Rose. Oughtred, Nathalie. Bedard, Alice. Vrielink ... - McGill University

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 273, No. 29, Issue of July 17, pp. 18435–18442, 1998 

© 1998 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. 

Identification of Amino Acid Residues in a Class I 

Ubiquitin-conjugating Enzyme Involved in Determining 

Specificity of Conjugation of Ubiquitin to Proteins* 

(Received for publication, March 2, 1998, and in revised form, May 1, 1998) 

Rose Oughtred‡§, Nathalie Bédard‡, Alice Vrielink, and Simon S. Wing‡** 

From the ‡Department of Medicine, Polypeptide Laboratory, McGill University, Montreal, Quebec H3A 2B2, Canada and 

the Department of Biochemistry and the Montreal Joint Centre for Structural Biology, McGill University, Montreal, 

Quebec H3G 1Y6, Canada 

The ubiquitin pathway is a major system for selective 

proteolysis in eukaryotes. However, the mechanisms underlying 

substrate selectivity by the ubiquitin system 

remain unclear. We previously identified isoforms of a 

rat ubiquitin-conjugating enzyme (E2) homologous to 

the Saccharomyces cerevisiae class I E2 genes, UBC4/ 

UBC5. Two isoforms, although 93% identical, show distinct 

features. UBC4-1 is expressed ubiquitously, 

whereas UBC4-testis is expressed in spermatids. Interestingly, 

although these isoforms interacted similarly 

with some ubiquitin-protein ligases (E3s) such as E6-AP 

and rat p100 and an E3 that conjugates ubiquitin to 

histone H2A, they also supported conjugation of ubiquitin 

to distinct subsets of testis proteins. UBC4-1 

showed an 11-fold greater ability to support conjugation 

of ubiquitin to endogenous substrates present in a testis 

nuclear fraction. Site-directed mutagenesis of the UBC4testis 

isoform was undertaken to identify regions of the 

molecule responsible for the observed difference in substrate 

specificity. Four residues (Gln-15, Ala-49, Ser-107, 

and Gln-125) scattered on surfaces away from the active 

site appeared necessary and sufficient for UBC4-1-like 

conjugation. These four residues identify a large surface 

of the E2 core domain that may represent an area of 

binding to E3s or substrates. These findings demonstrate 

that a limited number of amino acid substitutions 

in E2s can dictate conjugation of ubiquitin to different 

proteins and indicate a mechanism by which small E2 

molecules can encode a wide range of substrate 

specificities. 

The ubiquitin system is implicated in an ever expanding 

array of cellular processes that now range from DNA repair to 

cell cycle progression and muscle protein degradation (reviewed 

in Refs. 1–3). Ubiquitin is a highly conserved 76-residue 

protein whose many cellular functions are mediated by its 

covalent ligation to other proteins. Most of these functions arise 

from the ability of ubiquitination to lead to degradation of the 

* This work was supported in part by Medical Research Council 

Grant MT13341 (to A. V.) and Grant MT12121 (to S. S. W). The costs of 

publication of this article were defrayed in part by the payment of page 

charges. This article must therefore be hereby marked “advertisement” 

in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 

§ Recipient of the Eileen Peters McGill Major Fellowship. 

Recipient of a Chercheur Boursier award from the Fonds de la 

Recherche en Santé du Québec. 

** Recipient of a Clinician Scientist award from the Medical Research 

Council of Canada. To whom correspondence should be addressed: 

Dept. of Medicine, Polypeptide Lab., McGill University, Strathcona 

Bldg., 3640 University St., Suite W315, Montreal, Quebec H3A 2B2, 

Canada. Tel.: 514-398-4101; Fax: 514-398-3923; E-mail: cxwg@musica. 

mcgill.ca. 

This paper is available on line at http://www.jbc.org 18435 

selected protein. Indeed, ubiquitin-mediated proteolysis is responsible 

for the turnover of key regulatory proteins, including 

mitotic cyclins (cyclin B) (4, 5), cyclin-dependent kinases (Sic1 

and p27) (6, 7), and transcription factors (Mat2, c-Jun, and 

p53) (8–10). 

Recognition of specific substrates occurs at the level of conjugation, 

which is a multistep process involving three types of 

enzymes (11): a ubiquitin-activating enzyme (E1), 1 ubiquitinconjugating 

enzymes (UBCs or E2s), and, in many cases, ubiquitin-protein 

ligases (E3s). Initially, ubiquitin is activated by 

E1 through the ATP-dependent formation of a thiol ester bond 

between ubiquitin and E1 (12). The activated ubiquitin is then 

transferred via a thiol ester linkage to a cysteine residue of an 

E2 (reviewed in Ref. 13). Finally, the E2 itself, or more commonly 

in concert with an E3, ligates the ubiquitin via its 

carboxyl terminus to lysine residues of a protein substrate. 

Successive ubiquitin molecules may be added to lysine residues 

of the previous ubiquitin to produce a multi-ubiquitin chain. 

