A Homologue of CROC-1 in a Ciliated Protist (Sterkiella histriomuscorum)
Testifies to the Ancient Origin of the Ubiquitin-conjugating Enzyme Variant
Family
Eduardo Villalobo,*1 Loı̈c Morin,* Clara Moch,* Rachel Lescasse,* Michelle Hanna,†
Wei Xiao,† and Anne Baroin-Tourancheau*
*Laboratoire de Biologie Cellulaire 4, Université Paris-Sud and †Department of Microbiology and Immunology, University of
Saskatchewan, Saskatoon
Resting cysts of Sterkiella histriomuscorum (Ciliophora, Oxytrichidae) have been shown to contain messenger RNA,
one of which codes for a protein significantly similar to CROC-1. CROC-1 is a human regulatory protein capable
of transactivating the promoter of c-fos and belongs to a newly characterized family of ubiquitin-conjugating enzyme
(E2) variants (UEV). We have determined the corresponding macronuclear gene sequence, which is the first protistan
UEV sequence available. The phylogenetic analysis indicates the deep separation and solid clustering of all the
UEV sequences within the E2 tree showing the ancient origin of these regulatory genes and their high structural
conservation during evolution. Furthermore, overexpression of the ciliate UEV is able to rescue the Saccharomyces
cerevisiae mms2 null mutant from killing by DNA damaging agents, implying that the UEV family proteins are
functionally conserved. In S. histriomuscorum, expression of UEV is correlated with the growth of the cells as
transcripts are present in excysting and vegetative cells but are rapidly down-regulated during starvation. These
data support the high conservation of the UEV family in eukaryotes, and a regulatory role of the gene is discussed
in relation to known functions of UEVs. This analysis may promote the search for homologues of other regulatory
genes (metazoan regulators of differentiation) in ciliates.
Introduction
Ubiquitin (Ub), a small and highly conserved eukaryotic protein of 76 amino acids, is found free or conjugated to proteins of diverse functions (Jentsch 1992a).
Acceptors may be nuclear, cytoplasmic, or membrane
proteins, and their covalent binding to Ub primarily targets them into a degradation pathway via the 26S proteasome protease complex. This Ub-mediated proteolysis pathway, which has been extensively studied in the
past decade (reviewed in Ciechanover 1994), is involved
not only in the elimination of damaged or abnormal proteins but also in the rapid removal of functional proteins
implicated in a variety of regulated cellular mechanisms.
Many proteins involved in cell cycle, transcriptional regulation, oncogenesis, and other cellular processes have
been shown to be degraded via this pathway (see Deshaies 1995; Hershko 1997 and references therein). In
some cases, conjugation of Ub to cellular proteins can
also modulate their activity without entering a proteasome-mediated proteolyse pathway (reviewed in Jentsch
1992a). The Ub signaling pathway is therefore an important system participating in many diverse cellular
processes in eukaryotes.
The ubiquitination system consists of a multienzyme complex in which the conjugating enzyme (E2 or
Ubc) catalyses the covalent transfer of Ub to the lysine
residues of the substrate proteins. This ubiquitination
1 Present address: Departamento de Microbiologia, Facultad de
Biologia, Universidad de Sevilla, Spain.
Key words: ciliates, cyst, CROC-1, excystment, Sterkiella histriomuscorum, ubiquitin-conjugating enzyme.
Address for correspondence and reprints: Anne Baroin-Tourancheau, Laboratoire de Biologie Cellulaire 4, (UPRES-A 8080), Bâtiment 444, Université Paris-Sud, 91405 Orsay Cedex, France. E-mail:
[email protected].
Mol. Biol. Evol. 19(1):39–48. 2002
q 2002 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
step follows the conjugation of Ub to a conserved cysteine residue of the E2 proteins, which is critical for the
enzyme conjugation activity. Numerous genes encoding
E2 proteins have been identified in plants, animals, and
fungi, and they constitute a large family with a broad
range of biological functions. For instance, 13 E2s have
been characterized in Saccharomyces cerevisiae which
act on different sets of substrate proteins (Jentsch 1992a,
1992b). At the amino acid level, structural conservation
of the proteins is only observed in the central domain
and especially in the vicinity of the cysteine active site.
Recently, a novel family of genes that encode E2like proteins lacking the critical cysteine residue have
been identified (Sancho et al. 1998; Xiao et al. 1998)
and designated UEV for Ub-conjugating enzyme variant. The first report (Rothofsky and Lin 1997) concerned
the isolation of a human gene called croc-1 whose product has the capacity to activate the transcription of cfos. It appears that the biochemical activity of UEV is
required for the unique Ub Lys63 chain assembly mediated by UBC13 (Hofmann and Pickart 1999), and unlike the conventional Lys48 polyubiquitination (Chau et
al. 1989), the UBC13-UEV activity does not target protein degradation (Deng et al. 2000). In addition, differential expression of human UEV genes in proliferating
cells versus differentiating cells (Ma et al. 1998; Sancho
et al. 1998), in carcinogenic cells (Ma et al. 1998; Xiao
et al. 1998), and their roles in TRAF6-mediated NFkB
kinase activation (Deng et al. 2000) suggest that UEVs
may act as regulators in cellular differentiation and proliferation. UEV homologues in other multicellular organisms (plants, animals, fungi) have been reported;
however, the cellular processes in which they participate
may be different, as in yeast S. cerevisiae MMS2 has
been clearly implicated in the error-free branch of the
RAD6 DNA postreplication repair pathway (Broom39
40
Villalobo et al.
field, Chow, and Xiao 1998; Xiao et al. 1999, 2000;
Ulrich and Jentsch 2000).
From the observed conservation of this large E2
gene family in multicellular organisms, it is clear that
their common unicellular ancestor possessed a similar
molecular system. In protists, Ub genes have been isolated in several distant groups (e.g., diplomonads, ciliates, trichomonads) but the Ub-conjugating enzyme
family is poorly documented because only four E2 sequences are available: a partial genomic E2 sequence of
the parabasalid Trichomonas vaginalis (Keeling et al.
