(Received for publication, September 12, 1995; and in revised form, February 16, 1996)
From the
Covalent modification of proteins by attachment of multiubiquitin chains serves as an essential signal for selective protein degradation in eukaryotes. The specificity of ubiquitin-protein conjugation is controlled in part by a diverse group of ubiquitin-conjugating enzymes (E2s or UBCs). We have previously reported that the product of the wheat TaUBC7 gene recognizes ubiquitin as a substrate for ubiquitination in vitro, catalyzing the condensation of free ubiquitin into multiubiquitin chains linked via lysine 48 (van Nocker, S., and Vierstra, R. D.(1991) Proc. Natl. Acad. Sci. U. S. A. 88, 10297-10301). Based on this activity, this E2 may play a central role in the ubiquitin proteolytic pathway by assembling chains in vivo. Here, we describe the cloning and characterization of a three-member gene family from Arabidopsis thaliana (designated AtUBC7/13/14) encoding structural homologs of TaUBC7. Like TaUBC7, recombinant AtUBC7/13/14 proteins formed multiubiquitin chains in vitro. AtUBC7/13/14 mRNAs were found in all tissues examined, and unlike related UBCs from yeast, the levels of mRNA were not elevated by heat stress or cadmium exposure. Transgenic Arabidopsis were engineered to express increased levels of active AtUBC7, for the first time altering the level of an E2 in a higher eukaryote. Plants expressing high levels of AtUBC7 exhibited no phenotypic abnormalities and were not noticeably enriched in multiubiquitinated conjugates. These findings indicate that the in vivo synthesis of multiubiquitin chains is not rate-limited by the abundance of AtUBC7 and/or involves other, yet undefined components.
A variety of essential processes in eukaryotes are regulated by
selective protein breakdown via the ubiquitin-dependent proteolytic
pathway (Hershko and Ciechanover, 1992; Vierstra, 1993; Ciechanover,
1994). In this pathway, protein targets are first modified by
attachment of a chain of ubiquitin monomers, internally linked through
Lys of one ubiquitin and the carboxyl-terminal Gly
of the adjacent ubiquitin. This modification serves as a signal
for the subsequent breakdown of the target protein by the 26 S
proteasome complex (Hershko and Ciechanover, 1992). A 26 S proteasome
subunit which recognizes multiubiquitin chains has been described
recently and appears to have a high affinity for chains containing four
or more ubiquitins (Deveraux et al., 1994; van Nocker et
al., 1996). Substrates of the ubiquitin-dependent proteolytic
pathway include aberrant polypeptides and important cellular regulators
such as phytochrome A (Jabben et al., 1989), cyclins (Glotzer et al., 1991), p53 and c-Jun oncoproteins (Scheffner et
al., 1993; Treier et al., 1994), the yeast MAT
2
transcriptional regulator and G
protein Gpa1 (Hochstrasser et
al., 1991; Madura and Varshavsky, 1994), and components of the
NF-
B transcriptional complex (Palombella et al., 1994).
Ubiquitin conjugation is an ATP-dependent, multi-step process
requiring the sequential action of at least two enzymes (Hershko and
Ciechanover, 1992). Ubiquitin-conjugating enzymes (E2s) ()function by accepting activated ubiquitin from ubiquitin
activating enzymes (E1s) and transferring it to a target protein. This
transfer involves the formation of an ubiquitin-E2 thiol-ester
intermediate where ubiquitin is linked through Gly
to a
specific cysteine within E2. The bound ubiquitin then becomes
conjugated to a target protein via an isopeptide bond between
Gly
of ubiquitin and free lysl
-amino groups within
the target. In at least some cases, additional recognition factors,
termed ubiquitin-protein ligases (E3s), are required for substrate
recognition and ubiquitin transfer (Ciechanover, 1994; Scheffner et
al., 1995).
Proteins destined for degradation via the
ubiquitin-dependent proteolytic pathway become multiubiquitinated by
the attachment of a chain of ubiquitin monomers linked through
Gly
Lys
(Chau et al., 1989).
