Download - Wiley Online Library

RESEARCH ARTICLE
Ecm11protein of yeast Saccharomyces cerevisiae is regulated by
sumoylation during meiosis
Apolonija Bedina Zavec1, Aleksandra Comino1, Metka Lenassi2 & Radovan Komel1
1
Laboratory for Biosynthesis and Biotransformation, National Institute of Chemistry, Hajdrihova, Ljubljana, Slovenia; and 2Institute of Biochemistry,
Faculty of Medicine, University of Ljubljana, Vrazov Trg, Ljubljana, Slovenia
Correspondence: Apolonija Bedina Zavec,
Laboratory for Biosynthesis and
Biotransformation, National Institute of
Chemistry, Hajdrihova 19, SI-1000 Ljubljana,
Slovenia. Tel.: 1386 1 476 0200; fax: 1386 1
476 0300; e-mail: [email protected]
Received 15 March 2007; revised 19 July 2007;
accepted 26 July 2007.
First published online 20 September 2007.
DOI:10.1111/j.1567-1364.2007.00307.x
Editor: Ian Dawes
Keywords
SUMO; meiosis; Ecm11.
Abstract
Damaged regulation of the small ubiquitin–like modifier (SUMO) system contributes to some human diseases; therefore, it is very important to identify the
SUMO targets and to determine the function of their sumoylation. In this study, it is
shown that Ecm11 protein in Saccharomyces cerevisiae is modified by SUMO during
meiosis. It is known that Ecm11 is required in the early stages of yeast meiosis where
its function is related to DNA replication and crossing over. Here it is shown that the
level of Ecm11 protein is low in mitosis, but high in meiosis. The highest level of
Ecm11 is in the early-middle phase of sporulation. A specific site of sumoylation was
identified in Ecm11 at Lys5 and evidence is provided that sumoylation at this site
directly regulates Ecm11 function in meiosis. On the other hand, no relationship was
observed between sumoylation of Ecm11 and its role during vegetative growth. It
was shown that Ecm11 interacts with Siz2 SUMO ligase in a two-hybrid system;
although Siz2 is not essential for the Ecm11 sumoylation.
Introduction
Sumoylation is one of the covalent posttranslational modifications, such as acetylation, methylation and ubiquitylation, which plays an important role in controlling protein
function. SUMO modification affects many biological processes and is required for cell viability in yeast Saccharomyces
cerevisiae, nematodes and higher eukaryotes (Fraser et al.,
2000). Mammalian SUMO-1 is involved in a wide range of
important cellular processes: p53 and c-jun transcriptional
activation, signal transduction, inflammatory and immune
responses (Melchior, 2000), maintenance of genome integrity (Muller et al., 2001; Hickson, 2003), and recombination
(Shen et al., 1996). The SUMO protein is structurally similar
to ubiquitin and is a member of the ubiquitin-like proteins.
Sumoylation, like conjugation of all ubiquitin-like proteins,
occurs as the result of the sequential action of specific
enzymes: activating enzyme (E1), conjugating enzyme (E2)
and ligase (E3). In the processing of SUMO to react with the
target protein, SUMO is transferred from E1 to E2. E3
accelerates the rate of SUMO modification and confers
specificity and regulation of the sumoylation process.
SUMO is attached to a lysine (K) in the substrate, mostly
within the SUMO consensus sequence h-K-X-E/D, where h
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
represents a large hydrophobic residue (usually L, I or V)
and X is any residue (Johnson, 2004).