1996) and a complete E2 macronuclear sequence of, respectively, the ciliate Paramecium tetraurelia (Okano et
al. 1996), the slime mold Dictyostelium discoideum
(Clark et al. 1997), and the trypanosomatid Leishmania
major (Ivens et al. 1998). These four sequences are
highly divergent except in the central domain of the
molecule. Recently, while searching for dormant mRNA
in cysts of the ciliate Sterkiella histriomuscorum, we
have reported the isolation of a partial cDNA whose
deduced protein is most similar to the C-terminus of a
putative UEV from the plant Picea mariana (BaroinTourancheau et al. 1999). The human CROC-1 was also
among the top 10 matches in the Blast search. The identification of a UEV gene in a ciliate shows that the inactive variants are not specific to animals, plants, and
fungi but originated in unicellular ancestors. It is particularly interesting to identify UEV members in protists
with regard to the regulatory role(s) this subfamily has
evolved in metazoans, fungi, and plants and especially
with regard to the origin and evolution of regulatory
genes known to be involved in differentiation and
proliferation.
In this study, we characterized the complete macronuclear gene ShUEV and analyzed its expression during the life cycle. A phylogenetic analysis, including a
total of 45 E2 sequences, confirms the clustering of
ShUEV with the other UEVs. The deep genetic divergence between the different classes of proteins (inactive
E2 vs. active E2) suggests an early split in their functional assignment. To investigate further the structural
and functional conservation of ShUEV, we have shown
that the ciliate protein can substitute for the yeast UEV
(MMS2) for its DNA repair function. It thus emphasizes
both structural and functional conservation of the UEV
family during evolution.
Materials and Methods
Cell Culture, Encystment, and Excystment
A stock of St. histriomuscorum (strain BA) isolated
from a fresh water pond near Brest in France, was identified by W. Foissner. These cells were cultured at room
temperature in commercial mineral water (Volvic,
France) with Tetrahymena pyriformis or Chlorogonium
sp. as a food source. Encystment of cells was induced
by starvation. Manipulation of the cysts (encystment,
storage, excystment) was performed as described by Adl
and Berger (1997).
Cloning of the Macronuclear Gene
All molecular standard techniques, including RNA
extraction, screening of libraries, sequence analysis,
Southern and Northern hybridization were described by
Sambrook, Fritsch, and Maniatis (1989), Baroin-Tourancheau et al. (1999), and Villalobo et al. (2001).
Genomic Library
Genomic DNA was extracted from vegetative cells
isolated after the medium was depleted of food organisms (prestarved cultures) using standard procedures
(Sambrook, Fritsch, and Maniatis 1989). Ten micrograms of DNA was run in a 1% (w/v) agarose gel in
TAE (40 mM Tris-acetate, 2 mM EDTA) and DNA fragments in the 0.5- to 1.5-kb size fraction were subsequently electroeluted, phenol-chloroform extracted, and
precipitated in ethanol. The pellet was resuspended in
45 ml of 10 mM Tris, 0.1 mM EDTA, pH 8, and treated
with 2.5 U Bal31 (USB) at 318C for 10 min in a 50-ml
final volume of 25 mM Tris-HCl, pH 7.5, 50 mM CaCl2,
3 mM NaCl, 50 mM MgCl2. The reaction was stopped
by the addition of 50 mM EDTA and precipitated after
two extractions with phenol-chloroform. After reparation of the extremities with Klenow enzyme (USB), the
molecules were ligated overnight into SmaI-treated
pUC18 plasmid (Pharmacia) at 168C and used to transform competent Escherichia coli JM109. Screening of
the library was performed using as a probe the croc-1
cDNA clone previously isolated from a cDNA library
of cysts of St. histriomuscorum.
Inverse PCR
Total genomic DNA of St. histriomuscorum was
circularized following the conditions previously reported (Villalobo et al. 2001). A pair of internal outwardfacing primers was designed based on the sequences of
the genomic clones. The primers, 223 bp apart, were,
respectively—Forward: 59-TCAAGAATGAAATAGTT
GCTCAC-39, Reverse: 59-CATTCCAGTCAGTGAAT
GAG-39. An aliquot (about 100 ng) of the circularized
DNA was then used as the template in a 50-ml PCR
reaction with 5 U of Taq polymerase (Q-BioTaq from
Appligene-Quantum) and 30 cycles of denaturation at
948C for 30 s, annealing at 558C for 30 s, and polymerization at 728C for 1 min. The inverse-PCR products
were run on a 1% (w/v) agarose gel in TBE (90 mM
Tris, 90 mM boric acid, 2 mM EDTA, pH 8.3) and the
fragments of the expected size were cut out of the gel
and purified using the QIAquick gel extraction kit (Qiagen) according to the manufacturer’s instructions. DNA
was further cloned in SmaI-treated pUC18 plasmid at
168C.
Primer Extension
Ten micrograms of total RNA from cysts were reverse transcribed with primers specific to the St. histriomuscorum croc-1 sequence. Reactions were carried out
using (a-32P)dATP and an unlabelled primer. The se-
Structural and Functional Conservation of UEV Genes
quences of the primers and their location relative to the
macronuclear molecule (nucleotide numbers in parentheses) are, respectively—59-TGCAAGTCCATAGGAAACAC-39(225–206) and 59-CATTCCAGTCAGTGAATGAG-39(295–276). After a 30-min reaction at
508C with Superscript II reverse transcriptase (GIBCOBRL), the reaction was inactivated during 15 min at
708C. RNA was digested for 20 min at 378C with 5 U
RNase H (Amersham). Aliquots of the labeled primer
extension products were electrophoresed on a 6% acrylamide sequencing gel. Sequencing reactions were
carried out with the (295–276) primer mentioned above
using the Sequenase Version 2 kit (Amersham-Pharmacia) and were migrated next to the primer extension
products.
RT-PCR
Total RNA (3 mg) was reverse transcribed using
3.5 mM random hexamers pdN6 (Amersham) as primers
and 200 U M-MLV reverse transcriptase (Eurobio) in a
20-ml reaction containing 1 U of RNasin (Promega).
