M IN I R E V I EW

The process of kinetochore assembly in yeasts

Babhrubahan Roy, Neha Varshney, Vikas Yadav & Kaustuv Sanyal

Molecular Mycology Laboratory, Molecular Biology and Genetics Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India

Correspondence: Kaustuv Sanyal, Molecular

Mycology Laboratory, Molecular Biology and

Genetics Unit, Jawaharlal Nehru Centre for

Advanced Scientific Research, Jakkur Post,

Bangalore 560 064, India. Tel: +91 80

2208 2878; fax: +91 80 2208 2766;

e-mail: sanyal@jncasr.ac.in

Received 1 September 2012; revised 29

September 2012; accepted 1 October 2012.

DOI: 10.1111/1574-6968.12019

Editor: Derek Sullivan

Keywords

centromere; evolution; kinetochore

recruitment; spindle; microtubules; yeast.

Abstract

High fidelity chromosome segregation is essential for efficient transfer of the

genetic material from the mother to daughter cells. The kinetochore (KT),

which connects the centromere DNA to the spindle apparatus, plays a pivotal

role in this process. In spite of considerable divergence in the centromere DNA

sequence, basic architecture of a KT is evolutionarily conserved from yeast to

humans. However, the identification of a large number of KT proteins paved

the way of understanding conserved and diverged regulatory steps that lead to

the formation of a multiprotein KT super-complex on the centromere DNA in

different organisms. Because it is a daunting task to summarize the entire spec-

trum of information in a minireview, we focus here on the recent understand-

ing in the process of KT assembly in three yeasts: Saccharomyces cerevisiae,

Schizosaccharomyces pombe and Candida albicans. Studies in these unicellular

organisms suggest that although the basic process of KT assembly remains the

same, the dependence of a conserved protein for its KT localization may vary

in these organisms.

Introduction

The precise transmission of the genetic information from

one generation to the next during the mitotic cell cycle

is extremely important for a eukaryotic organism. This

process involves faithful duplication of the whole genome

during S phase followed by segregation of the duplicated

genome with high fidelity during mitosis. The molecular

mechanisms that ensure equal distribution of duplicated

chromosomes in mitosis require proper assembly of a

large multiprotein complex at the centromere (CEN),

known as the kinetochore (KT). The primary function of

a KT is to attach the chromosome to the dynamic plus

ends of spindle microtubules (MTs), a crucial step in seg-

regation of chromosomes. KTs are also associated with

the formation of heterochromatin at the centromeric/

pericentric regions and maintenance of cohesion between

sister chromatids till anaphase onset (Cleveland et al.,

2003; Cheeseman & Desai, 2008). Additionally, a KT is

involved in the recruitment of the spindle assembly

checkpoint machinery that monitors the KT-MT attach-

ment and initiates signals to prevent cell cycle progression

if an error persists. Once all the chromosomes are

bi-orientated, separation of two sister chromatids marks

the onset of anaphase. Any defect in the KT structure can

disrupt KT–MT interaction that may result in an unequal

distribution of chromosomes leading to aneuploidy.

Cellular events associated with themitotic cell cycle

In metazoan cells, the nuclear envelope breaks down

during mitosis that allows KT–MT interaction to facilitate

bi-oriented chromosomes to arrange on a plane known as

the metaphase plate (Nasmyth, 2001; Guttinger et al.,

2009). In contrast, the nuclear envelope never breaks

down in budding yeasts and thus cells undergo closed

mitosis without formation of a metaphase plate (Straight

et al., 1997; Sazer, 2005; De Souza & Osmani, 2007).

Existence of a metaphase plate is unlikely in Schizosac-

charomyces pombe and Candida albicans as well. Interest-

ingly, a semi-open mitosis has been reported recently in

fission yeast Schizosaccharomyces japonicus (Aoki et al.,

2011; Yam et al., 2011). The nuclear envelope breaks

down only during anaphase in this organism. The nuclear

envelope virtually breaks down by increasing its perme-

ability during both mitosis and meiosis in S. pombe as

well (Asakawa et al., 2010, 2011). These studies indicate

FEMS Microbiol Lett && (2012) 1–11 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

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that an intermediate type of cell division takes place in

fission yeasts.