The mechanism by which this intermolecular linkage is formed in
vivo is unknown, but at least two possibilities exist. In a two-
or multi-step process, a single ubiquitin is attached to a target
protein; this ubiquitin would then serve as the site for appending
additional ubiquitins. Alternatively, multiubiquitin chains could be
preformed by concatenation of free ubiquitin monomers; the resulting
chain would then be attached to the target in a single step (Chen and
Pickart, 1990; van Nocker and Vierstra, 1993). In support of the latter
possibility are the observations that (i) several types of E2s exist
that catalyze the formation of free multiubiquitin chains, at least in vitro; (ii) free chains can interact with E1 and various
E2s and be transferred en masse to targets in vitro with
kinetics similar to ubiquitin monomers; and (iii) large cellular pools
of free multiubiquitin chains exist in a variety of eukaryotes (Chen
and Pickart, 1990; van Nocker and Vierstra, 1991, 1993).
We have previously reported the cloning of a cDNA from wheat encoding an E2, TaUBC7, that is capable of forming free multiubiquitin chains in vitro (van Nocker and Vierstra, 1991). In an attempt to better elucidate the role of E2s such as TaUBC7 in multiubiquitin chain formation in vivo, we have cloned the corresponding genes from the plant Arabidopsis thaliana, which is better suited for genetic manipulations. Here, we report the isolation and characterization of a three-member gene family (designated AtUBC7/13/14) encoding E2 proteins with 71-76% identity to TaUBC7. Like TaUBC7, these E2s are capable of synthesizing multiubiquitin chains from free ubiquitin in vitro. Transgenic Arabidopsis were engineered to express high levels of AtUBC7. This ectopic expression did not phenotypically alter the plants nor increase the pool of multiubiquitin chains. From this, we conclude that multiubiquitin chain formation in vivo is not limited by AtUBC7/13/14 enzyme levels.
To isolate the AtUBC7-like
cDNAs AtUBC13 and AtUBC14, we screened an amplified,
-ZAP II cDNA library prepared with 0.5-1-kilobase pair
poly(A)
RNA isolated from 3-day-old etiolated,
hypocotyl/cotyledon tissue pretreated with ethylene (Schindler et
al., 1992). This screen utilized as the probe a 484-bp BglII fragment from the AtUBC7 cDNA. Genomic clones
of AtUBC7-like sequences were isolated from an
-EMBL 3
genomic library (gift of N. Crawford, University of California, San
Diego) by screening with probes derived from each of the AtUBC7/13/14 cDNAs. DNA from phage corresponding to positive
plaques was isolated according to the method of Sambrook et
al.(1989). In all cases, DNA sequence was determined from both
strands (Vieira and Messing, 1987). Nucleotide and amino acid sequence
analyses employed programs of the University of Wisconsin Genetics
Computer Group (UWGCG) Software Package (Devereux et al.,
1984).
Figure 1:
Nucleotide sequence alignment of cDNAs
for the members of the AtUBC7/13/14 gene family. The derived
amino acid sequence of AtUBC7 is shown above the corresponding
nucleotide sequence. The nucleotide sequences represent the longest
cDNAs isolated. Gaps in the sequence alignments are indicated by dots. The position of the 40-base, poly(A) tail
(A) present in the original AtUBC7 clone is
identified. Locations of introns present in the genomic clones are
indicated by arrowheads; the first four introns are present in
all three genes, whereas the fifth one is absent in AtUBC14.
The positions of oligonucleotide primers utilized in quantitative
RT-PCR (Fig. 5B) are underlined. Translation
stop codons (TGA) are indicated by an asterisk.