RNA was incubated for 5 min at 658C prior to the reverse-transcription step. After 50 min at 378C, the enzyme was inactivated at 958C for 5 min. The PCR step
was subsequently carried out using 1 ml of the RT reaction, a pair of specific primers, and 1.5 U of Taq polymerase in a 25-ml reaction. The sequences of the forward and the reverse primers and their location relative
to the macronuclear molecule (nucleotide number in parentheses) are, respectively—59-TTAGATATAACATGGTTGAAT-39(76–96) and 59-TCAATACATTTCACCATCTGC-39(579–559).
Expression of ShUEV in S. cerevisiae
The expression vector pYES2 (YEp, URA3, Invitrogen) was used to clone ShUEV under the control of
the galactose-inducible GAL1 promoter. The dTs of the
four internal stop codons within the ShUEV coding sequence (respectively, positioned at 190, 268, 400, 472
nt in the macronuclear sequence) were replaced by dC
prior to cloning. These substitutions were obtained
through four rounds of amplification-circularization with
four distinct pairs of corrected outward-facing primers
(186–169, 187–205; 396–379, 397–414; 465–447, 466–
483; 229–202, 266–283) performed on a 503-bp macronuclear amplification product. The last round allows
elimination of the 36-bp internal intron from the product. A final amplification with the pair (84–157, 579–
559) was done to give the amplification product free of
the 59 end intron. The 34-meric forward oligonucleotide
corresponds to nucleotide 84 through 157 of the macronuclear sequence minus the 40-nt intron. It was finally
cloned into pYES2 at the EcoRI site and the construct
was confirmed by sequencing.
Saccharomyces cerevisiae strain FY86 (MATahis3D200 ura3–52 leu2-D1 GAL1) and its mms2::LEU2 null
mutant FY86m2L were created and cultured as previously described (Xiao et al. 1998). Yeast cells were
transformed with pYES-ShUEV by a lithium acetate
method (Ito et al. 1983). For the gradient plate assay,
41
30 ml of molten YPD or YPGal agar were mixed with
the appropriate concentration of methyl methanesulfonate (MMS) to form the bottom layer; the gradient was
created by pouring the media into tilted square Petri
dishes. After brief solidification, the Petri dish was returned flat and 30 ml of the same molten agar without
MMS was poured to form the top layer. A 0.1-ml sample
was taken from an overnight culture, mixed with 0.9 ml
of molten 1% agar and immediately imprinted onto
freshly made gradient plates via a microscope slide. Gradients plates were incubated at 308C for the time indicated before taking photographs.
Construction of the Phylogenetic Trees
Management, formatting of the sequences for the tree
building programs, and construction of distance trees were
carried out using the MUST package (Philippe 1993). The
alignment of the sequences was manually performed with
the ED program of the package. The following sequences
(with their respective accession numbers) were included in
the analyses: P. tetraurelia, D5099; L. major, CAB75567;
D. discoideum, U67838; T. vaginalis, U38786; Picea mariana, AF051209, AAC32141; Arabidopsis thaliana,
AAD21451, L19354, L19355, U33757, U33758; Pisolithus tinctorius, L38756; Lycopersicon esculentum,
CAA58111; S. cerevisiae, P53152, CAA90451(YD6652),
K02962, NPp010462, NPp010344, S28951, AAB64489,
NPp010219, AAB67357, NPp013409, NPp010377,
NPp010339, P29340; Pichia pastoris, U12511; Caenorabditis elegans, A48145, T16646; Homo sapiens,
AAC05381, U39360, U39361, XPp004699, NPp003339,
NMp014501; Drosophila melanogaster, AA246265,
CAA44453, BAA34575, AAA28309, P35128; Mus musculus, NP-033484, AAG22084, AAG22085; Gallus gallus,
L77699; African swine fever virus, NPp042834. The terminal parts of the sequences are highly variable and can
only be aligned between closely related genes. Only unambiguously aligned regions were retained for phylogenetic analyses. A distance matrix was calculated considering all amino acid differences without weighting the
transition probabilities. This matrix served as a basis for
phylogenetic reconstructions using the neighbor-joining
procedure (Saitou and Nei 1987). Evaluation of the statistical validity of the nodes was performed by applying the
bootstrap procedure (1,000 replicates) on the neighborjoining method. The alignment was also used in a parsimony analysis. Parsimony analysis was done using PAUP
3.0 program for the Macintosh (Swofford, Illinois History
Survey, Champaign, Ill.). In parsimony trees, bootstrap
proportions were recorded from 100 resamplings.
Results
The characterization of several dormant mRNAs in
resting cysts of St. histriomuscorum (Baroin-Tourancheau et al. 1999) led us to isolate and identify a transcript encoding a carboxy terminal peptide of 116 amino
acids which shares 48% identity and 63% similarity with
the carboxy terminal part of the croc-1–like protein of
the plant (Picea mariana). The predicted sequence of
this transcript lacks the conserved cysteine residue of
42
Villalobo et al.
active E2. A longer 59 end transcript encoding a polypeptide of 133 aa with a scoring match of 51% identity
and 65% similarity was later isolated. We have used this
cyst cDNA as a hybridization probe to isolate the corresponding macronuclear gene.
Molecular Cloning and Sequence Analysis of ShUEV
In hypotrichous ciliates such as St. histriomuscorum (formerly known as Oxytricha trifallax), the somatic nucleus (macronucleus) is organized in tiny chromosomes ranging in size from 0.5 to 20 kb. In most
cases, each macronuclear molecule encodes a single
transcription unit flanked by its 59 and 39 noncoding
sequences and is ended by the telomeric 59-C4A4-39 direct repeats with short 39 overhangs. Probing a Southern
blot with high stringency yielded a single band at about
800 bp (data not shown). The screening of a genomic
library with this specific probe allowed us to isolate several truncated clones, all lacking 59 and 39 subtelomeric
and telomeric regions. In order to clone the missing telomeric region, we took advantage of the gene-sized DNA
molecules in using an inverse PCR-based strategy that
has been successfully applied in Histriculus cavicola
(Oxytrichidae) and in St. histriomuscorum (Perez-Romero et al. 1999; Villalobo et al. 2001). DNA molecules
covalently circularized by self ligation were amplified
using a pair of specific primers oriented toward the telomeres. A product of the expected size (550 bp), cloned
in pUC18, contained the 59 and 39 flanking regions up
to the 59-C4A4-39 terminal repeats. The deduced macronuclear molecule was obtained by assembling the sequences of the genomic and the inverse-PCR clones.