The microtubule organizing centres (MTOCs) remain

outside the nuclear envelope throughout the cell cycle in

metazoans. This allows KT–MT interaction to take place

only during mitosis. A metazoan KT is typically associ-

ated with multiple MTs (20–30 in humans; McDonald

et al., 1992). MTOCs, known as the spindle pole bodies

(SPBs) in yeast, remain attached to the nuclear envelope

throughout the cell cycle in Saccharomyces cerevisiae

(Byers & Goetsch, 1975). In addition, KTs remain

attached to the MTs throughout the cell cycle in this

organism. However, a temporary detachment of chromo-

somes from MTs occurs (for 1–2 min) at the time

of CEN DNA replication in S phase in S. cerevisiae

(Kitamura et al., 2007). After completion of CEN replica-

tion, a KT reassembles and reestablishes its attachment

with MTs. Subsequently, sister CENs precociously

separate from each other (Goshima & Yanagida, 2000;

Jaspersen & Winey, 2004; Sazer, 2005; Kitamura et al.,

2007; Tanaka & Tanaka, 2009). This transient detachment

between KT and MT may be specific in organisms (such

as S. cerevisiae and C. albicans) in which only one MT

interacts with a KT (Ding et al., 1993; Joglekar et al.,

2008; Thakur & Sanyal, 2011). Interestingly, SPBs in

S. pombe remain outside the nuclear envelope during

interphase. Following mitotic initiation, the duplicated

SPBs penetrate the nuclear membrane (Ding et al., 1997).

The KT is associated with 2–3 MTs in fission yeast

(Winey et al., 1995). Therefore, budding yeasts, fission

yeasts and metazoans exhibit obvious divergence in tim-

ing of commencement of KT–MT interaction, the num-

ber of MTs associated with a KT and the fate of the

nuclear membrane during the cell cycle.

The kinetochore structure

Early microscopy of mitotic chromosomes in human cells

revealed that the human KT is a tri-layered structure

(Brinkley & Stubblefield, 1966; McEwen et al., 2007):

inner and outer layers that are bridged by a middle layer.

Proteins that form the inner layer interact directly with

the CEN DNA, while the outer layer proteins form the

chromosomal attachment sites of the MT plus ends. Pro-

teins in the middle layer act as linkers between the inner

and outer KT (Cheeseman & Desai, 2008). However, in

unicellular organisms like yeasts, the structure of a KT

cannot be ascertained due to the small-sized cells. Immu-

nostaining of a KT protein in these organisms appears as

a single conspicuous focus of clustered KTs at nuclear

peripheral regions and close to spindle pole bodies

(Meluh et al., 1998; Takahashi et al., 2000; Sanyal &

Carbon, 2002). All KTs remain clustered together

throughout the cell cycle in S. cerevisiae (Anderson et al.,

2009; Duan et al., 2010) and C. albicans (Sanyal & Carbon,

2002; Roy et al., 2011; Thakur & Sanyal, 2011; Fig. 1a).

Clustered KTs are found in S. pombe as well except at

metaphase where multiple foci of KT proteins were

observed (Goshima et al., 1999; Tanaka et al., 2009;

Jakopec et al., 2012). Although the exact nature of KT

architecture in yeasts is uncertain, various genetic and

biochemical studies indicate the presence of functional

homologs of several KT proteins at distinct layers of a

human KT in these yeasts (Table 1). Determination of

relative positions of different proteins at the respective

KTs by ‘single molecule high-resolution colocalization’

demonstrates that axial localization of proteins at the KT

at distinct phases of mitosis in S. cerevisiae (Joglekar

et al., 2009) and humans is largely conserved (Wan et al.,

2009; Fig. 1b). However, such studies are yet to be carried

out in S. pombe and C. albicans. Nevertheless, the

difference in the cross-linking time of KT proteins of

(a)

(b)

Fig. 1. Spatial organization of different proteins/protein complexes at

the KT. (a) KTs are clustered at the nuclear periphery as demonstrated

by immunostained CENP-A/CaCse4 dot-like signals (red) on the DAPI-

stained nuclei (blue) in fixed cells of Candida albicans which were

spheroplasted and immunostained with anti-CaCse4 Ab (Sanyal &

Carbon, 2002) and DAPI. Imaging was performed with a 1009

magnification objective on a confocal laser scanning microscope (LSM

510 META, Carl Zeiss). The image was processed by ZEN 2008

software (LSM) to provide the three-dimensional view. (b) A model

showing the organization of different protein subcomplexes in a yeast

KT. Proteins are placed horizontally according to their probable 3D

location with respect to CEN DNA.