Figure 2: Amino acid sequence relationships between the members of the AtUBC7/13/14 protein family and other E2s. A, amino acid sequence alignment of the AtUBC7/13/14 proteins with wheat TaUBC7 and yeast ScUBC7. Residues which are identical or similar to AtUBC7 are outlined in black or gray, respectively. The 13-residue insertion characteristic of the AtUBC7-type E2s is underlined. The presumed active-site cysteine (Cys-89 for AtUBC7) is marked with an arrow. Numbers refer to the amino acid position for AtUBC7. B, dendrogram of amino acid sequence relationships among the AtUBC7/13/14 protein family, bovine E2-25 kDa, and other E2s. Distance along the horizontal axis separating two sequences is proportional to the divergence between the sequences. Members of the yeast ScUBC4/5 family, the Arabidopsis AtUBC8-12 family, and the human UBCH5a/b/c family are included as an example of closely related E2 protein families from different kingdoms. Sequences shown are Arabidopsis AtUBC7/13/14 (this work) and AtUBC8-12 (Girod et al., 1993), wheat TaUBC7 (van Nocker and Vierstra, 1991), yeast ScUBC7 (Jungmann et al., 1993), CDC34 (Goebl et al., 1988), and ScUBC4/5 (Seufert and Jentsch, 1990), human UBCH5a/b/c (Jensen et al., 1995), tomato UBC1 (K. Feussner and C. Wasternack, unpublished data), and bovine E2-25 kDa (Chen et al., 1991).
Figure 5:
Analyses of AtUBC7/13/14 expression. A, RNA gel blot analysis using total mRNA
isolated from Arabidopsis leaf, stem, or flower tissue. B, quantitative RT-PCR analysis of AtUBC7/13/14 mRNA
levels. Total RNA was isolated from the tissues indicated, converted to
cDNA, and used in quantitative PCR. PCR was initiated from cDNA (lane 1), serial dilutions of cDNA (1:10, lane 2;
1:100, lane 3; 1:1000, lane 4) or HO (lane 5). PCR utilized gene-specific oligonucleotide primers
corresponding to members of the AtUBC7/13/14 gene family (see Fig. 1) or the developmentally regulated agamous gene
(``Materials and Methods''; Yanofsky et
al.(1990)).
To isolate additional AtUBC7-like
cDNAs, an amplified -ZAP II A. thaliana cDNA
expression library was screened as above. This screen of 1
10
recombinant phage led to the isolation of six clones
identical to AtUBC7 except for the length of the 5` and 3`
non-translated sequence. All of these AtUBC7 clones had a 3`
non-coding region extending beyond the site of poly(A) addition found
in the original AtUBC7 clone. This screen also resulted in the
isolation of two cDNAs, designated AtUBC14; they were
identical except for the length of the 5` and 3` ends, and had 67%
nucleotide sequence identity to AtUBC7 (Fig. 1). (After
completion of this work, a cDNA identical to AtUBC14 was also
identified by random sequencing of an Arabidopsis cDNA library
(Genschik et al., 1994).) Following the isolation of a unique
genomic clone designated AtUBC13 (below), the
-ZAP II
cDNA expression library was screened as above using a 652-bp BclI/EcoRI fragment from the AtUBC13 genomic
clone as a probe. This screen of 1
10
recombinant
phage yielded three distinct cDNAs with complete nucleotide identity to AtUBC13 genomic sequence,
80% identity to AtUBC7, and
67% identity to AtUBC14 (Fig. 1). The AtUBC13 cDNAs differed only in the
length of 5` and 3` non-translated sequences (data not shown).
In an
attempt to isolate genomic clones of AtUBC7-like sequences, a
484-bp BglII fragment containing 28 bp of 5` non-coding and
nearly the entire coding region of AtUBC7 was used as a probe
to screen a -EMBL 3 library containing A. thaliana genomic DNA. Examination of 1.5
10
phage
resulted in the detection of a genomic sequence encompassing the entire
cDNA of AtUBC7. An additional clone, designated AtUBC13, was identified that contained AtUBC7-related
sequence (above). To identify a genomic equivalent of the AtUBC14 cDNA, the Arabidopsis genomic library was screened using
a 196-bp XbaI/HindIII fragment containing AtUBC14 3` non-translated sequence as a probe. This screen resulted in the
detection of a genomic sequence that included the entire cDNA sequence
of AtUBC14.