In S. cerevisiae the SUMO homologue is known as Smt3
and is encoded by a single essential gene, whereas in
vertebrates, four distinct paralogues (SUMO1-4) have been
identified. Because yeast cells with deleted SMT3 gene are
complemented by the human SUMO1 gene, it is assumed
that the SMT3 gene is a yeast orthologue of the human
SUMO1 (Johnson, 2004). In yeast SUMO conjugation
process, Siz1 and Siz2 SUMO ligases are required for the
majority of substrates (Johnson & Gupta, 2001), although
new E3 were found in yeast recently (Zhao & Blobel, 2005;
Cheng et al., 2006). The first SUMO-linked substrates
characterized in budding yeasts were septins (Cdc11, Cdc3,
and Shs1), but eliminating septin sumoylation has no
detectable phenotypic effect (Hochstrasser, 2002). Topoisomerase II, proliferating cell nuclear antigen (Pol30), Pds5
and Ysc4 (two proteins involved in chromosome cohesion),
and kinetochore proteins Ndc10, Bir1, Ndc80 and Cep3
were also shown to be sumoylated (Bachant et al., 2002;
Stead et al., 2003; D’Amours et al., 2004; Montpetit et al.,
2006). Recently, with a proteome-wide analysis of sumoylated proteins, many new potential sumoylated proteins
were found in budding yeast; however, in most of these
FEMS Yeast Res 8 (2008) 64–70
65
Yeast protein Ecm11 is sumoylated during meiosis
SUMO substrates, the biological role of their sumoylation is
not known.
Ecm11 is a protein of S. cerevisiae with a strong meiotic
phenotype (Zavec et al., 2004). Homozygous deletion of the
ECM11 gene causes delay in the process of meiosis, lower
efficiency of asci formation and lower spore viability. It was
concluded that Ecm11 affects meiotic DNA synthesis and
recombination. Cells with deleted ECM11 are also hypersensitive to calcofluor white, zymolase, papulacandin B and
hygromycin B, indicating defects in glucan biosynthesis
(Lussier et al., 1997). In a wide search of protein–protein
interactions, it was found that Ecm11 interacts with SUMO
(Smt3) in the two-hybrid system (Ito et al., 2000). Ecm11
protein has two lysine residues, K5 and K101, with corresponding surrounding sequence IKTE that could accept
SUMO.
In this paper it is shown that yeast Ecm11 belongs to the
group of sumoylated proteins. The SUMO-attachment site
in the Ecm11 protein and the biological role of Ecm11
sumoylation were identified.
Materials and methods
Gene disruption and site-directed mutagenesis
Genes ECM11, SIZ1 or SIZ2 were deleted and replaced by
the kanMX4 gene as described by Wach et al. (1996). Deleted
strains were generated by transforming the yeast strain with
linear PCR constructs containing the kanMX4 gene flanked
by terminal sequences homologous to the ECM11, SIZ1 or
SIZ2 gene. Replacement of the genes was verified by PCR
analysis with specific oligonucleotides.
For insertion of HA-tag (three HA) in the ECM11 gene
and for mutagenizing Lys5 and Lys101 of Ecm11 to Asn, the
‘Delitto perfetto’ system was used (Storici et al., 2001). Long
oligonucleotides of CORE cassette and oligonucleotides that
eliminate the CORE cassette were designed. Strains were
made by transformation, selection and counter selection
(URA3, kanMX4). Mutations were verified by sequencing.
Sequences of oligonucleotides are available upon request.
Calcofluor white sensitivity
The mutant strains in the logarithmic phase were plated on
various concentrations of calcofluor white in YPD and
observed for hypersensitivity relative to the isogenic wildtype strain, as described by Lussier et al. (1997).
Strains
The S. cerevisiae strains used for sporulation tests were as
follows: yC66 (MATa, can1-100, his3-11,15, leu1-12, lys2-1,
trp1-1, tyr1-2, ura3-1), yC67 (MATa, cyh2r, his3-11,15, leu1c,met13-c, trp1-1, tyr1-2, ura3-1) (G. Tevzadze); W303-1A
(MATa, ade2 can1-100r his3-11,15 leu2-3112 trp1-1 ura3-1),
W303-1B (MATa, ade2 can1-100r his3-11,15 leu2-3112 trp11 ura3-1) (YGSC, Berkely). In haploid strains, the ECM11
locus was mutated or disrupted; the mutated or disrupted
strains were then mated to give the homozygous diploid
strains. For the two-hybrid assay, yeast strain EGY48 (ura3,
his3, trp1, LexAop-leu2) was used.
Cloning experiments were performed with the Escherichia
coli strain DH5a, using standard media (Sambrook et al.,
1989).