ª 2012 Federation of European Microbiological Societies FEMS Microbiol Lett && (2012) 1–11Published by Blackwell Publishing Ltd. All rights reserved

2 B. Roy et al.

C. albicans with CEN chromatin indicates a structural

similarity between C. albicans (Sanyal et al., 2004; Roy

et al., 2011; Thakur & Sanyal, 2011) and metazoans KTs.

Timing of kinetochore assembly

Dynamics of assembly of KT proteins is dissimilar in

yeasts and metazoans. In metazoans, only the CEN-spe-

cific histone H3 variant and an inner KT-associated

super-complex, commonly known as constitutive centro-

mere-associated network, remain localized at the KT

throughout the cell cycle (Foltz et al., 2006; Liu et al.,

2006; Okada et al., 2006). Localization/delocalization

dynamics of middle and outer KT proteins is specific to

stages of the cell cycle. For example, a middle KT pro-

tein and a MT interacting protein are loaded at the KT

at late interphase and delocalize from the KT during

transition of late anaphase to telophase in metazoans

(Liu et al., 2006; Cheeseman & Desai, 2008; Cheeseman

et al., 2008). In contrast, proteins from all layers of a

KT exhibit constitutive localization at the CEN in S. ce-

revisiae (Meluh et al., 1998; Goshima & Yanagida, 2000)

and C. albicans (Sanyal & Carbon, 2002; Roy et al.,

2011; Thakur & Sanyal, 2011). All the outer KT proteins

of S. pombe localize at the CEN only during mitosis

except one component, which remains localized at the

Table 1. Kinetochore proteins/protein complexes in yeasts

Layers in

KT structure KT protein Complex

Organism

H. sapiens S. cerevisiae S. pombe C. albicans

Inner Ndc10 CBF3 – Ndc10 – –

Scm3 # HJURP Scm3 Scm3 Orf19.1668*

CENP-A # CENP-A Cse4 Cnp1 CaCse4

CENP-C # CENP-C Mif2 Cnp3 CaMif2

CENP-T CENP-S/T/W/X CENP-T Cnn1 SPBC800.13/Cnp20 N. A.

CENP-W CENP-W YDR374W-A/Wip1 SPAC17G8.15 N. A.

CENP-S CENP-S YDLO86-A/Mhf1 SPBC2D10.16 N. A.

CENP-X CENP-X YDL160C-A/Mhf2 SPCC576.12C/Mhf2 N. A.

Ctf19 Ctf19 CENP-P Ctf19 Fta2 N. A.

Okp1 CENP-Q Okp1 Fta7 N. A.

Mcm21 CENP-O Mcm21 Mal2 Ctf5/Orf19.3494*

Ame1 CENP-U Ame1 Mis17 N. A.

Mis6 CENP-I Ctf3 Mis6 Orf19.5701*

Sim4 CENP-K Mcm22 Sim4 N. A.

Mis15 CENP-N Chl4 Mis15 Chl4/Orf19.6851*

Fta1 CENP-L Iml3 Fta1 N. A.

Middle Mis12 Mis12/MIND Mis12 Mtw1 Mis12 CaMtw1

Dsn1 Dsn1 Dsn1 Mis13 N. A.

Nsl1 Nsl1 Nsl1 Mis14 Orf19.6537*

Nnf1 Nnf1 Nnf1 Nnf1 Orf19.2519*

Spc105 Spc105 Blinkin Spc105 Spc7 Orf19.4557*

Kre28 Zwint1 Kre28 – N. A.

Sos7 – – Sos7 N. A.