The final molecule was 787-bp long excluding both 39
terminal single stranded regions of the telomeres.
Examination of the sequence indicates a long open
reading frame starting at nucleotide 1 and ending at
TGA (position 577) which is the stop codon used in
Oxytricha species. In many ciliates, the standard stop
codons UAA and UAG are sense codons and encode
glutamine. The genetic code of St. histriomuscorum also
obeys this deviant rule because in the sequences of btubulin, several highly conserved glutamine residues are
encoded by TAA and TAG (data not shown). The nucleotide sequence matches that of the cDNA and shows
the presence of a small 36 bp intron in the macronuclear
gene (230–265 nt). Its A 1 T content (nearly 90%), with
the characteristic starting (GT) and ending (AG) dinucleotides is significantly much higher than the 60% A
1 T content of the surrounding coding region. Typically,
in hypotrichs, introns, 59 leader, and 39 trailer noncoding
DNA regions are AT rich (.70%) (Prescott 1994; Hoffman et al. 1995). This difference in A 1 T content renders the flanking domains easily distinguishable from
putative ORFs. Downstream of the TGA stop codon of
the predicted UEV amino acid sequence, the 39 trailer
of the macronuclear gene is indeed nearly 90% A 1 T.
Similarly, the 59 leader region is also recognized with a
high A 1 T content and putative TATA boxes. However,
the identification of its precise boundary is unexpectedly
difficult because no in-frame ATG initiation codon is
FIG. 1.—Determination of introns and the transcription start site.
A, The bands in lanes 1 and 2 correspond to the primer extension
signals of total RNA from cysts using the two specific primers located
(295–276) and (225–206) relative to the gene nucleotide sequence. The
sequence ladder on the right corresponds to the sequence of the inverse-PCR derived genomic clone obtained with primer (295–276).
Numbers on the right are the nucleotide distance from the primer. B,
1RT: RT-PCR amplification product from total RNA obtained with
the pair of primers (76–96 and 579–559) relative to the gene nucleotide
sequence. -RT: A control RNA sample treated in the same way but
without the reverse transcriptase enzyme. MW: DNA markers.
found upstream of the partial cDNA sequence. The first
in-frame ATG codon is located within the intron at position 244. To rule out any amplification or sequencing
artifacts, clones obtained from two different inversePCR experiments using different sets of primers have
been sequenced and were found to be identical. This
likely suggests the presence of a small intron very close
to the 59 end of the coding sequence. In searching for
such a putative intron, we have found that 11 nucleotides downstream of the ATG (87 nt, in-frame 13), the
nucleotide sequence (59-CAG/GTAAG-39) displays the
exact 59 junction sequence of a known intron in O. nova
(Hicke et al. 1990). Forty nucleotides downstream, the
corresponding exact (59-TAG/G-39) 39 junction sequence strongly supports the existence of a 59 end intron. Its A 1 T content (75%) is lower than the first
detected one but is still compatible with the high A 1
T content of the noncoding sequences in ciliates. Additional support for this second intron comes from the
determination of the transcription initiation site.
Mapping Introns and the Transcriptional Initiation Site
of ShUEV
Total RNA isolated from cysts was reverse-transcribed separately with two different primers bracketing
the 36-bp internal intron (see Materials and Methods).
In the cDNA, these two primers are expected to be 35
bp apart because of the removal of the intron. Figure 1A
displays the autoradiograph of an aliquot of the reverse-
Structural and Functional Conservation of UEV Genes
transcription reactions run in polyacrylamide gel. Firstly, the difference in length between the extension products was found to exactly equal the 35-bp distance between primers on the transcript. The splicing of the 36bp intron is thus confirmed, which excludes the ATG
within it (244 nt) to be the translation initiation site.
Secondly, two distinct experiments gave identical 59
ends. Assuming the existence of an additional 59-end
intron, we mapped the putative transcription start at a T
residue in the 59-ATAA-39 sequence 40 nt from the telomere. These features are in good agreement with recent
compilations of transcription initiation sites in hypotrichs (Ghosh et al. 1994; Hoffman et al. 1995) in which
it turns out that although no conserved promoter sequences (strict TATA boxes) are found, the vicinity of
the sites are high A 1 T regions with transcription starts
frequently observed close to the telomeres. From this
data, we designed a convenient primer in the putative
short (25 nt) 59-nontranslated region of the mRNA for
RT-PCR experiments to definitely solve the hypothesis
of the second intron.
The RT-PCR experiments performed on total RNA
from vegetative cells (or cysts) confirmed the removal
of the putative 59-end intron. Figure 1B shows that the
PCR product corresponds to a single band about 430 bp
in length when analyzed by agarose gel electrophoresis.
This amplicon was cloned in the pGEM-T plasmid and
its sequence was found to perfectly match the predicted
sequence of the three ligated exons. The entire gene
product is therefore 138 amino acids long with four of
eight glutamine residues encoded by the TAA or TAG
universal termination codons.
ShUEV Is a Structural and Functional Homologue of
UEV Family Proteins
The full-length ShUEV sequence was compared to
some E2 and UEV proteins taken from public databases
and the alignment is shown in figure 2. Except for the
extensions of the UEV proteins of human and Drosophila, all UEVs are of a very similar size (140 aa). The
sequence of St. histriomuscorum is readily aligned with
UEV proteins. By comparison, the alignment with other
E2s is limited to the central domain. As shown in figure
2, this conserved core between E2 and UEV is about 80
amino acids long.