Media and growth conditions
Strains were grown at 30 1C in standard yeast peptone
dextrose (YPD), YPA or minimal medium lacking appropriate amino acids. X-Gal plates were supplemented with
80 mg X-Gal mL 1. To induce sporulation, cells were grown
to 3 107 cells mL 1 YPA at 160 r.p.m., washed and transferred into half that volume of sporulation medium (SPM)
(0.3% potassium acetate, 0.02% raffinose) and shaken at
180 r.p.m. (Kassir & Simchen, 1991). Alternatively, sporulation was induced by replica plating patches of diploid
cells, grown overnight on YPD plates, onto 1% potassium
acetate agar plates. Sporulation efficiency was determined
microscopically by counting at least 200 cells sample 1.
FEMS Yeast Res 8 (2008) 64–70
Western immunoblotting
Cells were grown to the mid-log phase; pellets were washed
in 1 mL of 20% tricarboxylic acid (TCA) and frozen. They
were then resuspended in 1 mL of 20% TCA and lysed with
beads and three vortexings of 1 min each at 2 min intervals
on ice. The lysate was separated from beads, beads were
washed twice and the combined lysate centrifuged for
10 min at 16 000 g. The pellet was resuspended in Laemmli
buffer [50 mM Tris pH 6.8, 2% sodium dodecyl sulphate
(SDS), 10% glycerol, 2% b-mercaptoethanol] and heated at
100 1C for 3 min. Twenty microlitres of final sample was run
on 10% polyacrylamide gels. Proteins were transferred to
nitrocellulose membranes (Amersham Pharmacia Biotech).
Primary antibodies used for Western blots were anti-HA
(1 : 1000 dilution; Sigma). Blots were visualized with HRPconjugated secondary antibodies (Bio-Rad). SeeBlue Plus2
(Invitrogen) molecular weight markers were used.
Immunoprecipitation analysis
Cell lysates were prepared as described (Yaakov et al., 2003).
Protein concentration was measured by the Bradford method with Nanoquant reagent (Roth). After preclearing of
supernatants with G-sepharose (Sigma), 2 mg of rabbit antiHA or anti-Smt3 antibodies were added and the mixture
incubated at 4 1C overnight. The antibody complexes were
pulled down using G-sepharose, and washed five times with
the lysis buffer. The G-sepharose–protein complexes were
resuspended in 30 mL of 5 protein loading buffer
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
66
(Fermentas) and boiled for 5 min before loading. For the cell
lysate samples, an amount of lysate equal to the 25 mg of
protein was boiled for 5 min in the same loading buffer
before loading. Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) in 10% polyacrylamide
gel and transferred to polyvinylidine fluoride membrane
(Roth). Immunodetection with monoclonal mouse anti-HA
(sc-7392) and polyclonal anti-Smt3 (sc-11845) antibodies
(Santa Cruz Biotechnology) and polyclonal anti-mouse or
anti-goat secondary antibodies conjugated with HRP (Santa
Cruz Biotechnology) was performed using the enhanced
chemoluminescence detection system (Pierce).
Two-hybrid assay
The yeast strain EGY48, plasmids for the two-hybrid system
pEG202, pJG4-5, pSH18-34, pJK101 and pSH17-4, and
yeast genomic library ligated in pJG4-5 were provided by
Roger Brent’s laboratory (R.L. Finley and R. Brent, 1994).
LexA-ECM11 fusion derivative was generated from genomic
DNA by PCR, using modified primers, and cloned into the
XhoI and EcoRI sites of pEG202. The YNL137 gene was
amplified by PCR from genomic DNA and cloned into the
XhoI and EcoRI sites of pEG202. This construct served as a
negative control for two-hybrid interaction with positive
clones.
A.B. Zavec et al.
The yeast strain containing HA-tagged Ecm11 was grown
to the logarithmic phase and to different phases of meiosis
(Fig. 1a). Bands recognized by HA-antibodies were highly
specific, but weak, because HA-tagged ECM11 was expressed
under an endogenous promoter. During meiosis, the
amount of HA-tagged Ecm11 protein increased significantly
after 4 and 8 h in sporulation medium. The bands at
38 kDa probably represent the HA-Ecm11 protein
(34 kDa13 kDa), whereas the bands at 46 kDa probably
represent the HA–Ecm11–SUMO complex (34 kDa13
kDa111 kDa). Another band appears in samples taken after
4 h in sporulation medium, probably corresponding to a
complex of HA-Ecm11 with several covalently bound
SUMO proteins. After 12 h and especially after 24 h in
sporulation medium the amount of Ecm11 decreased. In
logarithmic phases of vegetative cell cycle the amount of
Ecm11 protein was below the level of detection.