Ndc80 Ndc80 Hec1 Ndc80 Ndc80 Ndc80

Nuf2 Nuf2 Nuf2 Nuf2 CaNuf2

Spc24 Spc24 Spc24 Spc24 Orf19.1484*

Spc25 Spc25 Spc25 Spc25 Orf19.6628*

Outer Dam1 Dam1 – Dam1 Dam1 CaDam1

Duo1 – Duo1 Duo1 CaDuo1/Orf19.1428*

Dad1 – Dad1 Dad1 CaDad1

Dad2 – Dad2 Dad2 CaDad2

Dad3 – Dad3 Dad3 CaDad3/Orf19.3871*

Dad4 – Dad4 Dad4 CaDad4/Orf19.596.2*

Spc34 – Spc34 Spc34 CaSpc34/Orf19.3788*

Ask1 – Ask1 Ask1 CaAsk1

Hsk3 – Hsk3 Hsk3 CaHsk3/Orf19.1482*

Spc19 – Spc19 Spc19 CaSpc19

*As annotated in www.candidagenome.org.

#, not applicable; N. A., not annotated; –, absent.

FEMS Microbiol Lett && (2012) 1–11 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Kinetochore assembly in yeasts 3

KT throughout the cell cycle (Liu et al., 2005; Sanchez-

Perez et al., 2005).

The organization of yeast centromeres

Organization of CENs in different fungi including several

yeast species can be classified into three categories: point,

large regional and small regional CENs (Roy & Sanyal,

2011; Sanyal, 2012). S. cerevisiae has short point CENs

(< 400 bp) with conserved DNA motifs for protein bind-

ing, and thus, they are genetically defined (Fitzgerald-

Hayes et al., 1982; Hieter et al., 1985). In contrast, S. pom-

be has longer regional CENs (� 40 kb) consisting of

repetitive as well as unique DNA elements (Clarke et al.,

1986; Nakaseko et al., 1987; Fishel et al., 1988; Takahashi

et al., 1992; Steiner et al., 1993; Baum et al., 1994; Wood

et al., 2002). C. albicans possesses small regional CENs

that span a 3- to 5-kb unique DNA sequence without any

repeat elements (Sanyal et al., 2004). Unlike point CENs,

regional CENs are epigenetically defined as they do not

possess any exclusive CEN-specific protein binding

sequence motifs (Steiner & Clarke, 1994; Baum et al.,

2006).

A series of experimental evidence gathered from (1)

in silico analysis, (2) genetic analysis of KT localization

interdependence, (3) biochemical purification of protein

complexes and (4) advanced microscopic observations

facilitate a comparative analysis of the process of KT

assembly in S. cerevisiae, S. pombe and C. albicans – each

having a distinct class of CENs as discussed above. Several

genetic and biochemical studies identified > 60 proteins

that are present at the KT in S. cerevisiae. In contrast, fewer

studies were performed on the KT proteins in C. albicans

and S. pombe. Thus, we mostly restrict this comparative

analysis to only a few KT protein families and their known

interacting partners that were studied in all three yeasts –the CENP-A, CENP-C, Mis12 and Dam1 complex. We

compare and contrast the processes that lead to KT–MT

interaction to facilitate chromosome segregation in these

organisms.

Centromeric chromatin properties inyeasts

CEN chromatin properties have been studied in different

yeasts. In S. cerevisiae, partial micrococcal nuclease

(MNase) digestion along with DNase I digestion of chro-

matin revealed that there are more distinct ladder patterns

at CEN chromatin as compared with that in bulk chroma-

tin (Bloom & Carbon, 1982). In this experiment, mapping

exact cleavage sites discovered a distinctly protected region

of 220–250 bp of CEN chromatin flanked by a highly phased

nucleosome structure with several nuclease sensitive sites.

On the other hand, S. pombe and C. albicans contain

unusual CEN chromatin. Partial MNase digestion yielded

canonical approximately 150-bp ladder patterns in bulk

chromatin, while smeary patterns were visible when

probed with core CEN regions in S. pombe (Polizzi &

Clarke, 1991; Song et al., 2008) and C. albicans (Baum

et al., 2006). Thus, CEN chromatin properties seem to be

different from canonical H3 chromatin. All CENs are

marked by a CEN-specific histone H3 variant – CENP-A.