The predicted protein products of the AtUBC13/14 genes were nearly identical to AtUBC7 in
terms of both apparent molecular mass and charge (AtUBC13: M = 18,822, pI = 5.34; AtUBC14: M
= 18,728, pI =
5.33). All three contain a conserved cysteine (residue 89 for AtUBC7 (Fig. 2A)) at a position similar to
that of the active-site cysteine in the yeast E2 RAD6, wheat TaUBC4, and Arabidopsis AtUBC1 (Sung et al.,
1990; Sullivan and Vierstra, 1991), implicating this cysteine in the
formation of the thiol-ester bond with ubiquitin. The region
surrounding this cysteine conformed to the E2 active site motif
HPN(I/V)(X)
GX(I/V/L)C(I/L)X(I/V)(I/L) that is
present in almost all E2s characterized to date. Additional cysteines
are present at positions 17 and 158 in all three E2s, and at position 6
in AtUBC13. AtUBC7/13/14 contains a conserved
13-residue sequence (GDDPXGYELASER) carboxyl-terminal to the active
site cysteine; a similar insertion is present also in wheat TaUBC7 and yeast ScUBC7 and ScUBC3 (CDC34).
Pairwise comparisons of AtUBC7/13/14 revealed a percent
deduced amino acid sequence identity of 86-96%. In contrast, AtUBC7/13/14 exhibited at most only 58% amino acid sequence
identity to the other Arabidopsis E2s that are fully
characterized to date (Bartling et al., 1993; Girod et
al., 1993; Sullivan et al., 1994). Because of the
structural similarity between AtUBC7, -13, and -14, we propose
that they constitute a functionally related family of E2s in Arabidopsis.
The complete nucleotide sequences of the AtUBC7/13/14 genomic clones were determined and their structures are depicted in Fig. 3. Five introns exist within the region corresponding to the cDNAs of AtUBC7 and -13, whereas four exist within AtUBC14. The introns in common interrupt the coding regions at identical positions and contain the consensus GT/AG intron borders, with the exception of the 5`-most intron of AtUBC7, which contained GC/AG borders (data not shown). AtUBC7/13/14 do not contain introns outside of the coding region, at least within the region corresponding to the cDNAs.
Figure 3: Genomic organization of AtUBC7/13/14. The regions shown correspond to the isolated genomic DNA fragments subjected to DNA sequence analysis. The boxed areas correspond to cDNA sequence; unshaded boxes indicate 5` and 3` non-translated regions; and shaded boxes denote the coding regions. Introns (shown as lines) are to scale. Restriction endonuclease sites utilized in subcloning of genomic fragments are indicated: B, BglII; R, EcoRI; H, HindIII. The position of the presumed active-site cysteine is indicated.
DNA gel blots of Arabidopsis DNA digested with a variety of restriction endonucleases revealed multiple fragments that hybridized to a coding region probe from AtUBC7 (Fig. 4). For each endonuclease used, three hybridizing fragments were identified, even at reduced stringency. Similar analyses using probes unique to AtUBC7, AtUBC13, or AtUBC14 showed that each probe specifically hybridized to one of the three fragments, indicating that AtUBC7/13/14 likely constitute the entire gene family in Arabidopsis (Fig. 4).
Figure 4:
Gel blots of Arabidopsis genomic DNA probed with AtUBC7/13/14 sequences. Arabidopsis DNA was digested to completion with the
restriction endonucleases indicated and subjected to DNA gel blot
analysis using P-labeled dCTP-labeled probes (see
``Materials and Methods''). The last three lanes of each
panel contain 40 pg of the respective cDNAs to demonstrate the
specificity of each probe.