In a previously published experiment Ecm11 had been
found to interact with Smt3 in two-hybrid system (Ito et al.,
2000). To prove the sumoylation of the Ecm11 protein with
an alternative approach, immunoprecipitation studies were
performed (Fig. 1b). With the HA reactivity, it was shown
Results
Ecm11 protein is sumoylated and its level is
significantly elevated during meiosis
The level of Ecm11 protein during the process of meiosis
was determined using Western blot analysis. Ecm11 was
epitope-tagged with three HA and located with anti-HA for
Western blot analysis (anti-Ecm11 antibodies are not available). The ECM11 gene in the haploid strains YCa and YCa
was tagged by site-directed mutagenesis. In these strains
ECM11-HA was expressed from its normal chromosomal
location. Strains were constructed with HA-tagged Ecm11 at
the C-terminus, at the N-terminus or with the epitope tag
inserted at the hydrophilic region in the middle of the gene.
The biological function of tagged Ecm11 in the vegetative
cell cycle was tested by the strain sensitivity on calcofluor
white, as described by Lussier et al. (1997). To test the
biological function of tagged Ecm11 in meiosis, the haploid
mutated strains were mated and used for tests of sporulation
efficiency, since it is known from previous studies that
strains with deleted ECM11 have a 20% lower sporulation
level (Zavec et al., 2004). The modified versions of Ecm11 on
both terminuses were nonfunctional. Ecm11 with an HA-tag
inserted in the middle of the protein was found to be
functional in vegetative cell cycle, but not in meiosis. The
last mutant was used to trace the level of Ecm11 in the cells.
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
Fig. 1. (a) Kinetics of Ecm11 formation and decay during sporulation.
HA-tagged Ecm11 protein was visualized by Western blot analysis, using
anti-HA antibodies. Cells with tagged Ecm11 in different phases of
meiosis (0, 4, 8, 12, 24 hours in sporulation medium), in logarithmic
phases of mitosis (log), and YC wild-type cells as a negative control (Ctl).
(b) Demonstration of HA-Ecm11 sumoylation during meiosis with immunoblot analysis of HA-immunoprecipitates. Whole cell lysates from
wild-type YC and isogenic mutant cells containing HA-tagged Ecm11
were immunoprecipitated using rabbit anti-HA and blotted for reactivity
with mouse anti-HA and goat anti-Smt3 antibodies. Samples taken after
4 and 8 h in sporulation medium served as controls for HA antibody
reactivity. (1) YC wild-type cells as negative control, (2) 4 h sporulation
sample, (3) 8 h sporulation sample, (4) anti-HA immunoprecipitate of 4 h
samples, (5) anti-HA immunoprecipitate of 8 h samples.
FEMS Yeast Res 8 (2008) 64–70
67
Yeast protein Ecm11 is sumoylated during meiosis
that HA-Ecm11 protein was efficiently immunoprecipitated
and with the Smt3 reactivity, the sumoylation was proven.
Beside the band at 46 kDa, a higher band 57 kDa,
probably representing the double sumoylation of HAEcm11 (34 kDa13 kDa111 kDa111 kDa), was detected
also by immunoprecipitation.
(a) 60
% of asci
50
40
30
20
Sumoylation of the N-terminus of Ecm11 is
essential for Ecm11 functioning in meiosis
The importance of Ecm11 sumoylation for progression
through meiosis was investigated by studying the effect of
mutation of predicted SUMO consensus sites in Ecm11 on
sporulation efficiency. Lysines K5 and K101 in the predicted
SUMO consensus sites were mutated to the uncharged
amino acid asparagine. Strains were constructed with mutations at K5, at K101 or at both lysines. The haploid strains
with mutated sumoylation sites were diploidized and sporulized. The kinetics and maximal level of sporulation of
mutant K101 did not differ significantly from those of the
wild-type strain. The strains mutated at K5 and at both
lysines, however, exhibited the same course of sporulation as
the strain with deleted ECM11 (Fig. 2a). Lys5 is clearly
essential for Ecm11 function in meiosis and is thus identified as the major in vivo SUMO attachment site on Ecm11
during meiosis. This was additionally confirmed by the
immunoprecipitation studies on cell extracts of wild-type
strain and K5, K101 and K5K101 mutant strains (Fig. 2b).