CENP-A molecules replace histone H3 molecules either

partially or fully at the CENs in all these three yeast spe-

cies (Meluh et al., 1998; Takahashi et al., 2000; Sanyal

et al., 2004; Burrack et al., 2011). The assembled KT pro-

teins at the CEN may also confer protection against

MNase (Song et al., 2008). A recent in vitro study sug-

gested that a complex of CENP-S-T-W-X forms a unique

structure of CEN chromatin (Nishino et al., 2012). The

homologs of these proteins were identified and character-

ized in different yeasts as well (Schleiffer et al., 2011;

Smith et al., 2011; Bock et al., 2012; Fukagawa, 2012).

Incorporation of this complex that form noncanonical

nucleosomes also may contribute to the unique structure

of CEN chromatin.

The inner kinetochore

CENP-A

Although the process of KT assembly has been shown to

be species specific, a common feature of the functional

CENs is the existence of a CEN-specific histone H3 vari-

ant, CENP-A (Meluh et al., 1998; Takahashi et al., 2000;

Sanyal & Carbon, 2002). Inner KT assembly is considered

to be initiated by CENP-A deposition. CENP-A recruit-

ment can occur through multiple pathways, which

involve several genetic and epigenetic factors. Recruitment

of CENP-A takes place at different stages of the cell cycle.

It occurs during S phase and anaphase in S. cerevisiae

(Pearson et al., 2004; Shivaraju et al., 2012), at S and G2

phases in S. pombe (Chen et al., 2003; Takayama et al.,

2008) and at least in anaphase in C. albicans (Shivaraju

et al., 2012). Further experimentation is required to

investigate whether CENP-A deposits at early S phase

when the CEN DNA is replicated in C. albicans (Koren

et al., 2010). An evolutionarily conserved nonhistone

DNA-binding chaperone Scm3/HJURP is an essential

component for KT assembly. This family of proteins has

the propensity to bind to the A-T rich CEN DNA and

contains a histone chaperone domain, which is required

for Cse4/H4 deposition in vivo (Xiao et al., 2011). Scm3

is required for CENP-A deposition at the CEN both in

S. cerevisiae and S. pombe (Camahort et al., 2007;

Mizuguchi et al., 2007; Stoler et al., 2007; Pidoux et al.,

ª 2012 Federation of European Microbiological Societies FEMS Microbiol Lett && (2012) 1–11Published by Blackwell Publishing Ltd. All rights reserved

4 B. Roy et al.

2009; Williams et al., 2009). Moreover, over-expression of

Scm3 results in a reduction in Cse4 at the CEN in S. cere-

visiae (Mishra et al., 2011). Although Scm3 is required

for Cse4 localization at the CEN, but its own localization

at the CEN is independent of Cse4 in both S. cerevisiae

and S. pombe (Williams et al., 2009; Luconi et al., 2011).

Similarly, another KT protein essential for CENP-A local-

ization is CENP-C. The localization of CENP-A is depen-

dent on CENP-C in both S. pombe (Tanaka et al., 2009)

and C. albicans (Thakur & Sanyal, 2012). In addition to

these proteins, epigenetic regulation of CENP-A deposi-

tion (reviewed in Roy & Sanyal, 2011) has been demon-

strated in S. pombe (Steiner & Clarke, 1994) and

C. albicans (Baum et al., 2006).

Ndc10, a part of the point CEN-specific CBF3 complex,

has been shown to influence the recruitment of most of

the KT proteins including CENP-A in S. cerevisiae (Ortiz

et al., 1999; Russell et al., 1999; Goshima & Yanagida,

2000; He et al., 2001; Janke et al., 2001, 2002). It is not

clear that Ndc10 is required only in S. cerevisiae because

an obvious homolog is not identified in S. pombe or

C. albicans. On the other hand, Ams2 at S phase (Chen

et al., 2003) and Hip1 at G2 phase (Takayama et al., 2008)

influence CENP-A loading in S. pombe. The cell cycle

phase–specific loading of CENP-A has also been shown to

be affected by Mis6 through Sim3 in S. pombe (Takahashi

et al., 2000; Dunleavy et al., 2007). Interestingly, proteins

from the middle and outer KT affect the localization of

CENP-A in C. albicans (Roy et al., 2011; Thakur & Sanyal,

2012). The Dam1 complex, a fungal-specific outer KT

protein complex, which has no known role in CENP-A

recruitment in S. cerevisiae or in S. pombe, influences the

localization and stability of CENP-A in C. albicans

(Thakur & Sanyal, 2012).