RNA gel-blot analysis of the AtUBC7/13/14 family detected 950-base mRNAs in all
tissues examined, including leaf, flower, silique, and stem (Fig. 5A; data not shown). Immunoblot analyses using
anti-ZmUBC7 immunoglobulins indicated that the corresponding
protein(s) were also present in these tissues (data not shown). The
great degree of nucleotide sequence homology between the AtUBC7/13/14 genes, minimal length and high A/T content of
non-homologous regions, and relatively low mRNA levels precluded the
use of gene-specific probes to detect individual expression patterns by
RNA gel blot analysis. Consequently, the expression patterns of the
three genes were analyzed individually using quantitative RT-PCR
employing gene-specific oligonucleotide primers (Fig. 5B). cDNA was generated simultaneously from
individual RNA pools to minimize sample variation, and PCR reactions
for all primer pairs were performed simultaneously to allow comparison
of the relative PCR product levels between RNA sources. To validate
this method, we analyzed mRNA levels of the homeotic gene agamous, which previously had been shown to be expressed in
flowers, but not in leaves (Yanofsky et al., 1990). This was
confirmed by the greater levels of agamous PCR product derived
from flower and silique RNA relative to that from leaf RNA
(
100-fold (Fig. 5B)). Primers specific to each of
the AtUBC7/13/14 genes produced RT-PCR products of the
expected size. Whereas levels of the AtUBC7 and AtUBC13 PCR products were invariant among leaf, flower, and silique
tissues, increased levels of the AtUBC14 PCR product, relative
to that of AtUBC7/13, were seen in leaves (Fig. 5B). We found no increase in expression of the AtUBC7/13/14 gene family following a heat stress (37 °C
for 2 h) sufficient to increase Arabidopsis HSP70 and HSP100 mRNA levels (data not shown). (In yeast, the ScUBC4/5 gene family is strongly responsive to heat stress
(Seufert and Jentsch, 1990).) Unlike yeast ScUBC5 and ScUBC7 (Jungmann et al., 1993), the AtUBC7/13/14 mRNAs did not accumulate following exposure of plants to levels of
cadmium (10 µM) that severely inhibited growth (data not
shown).
Figure 6:
Purification and activity of the AtUBC7/13/14 proteins synthesized in E. coli.A, purification of recombinant proteins. E2s were
purified from E. coli as described under ``Materials and
Methods,'' resolved by SDS-PAGE, and stained with Coomassie Blue. B, formation of thiol-ester intermediates between purified E2s
and I-labeled ubiquitin. Adducts were formed in a
reaction containing E1, MgATP, and the indicated E2. Reaction products
were assayed by non-reducing SDS-PAGE and autoradiography. The arrowhead to the right indicates the migration
position of free ubiquitin.
Like TaUBC7, the AtUBC7/13/14 E2s conjugated ubiquitin to ubiquitin in vitro, and following prolonged incubation could generate multiubiquitin chains containing as many as seven monomers (see Fig. 8B for the SDS-PAGE profile of chains assembled in vitro by AtUBC7). To compare accurately the kinetics of these three E2s in multiubiquitin chain formation, we assayed for the assembly of di-ubiquitin from free ubiquitin using equivalent amounts of E2 activity (as determined by thiol-ester assay) (Fig. 7). AtUBC14 was the most active (1.9 µmol of di-ubiquitin/min), followed by AtUBC7 (1.2 µmol/min) and AtUBC13 (0.7 µmol/min). These rates, however, were significantly lower than that of TaUBC7 under the same reaction conditions (3.8 µmol/min).
Figure 8:
Abundance and activity of AtUBC7
in transgenic Arabidopsis expressing AtUBC7 from the
constitutive cauliflower mosaic virus 35 S promoter. A,
extracts were subjected to SDS-PAGE and immunoblotting utilizing
anti-ZmUBC7 immunoglobulins. Tissues analyzed were root (R), seed (Se), leaf (L), stem (St), flower (F), and etiolated whole seedlings (Et). Tissue from wild-type plants are analyzed in lanes
labeled -, whereas tissue from a representative line expressing
high levels of AtUBC7 are analyzed in lanes labeled +. Lane 1 represents 15 ng of rAtUBC7 purified from E. coli. B, extracts as in A were subjected
to SDS-PAGE and immunoblotting utilizing anti-ubiquitin
immunoglobulins. Lane 1 represents 30 ng of multiubiquitin
chains synthesized in vitro (van Nocker and Vierstra, 1993).