First proteins were concentrated attached to Smt3 by
immunoprecipitation with rabbit anti-Smt3 antibodies,
then the proteins were separated and Western blot analysis
performed using goat anti-Smt3 antibodies. In the case of
K5 and K5K101 mutant strains no signals were observed
at 45 and 56 kDa bands corresponding to sumoylated
Ecm11 protein.
10
0
K101N
WT
K5N
K101N & K5N deleted
ECM11
strain
(b)
1
3
2
4
kDa
50
anti-Smt3
Fig. 2. (a) The effect on the efficiency of asci formation of mutations of
SUMO recognition sequences in Ecm11. The wild-type strain YC (WT),
the isogenic strains bearing one or both substitution mutations at K5 and
K101 in the ECM11 gene and the strain with deleted ECM11 were
sporulated for 3 days on solid media and the percentage of asci was
determined microscopically. The averages were obtained from three
measurements. (b) Demonstration of importance of K5 residue for
Ecm11 sumoylation with immunoblot analysis of Smt3-immunoprecipitates. Whole cell lysates from wild-type YC cells (1), K101 (2), K5 (3), and
K5K101 (4) mutant cells were immunoprecipitated using rabbit antiSmt3 and blotted for reactivity with goat anti-Smt3 antibodies.
The effect of Ecm11 sumoylation during
vegetative growth
Deletion of the ECM11 gene causes cells to become calcofluor white sensitive (Lussier et al., 1997); otherwise mutant
cells have the same generation time as wild-type strains
during vegetative growth (Zavec et al., 2004). To examine
possible effects of mutation of sumoylation sites in Ecm11
on vegetative cells, the sensitivity of the mutant strains to
calcofluor white was tested. Mutation in the predicted
SUMO sequences at K5 and K101, as well as in both of
them, did not affect the calcofluor white sensitivity of the
mutant strains, while a significant defect in growth on
calcofluor white plates was detected on the strain with
deleted ECM11 (Fig. 3). The same results were obtained
with the W303 and YC mutant strains.
FEMS Yeast Res 8 (2008) 64–70
Fig. 3. The effect of substitution mutations of SUMO recognition
sequences in Ecm11 on the sensitivity to calcofluor white: (1) K5N, (2)
K101N, (3) K5N and K101N, (4) deleted ECM11, (5) wild-type W303.
Protein Ecm11 interacts with Siz2 SUMO ligase in
the two-hybrid system
In order to identify proteins interacting specifically with
Ecm11, a two-hybrid screen was performed using Ecm11 as
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
68
A.B. Zavec et al.
a bait, and for interactions were searched with the yeast
genomic library. Only one positive clone exhibited galactose-dependent activation of both reporter genes. A DNA
fragment from the positive clone was sequenced and the
nucleotide query sequence was compared with a yeast
nucleotide sequence database by BLAST. The clone contained
80% of the SIZ2 ORF.
The deletion of neither SIZ2 nor SIZ1 affects
sporulation
To determine whether Siz2 is the only SUMO ligase for
Ecm11 protein during meiosis, a strain with deleted SIZ2
gene was constructed. Sporulation did not differ from that
of the isogenic wild type. A minor effect on sporulation level
was observed with deletion of the second yeast SUMO ligase
gene, SIZ1. Only deletions of both SUMO ligase genes
simultaneously strongly diminished asci formation (Fig. 4).