CENP-C

Members of the evolutionarily conserved CENP-C family

contain a c. 25-amino acid-long conserved region, known

as the CENP-C box, which is essential for its KT localiza-

tion (Meluh & Koshland, 1995; Yu et al., 2000; Suzuki

et al., 2004). CENP-C localization at the KT is mediated

by CENP-A in both S. cerevisiae (Westermann et al.,

2003) and S. pombe (Tanaka et al., 2009). CENP-C

requires Mis12 for its recruitment at the KT in both

S. cerevisiae (Westermann et al., 2003) and C. albicans

(Roy et al., 2011). Ndc10 and Nnf1 influence CENP-C

localization in S. cerevisiae (Meluh & Koshland, 1997;

Collins et al., 2005). However, the dependence of CENP-

C on Nnf1 has not been studied in S. pombe and C. albi-

cans. Interestingly, subunits of the Dam1 complex are

essential for CENP-C localization at the KT in C. albicans

(Thakur & Sanyal, 2012).

The middle kinetochore

The Ndc80-MIND-Spc105 (NMS) super-complex

The yeast counterpart of the KNL1-Mis12-Ndc80 (KMN)

network, identified in higher eukaryotes, consists of the

Ndc80 complex, MIND/Mis12 complex and Spc105/Spc7

complex.

The Ndc80 complex

The requirement of CENP-A for KT localization of the

Ndc80 complex is similar in budding yeasts, S. cerevisiae

(Collins et al., 2005) and C. albicans (Burrack et al.,

2011). Moreover, Cnn1/CENP-T and Ndc10 were

reported to influence the assembly of the Ndc80 complex

in S. cerevisiae (He et al., 2001; Janke et al., 2001;

Schleiffer et al., 2011; Bock et al., 2012; Nishino et al.,

2012). Middle KT components including Mis12 and Nnf1

were shown to affect the localization of this complex at

the KT (Westermann et al., 2003). In S. pombe, depen-

dence as well as localization of the Ndc80 complex is not

well established. The Dam1 complex subunits influence

the loading of Nuf2, a constituent of the Ndc80 complex

in C. albicans (Thakur & Sanyal, 2012).

The MIND/Mis12 complex

CENP-A plays an important role in recruiting Mis12 at

the KT both in S. cerevisiae (Pinsky et al., 2003; Wester-

mann et al., 2003; Collins et al., 2005) and C. albicans

(Burrack et al., 2011; Roy et al., 2011) but Mis12 and

CENP-A are independent of each other for their KT

recruitment in S. pombe (Takahashi et al., 2000).

Ndc10 is essential for the KT localization of each of

the constituents of the MIND complex in S. cerevisiae

(Goshima & Yanagida, 2000; Nekrasov et al., 2003; Pinsky

et al., 2003). KT localization of the Mis12 complex is

independent of Spc105 in S. cerevisiae (Pagliuca et al.,

2009) but Mis12, Mis13/Dsn1 and Mis14/Nsl1 require

Spc7 and Sos7 for their KT localization in S. pombe

(Kerres et al., 2007; Pagliuca et al., 2009; Jakopec et al.,

2012). Depletion of a subunit of the Dam1 complex

affects Mis12 localization in C. albicans (Thakur &

Sanyal, 2012).

The Spc105/Spc7 complex

The Spc105 complex of S. cerevisiae consists of two

subunits, which are Spc105 and Kre28. Ndc10 influences

KT recruitment of both the components of this complex

(Nekrasov et al., 2003; Pagliuca et al., 2009). The recruit-

ment of Spc105 at the KT is independent of the MIND

FEMS Microbiol Lett && (2012) 1–11 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Kinetochore assembly in yeasts 5

and Ndc80 complex in S. cerevisiae (Pagliuca et al.,

2009).

However, Spc7/Spc105 forms complex with Sos7, which

has been identified recently as a KT protein in fission

yeast S. pombe (Jakopec et al., 2012). Spc7 and Sos7 are

interdependent for their KT localization (Jakopec et al.,

2012). Both the proteins are dependent on Mis12 for

their loading at the KT (Kerres et al., 2007; Jakopec et al.,

2012).