The immunoreactive material at the top of the gel represents ubiquitin
attached to other cellular proteins. C, leaf extracts were
used in thiol-ester assays with I-labeled ubiquitin. Lane 1 represents wild-type plants; lane 2 represents
the AtUBC7 overexpressing line analyzed in A and B; and lane 3 represents a thiol-ester assay
utilizing 15 ng of rAtUBC7 purified from E.
coli.
Figure 7:
Time course for E2-catalyzed formation of
ubiquitin-ubiquitin (UBQ) conjugates. Conjugates were
formed in a reaction containing E1, MgATP, and 1 unit of the indicated
E2 (TaUBC7 (
), 0.19 µg; AtUBC7 (
),
0.14 µg; AtUBC13 (
), 0.16 µg; AtUBC14
(
), 0.16 µg), separated by SDS-PAGE, and quantitated by
direct
-counting of UBQ
following localization by
autoradiography. Mean values and S.E. were calculated based on three
replicates.
With the sense AtUBC7 vector, we obtained 11 independent lines producing increased
levels of AtUBC7 mRNA and protein (Fig. 8A and
data not shown). None of these lines exhibited co-suppression of the
wild-type AtUBC7/13/14 genes which can occur when endogenous
genes are ectopically expressed in Arabidopsis (Matzke and
Matzke, 1995). AtUBC7 protein from these lines comigrated with AtUBC7 produced in E. coli and was recognized by
anti-ZmUBC7 immunoglobulins (Fig. 8A).
Quantitative immunoblot analysis using purified, recombinant AtUBC7 as the standard indicated that plants homozygous for
the introduced DNA accumulated 7-fold more AtUBC7 protein
than non-transformed plants. This increase was evident in almost all
tissues examined, including roots, stems, leaves, inflorescence
meristems, flowers, siliques, and etiolated seedlings (Fig. 8A; data not shown). The only exception was seeds
where little accumulation of AtUBC7 was detected.
To
demonstrate that the AtUBC7 protein expressed in planta was enzymatically active, we partially purified AtUBC7
from transgenic leaf tissue and examined its ability to form a
thiol-ester adduct with I-labeled ubiquitin. Whereas the
amount of adduct formed by endogenous AtUBC7 in
non-transformed plants was below our limits of detection, in transgenic
lines producing high levels of AtUBC7 protein we detected a
ubiquitinated product that comigrated with authentic AtUBC7-ubiquitin thiol-ester adduct (Fig. 8C).
The specific activity of AtUBC7 expressed in the transgenic
plants was equal to, or slightly greater than, recombinant AtUBC7 produced in E. coli, as determined by
quantitative thiol-ester assays (data not shown).
To test the
hypothesis that AtUBC7/13/14 function in vivo to
assemble multiubiquitin chains, we compared the ubiquitin conjugate
profiles in cell lysates from non-transformed plants to those from AtUBC7-overexpressing plants by SDS-PAGE and immunoblotting.
In wild-type Arabidopsis extracts, free multiubiquitin chains
are the most abundant ubiquitin conjugates, easily detected as an
immunoreactive ladder differing by 8-kDa increments (van Nocker
and Vierstra, 1993). As seen in Fig. 8B, free
multiubiquitin chains containing from 2 to as many as 5 monomers could
be detected that comigrated with authentic Gly
Lys
chains synthesized in vitro using AtUBC7. The chains were abundant in all tissues examined,
including root, seed, leaf, stem, flower, and etiolated seedling (Fig. 8B). Surprisingly, in AtUBC7
overexpressing plants, no change was apparent in either the abundance
or distribution of free multiubiquitin chains or in the abundance of
other ubiquitinated proteins relative to that of the free ubiquitin
monomers (Fig. 8B). Transgenic plants producing high
levels of active AtUBC7 protein also were phenotypically
indistinguishable from non-transgenic plants by all assayed parameters
(see above).