In order to address whether ShUEV has conserved
the UEV activity throughout evolution, we wanted to
see if the ShUEV gene can replace one of its homologues in other organisms. One of the well-defined UEV
functions is the role of S. cerevisiae MMS2 in error-free
DNA postreplication repair (Broomfield, Chow, and
Xiao 1998; Xiao et al. 1999; Ulrich and Jentsch 2000;
Xiao et al. 2000). The mms2 null mutant enhances sensitivity to killing by DNA damaging agents such as
MMS and UV; expression of ShUEV rescued the mms2
cells from killing by MMS (fig. 3) and UV (data not
shown). As this functional complementation is observed
only on the galactose (inducing) plate (fig. 3B) but not
on the glucose (repressing) plate (fig. 3C), it confirms
43
FIG. 2.—Amino acid sequence alignment of St. histriomuscorum
protein with several UEV and E2 proteins. The UEV proteins correspond to the human proteins (UEV1A and hMMS2, an homologue of
yeast MMS2) and UEV related proteins from mouse, fly, yeast
(MMS2), and plant. The aligned E2 enzyme proteins are the yeast
(RAD6) and its homologue in Drosophila (DHR6) and the yeast
UBC13 and its human homologue (hUBC13). The active cysteine residue lacking in UEV proteins is in bold and shadowed. To the right is
the amino acid numbering of each sequence. H. sapiens: Homo sapiens; D. melanogaster: Drosophila melanogaster; A. thaliana: Arabidopsis thaliana; S. histriomuscorum: Sterkiella histriomuscorum; M.
musculus: Mus musculus; S. cerevisiae: Saccharomyces cerevisiae.
Star represents missing amino acids and dash amino acid identity.
that the effect is caused by the galactose-induced expression of ShUEV.
Phylogenetic Analyses Reveal Ancient Origin of the
UEV Family
The analysis includes a total panel of 45 proteins
comprising 33 active E2 enzymes and 12 UEV sequences from 13 and 9 different species, respectively. The
sequences are an average of 170 amino acids long, but
some are longer because of N- or C-terminal extensions.
Within some subfamilies, the high level of amino acid
conservation throughout the entire protein allows a
straightforward alignment. For the global tree, however,
the alignment requires a few insertion gaps and only the
conserved central domain is taken into account in the
analysis. The boundaries of the deleted characters are
indicated in the legend of the amino acid distance tree
shown in figure 4. The tree is resolved into several solid
clusters with bootstrap values (in %) close to or higher
than 60, one of which contains all the CROC-1 related
sequences. Indeed, the distance and parsimony treat-
44
Villalobo et al.
FIG. 3.—Functional complementation of yeast mms2 null mutation by ShUEV. Wild-type FY86 (lane 1), its isogenic mms2 null mutant FY86m2L (lane 2) and transformed pYES-ShUEV (lane 3) were
grown overnight and printed on to various plates. The plates were
photographed after a 41-h incubation at 308C. (A) Control YPGal plate
without MMS; (B) YPGal gradient plate containing 0.025% MMS; and
(C) YPD gradient plate containing 0.025% MMS. The arrow points to
the higher MMS concentration. Ten independent pYES-ShUEV transformants were analyzed with indistinguishable result and only one is
shown.
ments generate trees in which the inactive UEV and active E2 sequences are not scattered but clearly separated.
At the amino acid level, in addition to the lack of the
active Cys residue (see fig. 2), several signatures support
the clustering of UEV sequences especially at the Nterminus, the highly conserved peptide VPRXFRL(L/
Y)EEL. As shown in the tree, these sequences are united
and form a monophyletic cluster (the bootstrap value of
the node is 100) in which two subgroups are identified:
one branch comprising the metazoan representatives and
the other branch associating the plant, ciliate, and yeast
sequences. With both the phylogenetic tree construction
methods used (neighbor-joining, parsimony), the association of the sequence of St. histriomuscorum to MMS2
and plant sequences is systematically observed (bootstrap values are, respectively, 87 and 55). The relative
positions of the ciliate, yeast, and plant sequences are,
however, not robust and the branch forms a trifurcation.
This part of the tree remains unresolved even when larg-
er numbers of amino acids are analyzed. In analyses
restricted to the UEV subfamily, the dendrograms obtained from the entire protein sequences do not resolve
this node (of the 128 UEV aligned amino acids, there
are a total of 109 variable sites and 72 informative sites
for the parsimony analysis).
The 33 other E2 sequences are split into a set of
deeply diverging groups of sequences, all of which are
equally distant from the UEV sequences. The distance
tree basically recovered the clusters obtained by Keeling
et al. (1996) in an E2 phylogeny constructed from a
larger data set of 50 active E2 enzyme sequences. In
parsimony, the 84 informative sites support most (but
not all) of the resolved lineages of the distance tree. The
major resolved groups comprise the RAD6 protein of S.
cerevisiae and its homologues (Mus, Drosophila),
UBC8 of S. cerevisiae and its homologues, UBC7 of S.
cerevisiae associated with CDC34, two E2 proteins of
Arabidopsis (UBC13, UBC7), and an E2 sequence of
the African swine fever virus. This latter group is consistently recovered whatever the tree construction method used. A second clear assemblage weakly associates
several distinct and separated families. Among these
groups, one corresponds to UBC4 and UBC5 of S. cerevisiae and their homologues. A Dictyostelium Ubc is
associated with this family (bootstrap value above 55%).
Another one groups the UBC13 sequence of Saccharomyces and its homologues. Whatever the treatment,
the sequence of Paramecium is not affiliated with any
other sequence of the data set, whereas in the distance
tree, the sequence of Trichomonas is found consistently
associated with the sequences of Saccharomyces
(UBC12) (bootstrap value near 80).
