize andappear to be tightly juxtaposed. Although the autolysis of
the mother cells to four spored asci was remarkably
delayed, the morphology and viability of mcp3Δ sporesare normal (see Additional file 3).
The expression of the middle genes Meu1, Meu18, Meu23
and Meu28/Mde9/Spn5 (a meiotic septin) are abruptly
induced during meiosis, but their function remains
unknown [12]. Meu1 mRNA is one of a dozen meiosis-

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specific transcripts that is selectively removed by binding
to the cis-acting motif of Mmi1 if it is expressed during
vegetative growth [41]. Meu1 has five coiled-coil motifs in
the middle of the molecule and a nuclear localization
sequence (NLS) at its C-terminus, suggestive of a nuclear
function. However, disruption of meu1+ gene results in
no apparent phenotype (Shigehisa et al., unpublishedobservation). Expressions of Meu1, Meu18, Meu28 and
Mde4 are regulated by Mei4, a meiosis specific transcription factor [42]. Mcp1 and Mcp2 are meiosis-specific proteins with four and one coiled-coil domains, respectively,
and whose expressions are induced at the sporulation
phase of meiosis. Nonetheless, disruption of mcp1+ andmcp2+ genes causes no sporulation defect (our unpublished observation).

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lates horsetail chromosome movement by anchoring
dynein, and the repeat unit just after the coiled-coil motifs
is essential for its complete function. Meu14 regulates
FSM formation, and a single coiled-coil motif in the central region of the molecule appears to be important for its
function. Although the significance of the coiled-coildomain in these proteins is partly known, the roles ofcoiled-coil motifs for other meiosis specific proteinsremain unclear. More experiments are required to understand their roles during the progression through meiosis.

Competing interests
The author(s) declare that they have no competing interests.

Authors' contributions
Putative functions of late genes
The function of the two late genes is also unclear. Meu6
was identified as a gene whose expression is only upregulated during meiosis [12]. Meu6 harbors one coiled-coilmotif and an NLS, which suggests that it has a nuclear
role. Mpf1 was identified through a comprehensive
screening of meiosis specific RNA binding proteins. It
contains a RNA binding motif that belongs to the PUF
family and a coiled-coil motif at its N-terminus [43].

During the late phase of meiosis in Sz. pombe, several
coiled-coil proteins are recruited to the SPBs and playindispensable roles in FSM assembly [44], although theexpression of these proteins are not necessarily specific tomeiosis. For example, Spo15, a component of the spindle
pole body containing a coiled-coil motif, plays an essential role in the initiation of spore membrane formation,although its expression is detected in both meiotic andvegetative cells [45]. Sec9, a component of the SNARE(soluble N-ethyl-maleimide-sensitive factor attachment
protein receptor) complex, is required for both vegetative
growth and normal spore formation [46]. Expression of
the sec9+ gene is strongly induced during sporulation in a
Mei4 dependent manner, although sec9+ mRNA is also
detected in vegetative cells. In sec9 mutant cells, the FSM
was properly initiated near spindle pole bodies duringmeiosis II, but the subsequent membrane extension was
severely impaired.

A.O., T.T.S., D.O. and H.N. drafted the manuscript anddesigned the figures and table. All of the authors have readand approved the final manuscript.

Additional material
Additional File 1
Expression profiles of Mcp3 during meiosis of Sz. pombe. (A) Meiotic
expression of mcp3+ as assessed by Northern blot analysis. h+/h-(CD161) and h-/h- (CD16-5) diploid cells were subjected to nitrogen starvation,
which induces CD16-1 but not CD16-5 to enter meiosis (upper panels).
The cells were collected at 2 hour intervals and the total RNAs were blotted and probed with the mcp3+ ORF, respectively (lower panels). The
RNAs were also probed with the aro3+ ORF as a loading control. (B) Meiotic expression of Mcp3-9myc protein as assessed by Western blot analysis.
The h-/h-pat1-114 mcp4+-3ha mcp3+-9myc strain was induced to enter
meiosis synchronously by a temperature shift and the cells were collected
at 30 min intervals (upper panels) for protein extraction, blotting andprobing with anti-Myc antibody (lower panels). Meu13 expression was
also analyzed by using an anti-Meu13 antibody to help identify the meiotic stage at each timepoint. The tubulin levels were also examined as aloading control. At each timepoint in (A) and (B), the frequency of cellswith one, two, three or four nuclei was determined by counting at least
Hoechst 33342-stained 200 cells under a microscope. Thus, the upper
panels show the stage of meiosis.
Click here for file
[http://www.biomedcentral.com/content/supplementary/17471028-2-14-S1.tiff]

Conclusion and perspectives
As described above, coiled-coil motifs play pivotal roles
for the proper function of the Meu13-Mcp7 complex. Meiosis specific coiled-coil proteins, Meu13 and Mcp7, form
a complex probably through an interaction with their
coiled-coil motifs and play a pivotal role in meiotic
recombination. Mcp6/Hrs1 is required for proper astral
microtubule positioning to maintain the horsetail movement of chromosomes, and all of its interacting proteins
possess coiled-coil motifs. In addition, Mcp5/Num1 regu-

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8.

Additional File 2
Subcellular localization of Mcp3 during meiosis and sporulation. (A) A
homothallic haploid cell h90mcp3+-gfp was cultured in EMM with appropriate supplements and transferred to EMM-N to induce meiosis. Cells in
different stages of meiosis were stained with Hoechst 33342 to label DNA(blue) and Mcp3-GFP (green) was monitored under a fluorescence microscope. The merge of the two channels are shown in the rightmost panels.
Bar, 10 μm. (B) FM4-64 uptake in living mcp3+-gfp meiotic cells. Cells
were cultured in EMM medium containing appropriate supplements andtransferred to EMM-N medium to induce meiosis. Meiotic cells producing
Mcp3-GFP (green) were incubated with the fluorescent dye FM4-64(red) to visualize Golgi/endosomes. It appears that Mcp3-GFP is distinct
from the Golgi/endosome structures. Bar, 10 μm. (C) Mcp3 associates
transiently with Mcp4 during meiosis. A homothallic haploid strain bearing mcp3+-9myc mcp4+-3ha was induced to enter meiosis and then fixedchemically. Cells were stained with Hoechst 33342, anti-myc, and antiHA antibodies to visualize the DNA (blue), Mcp3-9myc (green), andMcp4-3HA (red) by fluorescence microscopy, respectively. Bar, 10 μm.
Click here for file
[http://www.biomedcentral.com/content/supplementary/17471028-2-14-S2.tiff]

Additional File 3
Autolysis of mcp3Δ asci is remarkably delayed compared to wild typeasci.Description: Wild type (mcp3+/mcp3+) asci autolyse 48 hrs after
meiotic induction on malt extract (ME) plates(left panel), while almost
no mcp3Δ/mcp3Δ asci are autolysed by this time (right panel). It is only
68 hrs after meiotic induction that most of the mcp3Δ/mcp3Δ asci areliberated from asci (data not shown). The mcp3+/mcp3Δ strain shows
the intermediate phenotype (middle panel).
Click here for file
[http://www.biomedcentral.com/content/supplementary/17471028-2-14-S3.tiff]

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Acknowledgements
We thank Dr. P. Hughes for critically reading the manuscript. We also
thank Mr. Akira Shigehisa for unpublished results regarding mcp2+ andmeu1+. This work was supported in part by grants-in-aid for Scientific
Research on Priority Areas "Applied Genomics", Scientific Research (S)from the Ministry of Education, Culture, Sports, Science and Technology ofJapan to H.N. T.T.S is a JSPS postdoctoral Fellow for Research Abroad.

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