Discussion
Sumoylation and the process of meiosis
Herein it was demonstrated that the Ecm11 protein in
S. cerevisiae is sumoylated during meiosis. There are many
known proteins that are sumoylated; however, only a few of
them have a role in meiosis. Only recently, it was shown that
SUMO1 has a role in mammalian spermatogenesis (Vigodner & Morris, 2005; Vigodner et al., 2006), SUMO in
budding yeast regulates synaptonemal complex formation
during meiosis (Cheng et al., 2006; Hooker & Roeder, 2006)
50
40
% of asci
Sumoylation is requisite modification for Ecm11
functioning in meiosis
Protein Ecm11 has a different function in meiosis and in
vegetative cells, and its modification with SUMO is important only in meiosis, but not during vegetative growth.
Mutation of the predicted sumoylation site K5 affects the
biological function of Ecm11 in meiosis. Mutation of K5 led
to the reduction of sporulation to the same level as in the
mutant with deleted ECM11 gene, while mutation of K101
does not affect sporulation level (Fig. 2a). Additionally,
no sumoylation was observed in the case of K5 or K5K101
mutated Ecm11 protein by immunoprecipitation (Fig 2b).
These results suggest, firstly, that the N-terminus of Ecm11
is modified by SUMO during meiosis and, secondly, that
sumoylation is essential for the biological role of Ecm11 in
meiosis.
The only known effect of deletion of ECM11 in vegetative
cells is that they become calcofluor white sensitive (Lussier
et al., 1997; Zavec et al., 2004). To examine the possible
effects of mutation of Ecm11 sumoylation sites on cells
during vegetative growth, the sensitivity of the mutant
strains was tested on calcofluor white. But no relationship
between sumoylation of Ecm11 and its role in cell wall
biogenesis was observed.
The level of Ecm11 protein and its sumoylation
status during meiosis
60
30
20
10
0
WT
Deleted SIZ2 Deleted SIZ1
Deleted
SIZ1 & SIZ2
strain
Fig. 4. The effect of deletion of SUMO ligases SIZ1 and SIZ2 on the
efficiency of asci formation of the YC isogenic strains. The strains were
allowed to sporulate for 3 days on solid media and the percentage of asci
was determined microscopically. The averages were obtained from three
measurements.
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
and has a role in meiotic recombination (Koshiyama et al.,
2006). These results represent additional confirmation that
sumoylation is important for the process of meiosis.
Western blot analysis revealed a marked increase in the level
of Ecm11 after 4 and 8 h in sporulation medium, followed
by a decrease in the later phases of meiosis (Fig. 1a). These
results are consistent with the mRNA measurement by
microarray hybridization, available on the Stanford Genome
Database. Based on the time in meiosis at which genes are
expressed in the process of meiosis (Vershon & Pierce,
2000), Ecm11 was classified into the early-middle group of
sporulation genes.
By immunoprecipitation, it was confirmed that Ecm11 is
sumoylated during meiosis and that Ecm11 interacts with
SUMO covalently. The HA antibodies recognized the HAtagged Ecm11, as well as additional 10 and 20 kDa larger
species, which correspond to the molecular mass of one or
two copies of mature SUMO (Fig. 1). Mono and double
sumoylation of Ecm11 was shown also with the anti-Smt3
reactivity on anti-HA immunoprecipitated samples. Multiple sumoylation was already observed for other sumoylated
proteins, identified in a study of posttranslational modifications in yeast proteome (Wykoff & O’Shea, 2005). It was
FEMS Yeast Res 8 (2008) 64–70
69
Yeast protein Ecm11 is sumoylated during meiosis
found that the majority of Ecm11 protein in the cell is
sumoylated during meiosis.