ShUEV Expression Is Specific to Growing Cells
In the absence of food, St. histriomuscorum form
resting dedifferentiated cysts which can transform back
to the vegetative form when food is restored. As previously observed (Baroin-Tourancheau et al. 1999),
croc-1–like transcripts are found in cysts and in vegetative cells, but the initially reported small difference in
size between cyst and vegetative transcripts is not confirmed here: the same length (about 600 nt) is observed
on Northern blots in both stages (fig. 5A). We note that
a faint signal corresponding to a 3-kb band can be observed in cysts several weeks old (fig. 5A), but not in
newly formed cysts (fig. 5B). As the cells undergo profound morphological transformations and metabolic
changes during the encystment-excystment cycle, we
have also examined on Northern blots the expression of
ShUEV in three different physiological states, namely,
→
FIG. 4.—Distance-matrix–based phylogenetic tree of Ub-conjugating enzymes amino acid sequences. The domain analyzed corresponds to
residues 51–102 and 105–138 of St. histriomuscorum in figure 2. All amino acid differences are taken into account in the distance matrix. Of
the 89 aligned amino acid sites, there are a total of 87 variable sites and 84 informative sites for the parsimony analysis. The tree is unrooted.
A consensus tree displaying identical groupings has been obtained after 1,000 bootstrap resamplings. The bootstrap percentages are indicated
above the branches. Scale bar equals 2.9% of site substitutions. The parsimony analysis using the same alignment yielded 6 trees and a strict
consensus was calculated. The groupings present in this 50% majority rule consensus tree are indicated with the bootstrap values recorded for
100 resamplings below the branches. The E2 and UEV clusters are indicated by brackets.
Structural and Functional Conservation of UEV Genes
45
46
Villalobo et al.
FIG. 5.—Expression analysis of the UEV gene. Northern blots of
DNase-treated total RNA isolated from (A) vegetative cells and several
weeks-old cysts and from (B) starved cells, newly formed cysts, and
excysting cells. A negative-control hybridization with RNA from Tetrahymena is also shown in (A). The membranes in the top panel (hybridized with a ShUEV cDNA probe) were stripped and rehybridized
with an 18S rDNA probe to control for the amount of RNA loaded
(bottom panels). RNA markers are shown on the left. The arrow in
(A) indicates the high molecular weight faint band observed when cysts
are kept dormant for at least several weeks.
starved cells, cysts, and excysting cells. As shown in
figure 5B, expression of ShUEV is drastically diminished
in starved cells. The down-regulation appears to be rapid
and can be observed in prestarved cells (i.e., fed cells
collected in the absence of food, data not shown). In
contrast, there is no apparent alteration in the accumulation of transcripts during the process of excystment.
Discussion
The fact that E2s and their closely related inactive
counterparts participate in numerous fundamental cellular processes strongly suggests that the Ub-targeting
device represents an ancient and key system in the evolutionary history of eukaryotes. As a great part of the
eukaryotic evolution was spent developing and diversifying the unicellular status, it should be possible to trace
the E2 component back to protists. Here we have characterized a new member of the ciliate E2 gene family
that very interestingly can be assigned to a specific inactive variant subfamily.
Along with the sequence of Paramecium (Okano
et al. 1996), ShUEV is the second available ciliate member of the E2 gene family, and it encodes the first inactive E2 protein isolated in protists to be positioned in
the Ub-conjugating tree. Our analysis re-examines the
data previously obtained by Keeling et al. (1996) and
brings new information. E2 enzymes are encoded by a
multigenic family, and the intermingling of protist and
nonprotist species in the global topology of the tree supports an early origin of the different E2 copies in the
evolutionary history of the eukaryotes. The molecular
tree shows several robust monophyletic clusters, one of
which contains all known UEVs, including ShUEV. It
thus confirms that UEVs constitute a coherent family as
proposed in several recent studies (Sancho et al. 1998;
Xiao et al. 1998). Moreover, the sequence from a ciliated
protist in the UEV cluster implies that genes encoding
UEV are ancient, not specific to the multicellular lineages, and may have originated early in the eukaryotes.
This point is also illustrated by the fact that the E2 sequence of Paramecium, the other ciliate representative
of the data set, branches off in a distinct group of sequences in contradiction with the monophyly of the ciliates. The duplication event leading to these different
subfamilies thus precedes the divergence of the group
of ciliates from the other eukaryotes. It also confirms
that the E2 family is multigenic in ciliates. Homologous
E2 copies of St. histriomuscorum (or of other ciliate
representatives) therefore likely exist in other well-defined clusters. In St. histriomuscorum, the large distance
between the isolated UEV gene and its expected distant
E2 counterpart(s) is suggested by the fact that Southern
blot hybridization at high stringency revealed only the
croc-1–like gene at 800 bp. This is also true of the
Northern blots as the positive expression is associated
with a single type of transcript (600 bp). The faint band
corresponding to a much larger transcript could also result from an E2 distant member, although the reason for
its absence in newly formed cysts is not clear.
At the functional level, the deep separation of the
UEV from the other members of the E2 family suggests
an early and specific functional assignment to the E2
variant subfamily. Evidence exists for the functional
conservation from yeast to humans. Indeed, a conserved
core functional structure between mammalian and yeast
proteins has been highlighted by showing that the human CROC-1B core domain (after N-terminal truncation) and hMMS2 complement the yeast mms2 (Xiao et
al. 1998). Conversely, yeast MMS2 is able to mediate
the activation of human c-fos promoter (Xiao et al.
1998). Given the large genetic distance between protists
and multicellular organisms, the existence of a ciliate
homologue (as distantly related to other UEV members
as its yeast homologue) could extend this functional
conservation to many protistan phyla and throughout the
entire eukaryotic kingdom. Our results showing that
ShUEV corrects the DNA repair defect of S. cerevisiae
mms2 null mutant cells bring strong support to a conserved mode of action of all UEV family proteins during
evolution.