The outer kinetochore

The Dam1 complex

The Dam1 complex is essential for cell viability and local-

ized at the KT throughout cell cycle in both budding

yeasts, S. cerevisiae (Hofmann et al., 1998; Cheeseman

et al., 2001a, b; Enquist-Newman et al., 2001) and

C. albicans (Burrack et al., 2011; Thakur & Sanyal, 2011).

CENP-A influences the KT recruitment of this complex

in both the budding yeasts (Collins et al., 2005; Burrack

et al., 2011).

In contrast to budding yeasts, the Dam1 complex is

nonessential for cell viability in fission yeast S. pombe.

Moreover, except Dad1, other subunits of this complex

localize at the KT only during mitosis in S. pombe (Liu

et al., 2005; Sanchez-Perez et al., 2005). The recruitment

of the Dam1 complex is affected by Ndc10, Mis12 and

Ndc80 in S. cerevisiae (He et al., 2001; Li et al., 2002;

Scharfenberger et al., 2003; Collins et al., 2005; Pagliuca

et al., 2009), whereas localization of the Dam1 complex is

controlled by the Mis6 complex proteins in S. pombe (Liu

et al., 2005; Sanchez-Perez et al., 2005).

Moving forward

In this review, we compared the process and sequence of

events during KT assembly in three different ascomyce-

tous yeasts, each carrying a specific type of CEN. While

similarities and differences in KT assembly in these

organisms are evident, some key questions need to be

experimentally addressed.

Ndc10 is the key determinant in KT assembly in

S. cerevisiae. Is there a functional homolog of Ndc10 in

organisms (such as C. albicans and S. pombe) possessing

sequence-independent regional CENs? The requirement

of Scm3 for loading of CENP-A is found to be similar

in S. cerevisiae and S. pombe but not yet studied in

C. albicans. The localization dependence between Ndc80

and CENP-A has been examined in S. cerevisiae and

C. albicans but not in S. pombe. The roles of an inner

KT protein Mis6/Ctf3 and a middle KT protein Spc105/

Spc7 in KT assembly have been studied in S. cerevisiae

and S. pombe. The identification and characterization of

the functional homologs of these proteins in C. albicans

will improve our knowledge of KT assembly in this

yeast.

The requirement of the Dam1 complex for assembly of

a KT also differs between two budding yeasts, S. cerevisi-

ae and C. albicans. The Dam1 complex requires compo-

nents of inner and middle KT for its KT localization in

S. cerevisiae but not vice versa. In contrast, depletion of

the Dam1 complex results in the disruption of KT archi-

tecture and destabilization of CENP-A in C. albicans

(Thakur & Sanyal, 2012). What will be the consequence

of the Dam1 complex depletion on KT architecture and

stability of KT proteins in S. cerevisiae and S. pombe? In

S. cerevisiae, Dam1 can form MT attachment site if it is

targeted by tethering to an ectopic noncentromeric DNA

sequence (Kiermaier et al., 2009; Lacefield et al., 2009). It

will also be interesting to study what happens if Dam1 is

targeted to such an ectopic location in S. pombe or

C. albicans where the CEN formation is epigenetically

regulated.

Points to ponder

It is important to note that the localization dependence

studies were not performed uniformly as the sensitivity of

quantitative measurement techniques improved signifi-

cantly over the years. Moreover, the methods used to

assay KT localization dependence are sometimes not

mentioned clearly, and in many occasions, the methods

are rather qualitative than quantitative. For example, the

CENP-A independent localization of Mis12 at the CEN in

fission yeast has been claimed based on an experiment

that was not shown (Takahashi et al., 2000). Unfortu-

nately, this information was cited in several subsequent

publications. This unconfirmed observation was some-

times even considered as a variant feature of fission yeast.

Similar observations have been reported in localization

dependence studies performed in other organisms as well

(Cheeseman et al., 2004; Przewloka et al., 2007). These

questions should be readdressed with the help of more

sensitive assays in uniform experimental conditions in a

variety of model systems. The outcome of these experi-

ments will help us to precisely compare and contrast the

KT structure and its function across species. The con-

trasting results of an identical question can occur due to

the differences in experimental conditions or measure-

ment techniques. For an example, localization dependence

of Dsn1 on Mtw1 in S. cerevisiae is contradictory in two

reports (De Wulf et al., 2003; Pinsky et al., 2003). More

quantitative assays to determine the actual scenario

are required in such cases to resolve these apparent

discrepancies.