Although the biochemical mechanisms of the pathway are 

becoming well defined, the molecular mechanisms by which 

substrates are selected by the ubiquitin-conjugating apparatus 

remain unclear. E3s are important for recognition and binding 

of the substrate (14). E3s may serve as docking proteins that 

bind both specific substrates and E2s (14), thereby permitting 

the transfer of ubiquitin from an E2 to a substrate. For example, 

the E3 SCF Cdc4 binds the E2 molecule Cdc34 and a specific 

substrate, Sic1, simultaneously, thereby facilitating the transfer 

of ubiquitin from Cdc34 to Sic1, an inhibitor of the yeast 

S-phase cyclin-dependent kinase Cln1-Cdc28 (15, 16). Alternatively, 

E3s may function as the final intermediate in the ubiquitin 

thiol ester cascade (17). The E3 E6-AP (E6-associated 

protein) forms a thiol ester linkage with ubiquitin prior to 

catalyzing the ubiquitination of p53 in the presence of the viral 

E6 protein (17). The catalytically active cysteine in E6-AP is 

found within its carboxyl terminus domain, and a number of 

putative E3s have been identified based on the presence of such 

HECT (homology to E6-AP carboxyl terminus) domains (18). 

Although E3s bind substrates, E2s may also be involved in 

substrate recognition either by conjugating substrates directly 

or probably more commonly by interacting only with specific 

E3s. Indeed, yeast genetic studies have revealed a variety of 

functions for different E2s indicating that they can direct conjugation 

of ubiquitin to specific substrates. For example, UBC2 

(RAD6) is required for DNA repair (19), whereas UBC4/UBC5 

1 The abbreviations used are: E1, ubiquitin-activating enzyme; E2 

and UBC, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; 

PCR, polymerase chain reaction; GST, glutathione S-transferase; DTT, 

dithiothreitol; AMP-PNP, 5-adenylyl imidodiphosphate; RM-Ub, reductively 

methylated ubiquitin.

18436 

FIG. 1. Comparison of rat UBC4-1, 

UBC4-testis, and other prototype 

class I E2s. Rat isoforms UBC4-1 and 

UBC4-testis are homologous to S. cerevisiae 

UBC4. The 11 amino acids that differ 

between UBC4-1 and UBC4-testis are underlined. 

The critical residues that confer 

a rat UBC4-1 phenotype on UBC4-testis 

are indicated with asterisks. The protein 

sequences of the mammalian homologues 

of yeast Rad6, HHR6A and HHR6B, and 

S. cerevisiae Ubc7 are also depicted. The 

active-site cysteine is indicated by an 

arrowhead. 

are required for the degradation of short-lived and abnormal 

proteins (20). 

Differences in E2 function evidently reflect differences in E2 

structure. E2 enzymes have been divided into four structural 

classes based on amino acid sequence comparison (21). Class I 

enzymes (e.g. Ubc4 and Ubc5) (20) consist of a conserved catalytic 

core domain of 150 amino acids that contains the activesite 

cysteine involved in ubiquitin transfer. Class II enzymes 

(e.g. Ubc2/Rad6 and Ubc3/Cdc34) (19, 22) have extra C-terminal 

extensions or tails attached to the core domain, whereas 

class III enzymes (e.g. UbcH6 and UbcD2) (23, 24) have attached 

N-terminal tails. Finally, class IV enzymes (e.g. E2-C) 

(25) possess both C- and N-terminal extensions. 

Jentsch et al. (21) speculated that E2 extensions play either 

a direct role in substrate recognition or else an indirect role 

through their interaction with E3s. While C- and N-terminal 

extensions may participate in specifying the interaction of E2s 

with E3s and/or substrates, a number of studies have indicated 

that specificity elements also reside within the E2 core. For 

example, the class II enzyme RAD6 is required for DNA repair, 

induced mutagenesis, and sporulation in yeast (19) and is 

capable of polyubiquitinating histones in vitro (26). However, 

removal of the polyacidic tail of Rad6 results only in loss of its 

sporulation function (27) and histone-polyubiquitinating activity 

(26). The core domain is sufficient for performing its DNA 

repair function. Since this truncated Rad6 containing only the 

core domain exhibits a distinct phenotype from the class I (core 

domain only) E2s, Ubc4 and Ubc5, which function in the turnover 

of short-lived and abnormal proteins (20), the core domain 

must possess determinants of function and by inference substrate 

specificity. More recently, the C-terminal tail of E2–25K 

was found to be necessary, but not sufficient, for some of its E2 

functions, indicating that the tail depends on structural features 

in the core for its function (28). These and other results 

suggest that although E2 core domains are highly conserved, 

they possess unique structural features that are critical for 

individual E2 function and specificity. 

Recently, we cloned and characterized a family of mammalian 

class I E2s homologous to S. cerevisiae Ubc4/Ubc5 (29, 30). 

E2 Residues Determining Substrate Specificity 

Two isoforms of rat UBC4, 2 although 93% identical, show distinct 

features. Rat UBC4-testis possesses an acidic pI and 

shows testis-specific RNA expression that is specifically induced 

in the developing spermatids (30), whereas rat UBC4-1 

has a basic pI and is expressed ubiquitously (29). Therefore, 

although the high degree of sequence similarity might suggest 

that these isoforms are redundant, the highly regulated and 

cell-specific expression suggested a unique role for the UBC4testis 

isoform. 