Multiubiquitin chains linked through Gly
Lys
function as a strong signal in directing proteolysis
when attached to various target proteins (Chau et al., 1989;
Ciechanover, 1994). Despite its importance to basic cell biology, the
mechanism by which target proteins become multiubiquitinated is not yet
known. We have previously reported the isolation and molecular
characterization of TaUBC7, a ubiquitin-conjugating enzyme
from wheat capable of forming multiubiquitin chains linked exclusively
through Gly
Lys
from free ubiquitin in vitro (van Nocker and Vierstra, 1991). Here, we report the
isolation and characterization of both cDNA and genomic clones
comprising a family of three transcribed genes from Arabidopsis, designated AtUBC7, -13, and
-14, encoding structural homologs of TaUBC7 that are
also capable of multiubiquitin chain formation in vitro. All
three recombinant Arabidopsis E2s exhibited slightly different
kinetics in this reaction that were significantly lower than that of TaUBC7. However, because our studies employed a recombinant
wheat E1, the lower rate of chain formation by the Arabidopsis E2s compared with TaUBC7 may reflect a preference for
interacting with a more closely related E1.
DNA gel blot analysis under conditions of low stringency and extensive screenings of both genomic and cDNA expression libraries indicate that AtUBC7/13/14 constitute the entire gene family in Arabidopsis. This genomic organization is typical of that seen for other Arabidopsis E2s; the AtUBC1 and AtUBC4 families contain three members each (Sullivan et al., 1994), and the AtUBC8 family contains at least five members (Girod et al., 1993). Chromatographic analyses of E2s from wheat suggest that multiple isoforms of TaUBC7 likely exist in this species as well, but whether they are encoded by different genes has not been determined (van Nocker and Vierstra, 1991).
The AtUBC7/13/14 cDNAs
were predicted to be nearly full-length by comparison with the mRNA
lengths of 950 bases. For each cDNA isolated, heterogeneity was
observed in the length of both the 5` and 3` non-translated regions.
Some, or all, of the variability in 3` sequence length was an artifact
of library construction. (
)However, at least for AtUBC7, the presence of two poly(A) addition sites was
evident. Both RNA gel blot analysis and RT-PCR indicate that AtUBC7/13/14 are actively transcribed in most, if not all, Arabidopsis tissues. In leaves, flowers, siliques, and stems, AtUBC7/13/14 mRNAs accumulate to approximately the same
abundance and are present at the same ratio, with the exception of AtUBC14, which is relatively more abundant in leaves. The AtUBC7/13/14 genes are also expressed in etiolated seedlings,
as this was the source of the tissue from which the cDNA libraries were
created. The wide tissue distribution of AtUBC7/13/14 proteins
indicate that this family of E2s has a pervasive role in basic cellular
functions. The corresponding mRNAs do not accumulate in response to
heat stress or cadmium exposure. This data suggest that the
corresponding E2s are either not involved in the response to these
stresses, or are already at sufficient levels to carry out a
stress-related function.
AtUBC7/13/14 encode acidic,
19-kDa proteins of 166-167 amino acids with 76% sequence
identity to wheat TaUBC7 and 50% identity to yeast ScUBC7. They contain the
150-amino acid core domain
identified in all E2s characterized to date that includes an
active-site cysteine for ubiquitin transfer. In AtUBC7/13/14,
the core domain is interrupted by a short (
13 amino acids)
insertion near the active site. Among 27 other E2s characterized to
date at the molecular level, similar internal insertions are found only
in yeast ScUBC3 (CDC34) and ScUBC7, and TaUBC7 from wheat (Goebl et al., 1988; van Nocker and
Vierstra, 1991; Jungmann et al., 1993). Comparison of the
amino acid sequence of these proteins with that of AtUBC1 and
yeast ScUBC4, whose crystallographic structures have been
determined (Cook et al., 1993), suggests that this internal
sequence extends outward as a loop from the surface of the protein and
may not be involved in intramolecular interactions. This loop may
provide the necessary binding site for ubiquitin recognition during
chain formation. AtUBC7/13/14 proteins show the greatest
structural dissimilarity to yeast ScUBC7 and wheat TaUBC7 at the amino-terminal region. This amino-terminal
heterogeneity is typical of that found among E2s from Arabidopsis and other organisms. Based on the crystal structure of AtUBC1 and ScUBC4, the extreme amino terminus likely
extends away from the core of the protein (Cook et al., 1993)
and may interact with E1 based on amino-terminal deletion analyses
(Sullivan and Vierstra, 1991).