ª 2012 Federation of European Microbiological Societies FEMS Microbiol Lett && (2012) 1–11Published by Blackwell Publishing Ltd. All rights reserved

6 B. Roy et al.

Concluding remarks

It is evident that although most of the proteins assemble at

the CEN are functionally conserved across species, the CEN

DNA is diverged even in closely related species. Compara-

tive genomic analyses in different yeasts revealed that the

CEN DNA is hyper-variable even in closely related species

(Bensasson et al., 2008; Padmanabhan et al., 2008; Rhind

et al., 2011). The phenomenon of hyper-variability of the

DNA sequence at the CEN despite its conserved function in

chromosome segregation was previously designated as the

‘centromere paradox’ (Henikoff et al., 2001).

In this review, we analysed the similarities and differ-

ences in the process of KT assembly in yeasts. While the

(a) (b) (c)

Fig. 2. The recruitment of proteins during KT assembly in yeasts. The recruitment of various proteins and their interaction during formation of

the KT structure has been shown in (a) Saccharomyces cerevisiae, (b) Schizosaccharomyces pombe and (c) Candida albicans. Top panels show

relative presence of various KT proteins from outer to inner layers (top to bottom). Arrows indicate the localization dependence of a protein/

protein complex to another during the assembly process. Arrowheads point towards the component, which is dependent on the other protein.

Bottom panels show composition of various major KT complexes. Dotted arrows show intracomplex dependency. The double-headed arrows

indicate mutual dependency between proteins and protein complexes. The interdependence of various KT proteins shown here is based on the

data assembled from the literature – for the S. cerevisiae KT (Meluh & Koshland, 1997; Ortiz et al., 1999; Russell et al., 1999; Goshima &

Yanagida, 2000; He et al., 2001; Janke et al., 2001, 2002; Li et al., 2002; De Wulf et al., 2003; Nekrasov et al., 2003; Pinsky et al., 2003;

Scharfenberger et al., 2003; Westermann et al., 2003; Collins et al., 2005; Camahort et al., 2007; Mizuguchi et al., 2007; Pagliuca et al., 2009;

Schleiffer et al., 2011; Bock et al., 2012; Nishino et al., 2012), for the S. pombe KT (Takahashi et al., 2000; Chen et al., 2003; Hayashi et al.,

2004; Liu et al., 2005; Saitoh et al., 2005; Sanchez-Perez et al., 2005; Kerres et al., 2006, 2007; Dunleavy et al., 2007; Takayama et al., 2008;

Pidoux et al., 2009; Tanaka et al., 2009; Williams et al., 2009; Jakopec et al., 2012) and for the C. albicans KT (Burrack et al., 2011; Roy et al.,

2011; Thakur & Sanyal, 2011, 2012).

FEMS Microbiol Lett && (2012) 1–11 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Kinetochore assembly in yeasts 7

organization of a KT is conserved, there appears to be

subtle divergence in regulation of KT assembly in these

organisms. Whether this process has evolved uniquely in

different organisms to keep pace with the fast evolving

CEN DNA is not clear. If the process of KT assembly

occurs in a step-wise manner, a master regulator may

control the recruitment of most of the KT proteins. In

fact, an inner KT protein Ndc10 plays the central role in

S. cerevisiae (Fig. 2a), while the middle KT proteins –Mis6 and Spc7 – play governing roles to a great extent in

S. pombe (Fig. 2b). This process is remarkably diverged

with a complex interdependence among many essential

KT proteins from various layers in C. albicans (Fig. 2c).

Unravelling this fascinating molecular mechanism of KT

assembly in many organisms will improve our under-

standing of how the KT assembly pathways coevolved

with the CEN DNA during speciation.

Acknowledgements

We thank B. Suma (Central instrumentation facility,

Molecular Biology and Genetics Unit, Jawaharlal Nehru

Centre for Advanced Scientific Research) for confocal

microscopy and image processing. We are thankful to the

members of Sanyal laboratory for insightful comments.

We express our regret to our colleagues whose work

could not be cited due to space limitations.

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FEMS Microbiol Lett && (2012) 1–11 ª 2012 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

Kinetochore assembly in yeasts 11