Siz2 is SUMO ligase for Ecm11
These results showed that Ecm11 interacts with the SUMO
ligase Siz2 in a two-hybrid system. In all organisms examined so far, single activating (E1) and conjugating (E2)
enzymes for sumoylation have been detected, but multiple
ligases (E3), indicating that the latter determine substrate
specificity (Johnson, 2004). There are more E3 ligases in
budding yeast, but Siz1 and Siz2 are required for most
SUMO conjugation, because double siz1/siz2 deletion results in the elimination of c. 99% of the SUMO conjugates
(Johnson & Gupta, 2001). It was found that the SIZ2
deletion had no effect on sporulation regarding the isogenic
wild type, so that Siz2 is clearly not essential for Ecm11
sumoylation. Deleting the SIZ1 gene also had minor effect
on sporulation regarding the isogenic wild type. Yeast
mutants lacking both SUMO ligases, Siz1 and Siz2, have an
unusual phenotype, with sectors of enlarged cells that are
arrested in the G2/M phase (Chen et al., 2005). In this
context, this result with sporulation test was as expected:
simultaneous deletion of both SUMO ligase genes, SIZ1 and
SIZ2, had much greater effect on sporulation than deletion
of ECM11, and strongly diminished asci formation (Figs 2
and 4). These results confirm those described by Johnson &
Gupta (2001). They found that mutants with deleted SIZ1 or
SIZ2 mated efficiently, and that single mutants sporulated
with the same efficiency as the isogenic wild type to produce
viable spores; however, in their experiment the double
mutant had not been tested for sporulation. It can be
surmised that Siz2 is the major ligase for Ecm11 protein,
but could be replaced by some other SUMO ligase, most
probably Siz1, when Siz2 is missing.
Conclusions
Many proteins in budding yeast have been reported to be
modified by SUMO, but the experimental evidence for the
biological role of sumoylation in most of these cases has
proven inconclusive. Ecm11 protein of S. cerevisiae is one in
the growing list of proteins modified by SUMO and it could
be shown that sumoylation is essential for its function in
meiosis.
Acknowledgements
The authors thank Dr Roger Brent for yeast strain, plasmids
and yeast genomic library. Dr Franc Avbelj and Dr Roger
Pain are thanked for helpful discussions. The work was
supported by grant P1-0104 from the Slovenian Research
Agency, which is gratefully acknowledged.
FEMS Yeast Res 8 (2008) 64–70
Dedication
This paper is dedicated to the memory of Dr Aleksandra
Comino, who contributed substantially to design of this
study. She died much too young.
References
Bachant J, Alcasabas A, Blat Y, Kleckner N & lledge SJ (2002) The
SUMO-1 isopeptidase Smt4 is linked to centromeric cohesion
through SUMO-1 modification of DNA topoisomerase II. Mol
Cell 9: 1169–1182.
Chen XL, Reindle A & Johnson ES (2005) Misregulation of 2 mm
circle copy number in a SUMO pathway mutant. Mol Cell Biol
25: 4311–4320.
Cheng CH, Lo YH, Liang SS, Ti SC, Lin FM, Yeh CH, Huang HY
& Wang TF (2006) SUMO modifications control assembly of
synaptonemal complex and polycomplex in meiosis of
Saccharomyces cerevisiae. Genes Dev 20: 2067–2081.
D’Amours D, Stegmeier F & Amon A (2004) Cdc14 and
condensin control the dissolution of cohesin-independent
chromosome linkages at repeated DNA. Cell 117: 455–469.
Finley RL Jr & Brent R (1994) Interaction mating reveals binary
and ternary connections between Drosophila cell cycle
regulators. Proc Natl Acad Sci USA 91: 12980–12984.
Fraser AG, Kamath RS, Zipperlen P, Martinez-Campos M,
Sohrmann M & Ahringer J (2000) Functional genomic analysis
of C. elegans chromosome I by systematic RNA interference.
Nature 408: 325–330.
Hickson ID (2003) RecQ helicases: caretakers of the genome. Nat
Rev Cancer 3: 169–178.
Hochstrasser M (2002) New structural clues to substrate
specificity in the ‘ubiquitin system’. Mol Cell 9: 453–454.
Hooker GW & Roeder GS (2006) A Role for SUMO in meiotic
chromosome synapsis. Curr Biol 16: 1238–1243.
Ito T, Tashiro K, Muta S, Ozawa R, Chiba T, Nishizawa M,
Yamamoto K, Kuhara S & Sakaki Y (2000) Toward a
protein–protein interaction map of the budding yeast: a
comprehensive system to examine two-hybrid interactions in
all possible combinations between the yeast proteins. Proc Natl
Acad Sci USA 97: 1143–1147.
Johnson ES (2004) Protein modification by SUMO. Annu Rev
Biochem 73: 355–382.
Johnson ES & Gupta AA (2001) An E3-like factor that promotes
SUMO conjugation to the yeast septins. Cell 106: 735–744.