Given the strong amino acid conservation between the AtUBC7/13/14 proteins and ScUBC7, it is tempting to speculate that these proteins are functionally analogous, in spite of the fact that AtUBC7/13/14 mRNAs do not accumulate following exposure of the plant to cadmium. However, inferring function based on structural conservation is complicated by the presence of at least one other yeast E2 exhibiting homology to AtUBC7/13/14, the cell cycle regulator CDC34. Although CDC34 differs in overall structure (containing a negatively charged domain appended to the carboxyl terminus of the core), within the core region CDC34 exhibits as great an amino acid sequence similarity (68%) to AtUBC7 as does ScUBC7 (66%).
Ectopic expression of AtUBC7 mRNA in transgenic Arabidopsis resulted in the accumulation of high levels of
active protein in most tissues examined. However, no change in
ubiquitin conjugate profiles were seen, nor were any phenotypic effects
evident. We offer several explanations to account for this. First, AtUBC7 may function in vivo in capacities other than
in multiubiquitin chain formation. Second, it is possible that the AtUBC7/13/14 family comprises only one of several E2 isoforms
that contribute to the cellular pool of multiubiquitin chains in Arabidopsis. For example, in addition to TaUBC7 and AtUBC7/13/14, the ability to synthesize multiubiquitin chains in vitro is common to at least one other E2, bovine
E2-25 kDa. However, the plant and bovine E2s are not structural
homologs as they differ in size and share less than 34% overall amino
acid identity (Fig. 2B). A potential plant counterpart
of E2-25 kDa has been identified from a tomato cDNA library
(exhibiting 48% identity to E2-25 kDa; Fig. 2B), but whether it is also capable of making
multiubiquitin chains is unknown. Thus, an increase in AtUBC7
level might not lead to a proportional increase in multiubiquitin chain
levels. Third, the formation of multiubiquitin chains may not be
limited by the enzyme(s) required for ubiquitin polymerization, but by
available quantities of free ubiquitin monomers. Fourth, other enzymes (e.g. other E2s and E3s) may be required for chain formation in vivo in addition to the AtUBC7/13/14 and may be
limiting. It is tempting to speculate that the members of the AtUBC7/13/14 family may act in concert or with other E2s/E3s
to promote chain formation. In support of this, it appears that AtUBC7 physically interacts with AtUBC13 and AtUBC14 in vivo, (
)and evidence is
accumulating for the concerted action of multiple yeast E2s in the
multiubiquitination and degradation of MAT
2 (Chen et al.,
1993).
Future studies of the role of the AtUBC7/13/14 family in multiubiquitination will depend on creating or
identifying mutants deficient in these E2s in Arabidopsis.
Although high level expression in transgenic Arabidopsis can
often result in co-suppression of the endogenous genes (Matzke and
Matzke, 1995), we detected no suppression of the endogenous AtUBC7/13/14 genes in any transgenic line. Nor were we able to
reduce the levels of AtUBC7 protein through high level
expression of antisense mRNAs. An alternative approach would be to
interfere with the function of AtUBC7/13/14 E2s by expressing
structurally altered forms. A particularly attractive possibility in
this regard would be a Cys Ser substitution at the active site.
This substitution in yeast RAD6 and AtUBC1 still allows
ubiquitin to bind to the E2s, in this case through an ester linkage,
but precluded transfer of the bound ubiquitin to substrates (Sung et al., 1990; Sullivan and Vierstra, 1993). Bailly et al. (1994) have shown that such inactive E2s can act in a dominant
negative manner when expressed to high levels in yeast.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U33757 [GenBank](AtUBC7), U33758 [GenBank](AtUBC13), and U33759 [GenBank](AtUBC14).