It is now clear that yeast and mammalian UEVs act
as positive regulators of a specific E2 enzyme UBC13,
through heterodimer formation leading to a unique polyubiquitin chain assembly process (Hofmann and Pickart 1999). The fact that expression of ShUEV complements the yeast mms2 mutant indicates that ShUEV
must interact with yeast UBC13 in the host cell. On the
other hand, despite massive overexpression of ShUEV,
it does not restore mms2 mutants to the wild type level,
which is in contrast to the overexpression of human
(Xiao et al. 1998) and mouse (Franko, Ashley, and Xiao
Structural and Functional Conservation of UEV Genes
2001) UEV genes in yeast cells. Although the compromised activity of ShUEV in heterologous cells may be
attributed to several reasons, we consider a reduced level
of interaction between ShUEV and yeast UBC13 to be
the most likely scenario. The crystal structure of
hMMS2-hUBC13 complex indicates that the two proteins interact through several hydrophobic bonds involved in a number of amino acid residues (Moraes et
al. 2001). ShUEV contains up to 15 nonconserved residues that are identical among yeast, human, and mouse
UEVs, some of which may contribute to the interface.
In St. histriomuscorum, we do not know yet if ShUEV
interacts with a UBC13-like partner; however, it is striking to note that UEV and UBC13 clusters are by far the
best-resolved parts of the tree. All known UBC13 homologues belong to a solid branch with which a protistan representative (L. major) is strongly affiliated. This
raises the possibility that in this highly conserved E2
subfamily, ciliate homologues (notably St. histriomuscorum) exist and that the two deeply separated families
(UBC13 and UEV) coevolved to maintain the functional
association to each other.
Human cells contain at least two UEV family genes
and three proteins (hMMS2, CROC-1A, and CROC-1B)
and they appear to have distinct cellular activities.
Whereas CROC-1 is implicated in differentiation and
proliferation (Rothofsky and Lin 1997; Ma et al. 1998;
Sancho et al. 1998; Deng et al. 2000), hMMS2 appears
to be specific for DNA postreplication repair (Li et al.
2001). Although our complementation data imply that
ShUEV is able to support UBC13-mediated Lys63 polyubiquitination in yeast cells and plays a role in maintaining genome integrity, we do not know to what extent
the ubiquitination process is similar between yeast and
St. histriomuscorum. Nevertheless, it is interesting to
note that there is a differential expression of ShUEV in
dividing (vegetative) versus nondividing (starved) population. In hypotrichs, starvation is accompanied by a
general decrease of transcriptional activity, leading to a
differential down-regulation of many transcripts depending on their relative stability (Brandt and Klein 1995).
The observed rapid down-regulation of ShUEV is therefore not a general feature of the mRNA population and
implies that this transcript is rapidly destroyed in nongrowing cells. Its up-regulation in cysts, excysting cells
and vegetative cells agrees with our recent proposal that
many transcripts in cysts are stored to act later during
excystment and vegetative growth of the cells (Villalobo
et al. 2001). This differential pattern of expression could
suggest a contribution of the ciliate UEV gene in the
regulatory processes involved in the control of cell cycle
and cellular growth. Experimental work aimed to disrupt
the gene function in St. histriomuscorum is under way.
The nucleotide sequence accession numbers are
AF139024 and AF382210.
Acknowledgments
The authors thank Alison Shelmerdine for her help
with the preparation of the manuscript. This work was
supported by the Centre National de la Recherche Scien-
47
tifique and the Université Paris-Sud to A.B.-T. and The
Canadian Institutes of Health Research operating grant
MOP-38104 to W.X. E.V. was the recipient of a Marie
Curie post-doctoral fellowship from the EC (contract
number ERBFMBICT 983187).
LITERATURE CITED
ADL, S. M., and J. D. BERGER. 1997. Timing of life cycle
morphogenesis in synchronous samples of Sterkiella histriomuscorum I. The vegetative cell cycle. Eur. J. Protistol.
33:99–109.
BAROIN-TOURANCHEAU, A., L. MORIN, T. YANG, and R. PERASSO. 1999. Messenger RNA in dormant cells of Sterkiella
histriomuscorum (Oxytrichidae): identification of putative
regulatory gene transcripts. Protist 150:137–147.
BRANDT, A., and A. KLEIN. 1995. Transcription rates and transcript stabilities of macronuclear genes in vegetative Euplotes crassus cells. J. Eukaryot. Microbiol. 42:691–696.
BROOMFIELD, S., B. L. CHOW, and W. XIAO. 1998. MMS2,
encoding a ubiquitin-conjugating enzyme-like protein, is a
member of the yeast error-free postreplication repair pathway. Proc. Natl. Acad. Sci. USA 95:5678–5683.
CHAU, V., J. W. TOBIAS, A. BACHMAIR, D. MARRIOTT, D. J.
ECKER, D. K. GONDA, and A. VARSHAVSKY. 1989. A multiubiquitin chain is confined to specific lysine in a targeted
short-lived protein. Science 243:1576–1583.
CIECHANOVER, A. 1994. The ubiquitin-proteasome proteolytic
pathway. Cell 79:13–21.
CLARK, A., A. NOMURA, S. MOHANTY, and R. A. FIRTEL. 1997.
A ubiquitin-conjugating enzyme is essential for developmental transitions in Dictyostelium. Mol. Biol. Cell 8:1989–
2002.
DENG, L., C. WANG, E. SPENCER, L. YANG, A. BRAUN, J. YOU,
C. SLAUGHTER, C. PICKART, and Z. J. CHEN. 2000. Activation of the IkappaB kinase complex by TRAF6 requires
a dimeric ubiquitin-conjugating enzyme complex and a
unique polyubiquitin chain. Cell 103:351–361.
DESHAIES, R. J. 1995. Make it or break it: the role of ubiquitindependent proteolysis in cellular regulation. Trends Cell
Biol. 5:428–434.
FRANKO, J., C. ASHLEY, and W. XIAO. 2001. Molecular cloning
and functional characterization of two murine cDNAs
which encode Ubc variants involved in DNA repair and
mutagenesis. Biochem. Biophys. Acta 1519:70–77.
GHOSH, S., J. W. JARACZEWSKI, L. A. KLOBUTCHER, and C. L.
JAHN. 1994. Characterization of transcription initiation,
translation initiation, and poly(A) addition sites in the genesized macronuclear DNA molecules of Euplotes. Nucleic
Acids Res. 22:214–221.
HERSHKO, A. 1997. Roles of ubiquitin-mediated proteolysis in
cell cycle control. Curr. Opin. Cell Biol. 9:788–799.