Kassir Y & Simchen G (1991) Monitoring meiosis and sporulation
in Saccharomyces cerevisiae. Methods Enzymology 194: 94–110.
Koshiyama A, Hamada FN, Namekawa SH, Iwabata K, Sugawara
H, Sakamoto A, Ishizaki T & Sakaguchi K (2006) Sumoylation
of a meiosis-specific RecA homolog, Lim15/Dmc1, via
interaction with the small ubiquitin-related modifier
(SUMO)-conjugating enzyme Ubc9. FEBS J 273: 4003–4012.
Lussier M, White AM, Sheraton J et al. (1997) Large scale
identification of genes involved in cell surface biosynthesis and
architecture in Saccharomyces cerevisiae. Genetics 147:
435–450.
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
70
Melchior F (2000) SUMO – nonclassical ubiquitin. Annu Rev Cell
Dev Biol 16: 591–626.
Montpetit B, Hazbun TR, Fields S & Hieter P (2006) Sumoylation
of the budding yeast kinetochore protein Ndc10 is required for
Ndc10 spindle localization and regulation of anaphase spindle
elongation. J Cell Biol 174: 653–663.
Muller S, Hoege C, Pyrowolakis G & Jentsch S (2001) SUMO,
ubiquitin’s mysterious cousin. Nat Rev Mol Cell Biol 2:
202–210.
Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning: A
Laboratory Manual. Cold Spring Harbour Laboratory Press,
New York.
Shen ZP, Pardington-Purtymun E, Comeaux JC, Moyzis RK &
Chen DJ (1996) Associations of UBE2I with RAD52, UBL1,
p53, and RAD51 proteins in a yeast two-hybrid system.
Genomics 37: 183–186.
Stead K, Aguilar C, Hartman T, Drexel M, Meluh P & Guacci V
(2003) Pds5p regulates the maintenance of sister chromatid
cohesion and is sumoylated to promote the dissolution of
cohesion. J Cell Biol 163: 729–741.
Stelter P & Ulrich DH (2003) Control of spontaneous and
damage-induced mutagenesis by SUMO and ubiquitin
conjugation. Nature 425: 188–191.
Storici F, Lewis LK & Resnick MA (2001) In vivo site-directed
mutagenesis using oligonucleotides. Nat Biotech 19: 773–776.
Vershon AK & Pierce M (2000) Transcriptional regulation of
meiosis in yeast. Curr Opin in Cell Biol 12: 334–339.
2007 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
c
A.B. Zavec et al.
Vigodner M & Morris PL (2005) Testicular expression of
small ubiquitin-related modifier-1 (SUMO-1) supports
multiple roles in spermatogenesis: silencing of sex
chromosomes in spermatocytes, spermatid microtubule
nucleation, and nuclear reshaping. Dev Biol 282: 480–492.
Vigodner M, Ishikawa T, Schlegel PN & Morris PL (2006)
SUMO-1, human male germ cell development, and the
androgen receptor in the testis of men with normal and
abnormal spermatogenesis. Am J Physiol Endocrinol Metab
290: E1022–E1033.
Wach A (1996) PCR synthesis of marker cassettes with long
flanking homology regions for gene disruptions in
S. cerevisiae. Yeast 12: 259–265.
Wykoff DD & O’Shea EK (2005) Identification of sumoylated
proteins by systematic immunoprecipitation of the budding
yeast proteome. Mol Cell Proteomics 4: 73–83.
Yaakov G, Bell M, Hohmann S & Engelberg D (2003)
Combination of two activating mutations in one HOG1 gene
forms hyperactive enzymes that induce growth arrest. Mol Cell
Biol 23: 4826–4840.
Zavec AB, Lesnik U, Komel R & Comino A (2004) The
Saccharomyces cerevisiae gene ECM11 is a positive effector of
meiosis. FEMS Microbiol Lett 241: 193–199.
Zhao X & Blobel G (2005) A SUMO ligase is part of a nuclear
multiprotein complex that affects DNA repair and
chromosomal organization. Proc Natl Acad Sci USA 102:
4777–4782.
FEMS Yeast Res 8 (2008) 64–70