HICKE, B. J., D. W. CELANDER, G. H. MACDONALD, C. M.
PRICE, and T. R. CECH. 1990. Two versions of the gene
encoding the 41-kilodalton subunit of the telomere binding
protein of Oxytricha nova. Proc. Natl. Acad. Sci. USA 87:
1481–1485.
HOFFMAN, D. C., R. C. ANDERSON, M. L. DUBOIS, and D. M.
PRESCOTT. 1995. Macronuclear gene-sized molecules of hypotrichs. Nucleic Acids Res. 23:1279–1283.
HOFMANN, R. M., and C. M. PICKART. 1999. Noncanonical
MMS2-encoded ubiquitin-conjugating enzyme functions in
assembly of novel polyubiquitin chains for DNA repair.
Cell 96:645–653.
ITO, H., Y. FUKUDA, K. MURATA, and A. KIMURA. 1983. Transformation of intact yeast cells treated with alkali cations. J.
Bacteriol. 153:163–168.
48
Villalobo et al.
IVENS, A. C., S. M. LEWIS, A. BAGHERZADEH, L. ZHANG, H.
M. CHAN, and D. F. SMITH. 1998. A physical map of the
Leishmania major Friedlin genome. Genome Res. 8:135–
145.
JENTSCH, S. 1992a. The ubiquitin-conjugation system. Annu.
Rev. Genet. 26:179–207.
JENTSCH, S. 1992b. Ubiquitin-dependent protein degradation:
a cellular perspective. Trends Cell Biol. 2:98–103.
KEELING, P. J., A. L. DOHERTY-KIRBY, E. M. TEH, and W. F.
DOOLITTLE. 1996. Linked genes for calmodulin and E2
ubiquitin-conjugating enzyme in Trichomonas vaginalis. J.
Eukaryot. Microbiol. 43:468–474.
LI, Z., W. XIAO, J. J. MCCORMICK, and V. M. MAHER. 2001.
Expression of hMMS2 antisense in human cells decreases
the frequency of UV-induced recombination and increases
the frequency of induced mutations. Proc. Am. Assoc. Cancer Res. 42:420.
MA, L., S. BROOMFIELD, C. LAVERY, S. L. LIN, W. XIAO, and
S. BACCHETTI. 1998. Up-regulation of CIR1/CROC1 expression upon cell immortalization and in tumor-derived human cell lines. Oncogene 17:1321–1326.
MORAEO, T. F., R. A. EDWARDO, S. MCKENNA, L. PASTUSHOK,
W. XIAO, M. J. N. GLOVER, and M. J. ELLISON. 2001. Crystal structure of the human ubiquitin congregating enzyme
complex; h Mm S2phU6C13. Nature Structual Biol. 8:669–
673.
OKANO, S., H. TOKUSHIMA, Y. NAKAOKA, and K. SHIMIZU.
1996. Cloning of a novel ubiquitin-conjugating enzyme
(E2) gene from the ciliate Paramecium tetraurelia. FEBS
Lett. 391:1–4.
PEREZ-ROMERO, P., E. VILLALOBO, C. DIAZ-RAMOS, P. CALVO,
and A. TORRES. 1999. Actin of Histriculus cavicola: characteristics of the highly divergent hypotrich ciliate actins.
J. Eukaryot. Microbiol. 46:469–472.
PHILIPPE, H. 1993. MUST, a computer package of management
utilities for sequences and trees. Nucleic Acids Res. 21:
5264–5272.
PRESCOTT, D. M. 1994. The DNA of ciliated protozoa. Microbiol. Rev. 58:233–267.
ROTHOFSKY, M. L., and S. L. LIN. 1997. CROC-1 encodes a
protein which mediates transcriptional activation of the human FOS promoter. Gene 195:141–149.
SAITOU, N., and M. NEI. 1987. The neighbor-joining method:
a new method for reconstructing phylogenetic trees. Mol.
Biol. Evol. 4:406–425.
SAMBROOK, J., E. F. FRITSCH, and T. MANIATIS. 1989. Molecular cloning—a laboratory manual. 2nd edition. Cold
Spring Harbor Laboratory Press, New York.
SANCHO, E., M. R. VILA, L. SANCHEZ-PULIDO et al. (16 coauthors). 1998. Role of UEV-1, an inactive variant of the
E2 ubiquitin-conjugating enzymes, in in vitro differentiation
and cell cycle behavior of HT-29-M6 intestinal mucosecretory cells. Mol. Cell. Biol. 18:576–589.
ULRICH, H. D., and S. JENTSCH. 2000. Two RING finger proteins mediate cooperation between ubiquitin-conjugating
enzymes in DNA repair. EMBO J. 19:3388–3397.
VILLALOBO, E., C. MOCH, R. PERASSO, and A. BAROIN-TOURANCHEAU. 2001. Searching for excystment-regulated genes
in Sterkiella histriomuscorum (Ciliophora, Oxytrichidae): a
mRNA differential display analysis of gene expression in
excysting cells. J. Eukaryot. Microbiol. 48:382–390.
XIAO, W., B. L. CHOW, S. BROOMFIELD, and M. HANNA. 2000.
The Saccharomyces cerevisiae RAD6 group is composed of
an error-prone and two error-free postreplication repair
pathways. Genetics 155:1633–1641.
XIAO, W., B. L. CHOW, T. FONTANIE, L. MA, S. BACCHETTI, T.
HRYCIW, and S. BROOMFIELD. 1999. Genetic interactions
between error-prone and error-free postreplication repair
pathways in Saccharomyces cerevisiae. Mutat. Res. 435:1–
11.
XIAO, W., S. L. LIN, S. BROOMFIELD, B. L. CHOW, and Y. F.
WEI. 1998. The products of the yeast MMS2 and two human homologs (hMMS2 and CROC-1) define a structurally
and functionally conserved Ubc-like protein family. Nucleic
Acids Res. 26:3908–3914.
GEOFFREY MCFADDEN, reviewing editor
Accepted August 27, 2001