Therefore, we characterized carefully the abilities of rat 

UBC4-1 and UBC4-testis to support conjugation of ubiquitin in 

vitro to different subsets of testis proteins. Rat UBC4-1 shows 

an 11-fold greater ability to support conjugation of ubiquitin to 

endogenous substrates present in a testis nuclear fraction (30). 

We also determined whether these two isoforms interact differentially 

with other E3s. In addition, since rat UBC4-1 and 

UBC4-testis differ by only 11 amino acids (Fig. 1) and they are 

highly similar to yeast Ubc4, whose crystal structure has been 

solved (31), this provided a unique opportunity to identify, by 

site-directed mutagenesis of UBC4-testis, regions of the E2 

core domain responsible for the observed difference in substrate 

specificity. 

EXPERIMENTAL PROCEDURES 

Site-directed Mutagenesis—pET-11d (Novagen)-based Escherichia 

coli expression plasmids encoding rat UBC4-1 or UBC4-testis have 

been described (29, 30). Mutagenesis of selected residues in UBC4testis 

to those in UBC4-1 was performed using the Chameleon doublestranded 

site-directed mutagenesis kit (Stratagene) according to the 

manufacturer’s instructions. Briefly, separate mutagenic primers3 encoding 

site-specific mutations in UBC4-testis and a selection primer 

were annealed to denatured UBC4-testis-containing pET-11d plasmids, 

2 Previously, the rat homologue of yeast Ubc4 was referred to as 

E217KB and included isoforms 2E and 8A (29, 30). For purposes of clarity 

and to conform to a trend by workers in the field to name E2s after their 

apparent yeast homologues, isoform 2E will henceforth be referred to as 

rat UBC4-1, and isoform 8A as rat UBC4-testis. The nucleotide sequences 

of UBC4-1 and UBC4-testis have been submitted to Gen- 

BankTM with accession numbers U13177 and U56407. 

3 Oligonucleotide sequences and detailed PCR conditions are available 

on request.

and the mutant DNA strand was extended with T7 DNA polymerase 

and ligated with T4 DNA ligase. The selection primer, located 2 

kilobases from the mutagenic primers, changed the unique AccI restriction 

site on pET-11d to a unique KpnI restriction site on the mutant 

plasmid strand, thereby permitting selection of intact mutant plasmids 

by digestion with AccI. Initially, separate mutant UBC4-testis constructs 

with a D55H, E68A, or double D55H/E68A substitution were 

generated. Similarly, subsequent mutations were added separately or 

in combination onto the initial UBC4-testis D55H/E68A double mutant 

in the following order: Gln-15, Gln-125, Ala-49, or Ser-107. The intact 

mutant plasmids were transformed into E. coli XL1-Blue cells (Stratagene). 

All plasmids were sequenced to confirm the presence of the 

desired mutation using the fmol TM DNA sequencing System (Promega). 

Subtractive mutagenesis was then performed in a similar manner to 

define the minimal UBC4-1 residues on UBC4-testis that were necessary 

and sufficient for the UBC4-1-like conjugating activity. 

The converse mutagenesis of the four critical residues (Arg-15, Val- 

49, Cys-107, and Arg-125) of UBC4-1 to those of UBC4-testis was 

performed via PCR amplification (32). Mutant UBC4-1 fragments were 

generated using UBC4-1-containing pET-11d as a template, primers 

bearing the relevant base substitutions, and sense or antisense oligonucleotides 

encoding the amino and carboxyl termini of the protein as 

well as restriction sites to permit cloning into the NcoI and BamHI sites 

of pET-11d. The mutant UBC4-1 fragments were then purified by 

agarose gel electrophoresis and incorporated into full-length mutant 

UBC4-1 inserts via a second round of PCR amplification using the 

separate mutant UBC4-1 fragments as a template and both the 5-NcoI 

and the 3-BamHI primers. The PCR products were purified on a 

QIAquick PCR purification column (QIAGEN Inc.), digested with NcoI 

and BamHI, and then ligated into a pET-11d vector that had been 

digested with the same enzymes. Purified plasmids were transformed 

into XL1-Blue cells, and individual positive clones were sequenced 

using the fmol TM DNA sequencing system to confirm the presence of the 

desired mutation. 

Preparation of Proteins—The pGEX-ubiquitin plasmid encoding the 

GST-ubiquitin fusion protein (33) was expressed in E. coli strain DH5 

and induced with 0.1 mM isopropyl--D-thiogalactopyranoside for 2hat 

37 °C. Bacterial cell pellets resuspended in phosphate-buffered saline 

and 1% Triton X-100 were lysed by sonication and clarified by centrifugation 

at 12,000 g. Glutathione-Sepharose (Amersham Pharmacia 

Biotech) was added to the supernatant, and the mixture was rotated 

overnight at 4 °C. Beads were washed in phosphate-buffered saline and 

1% Triton X-100 and eluted in 20 mM glutathione in phosphate-buffered 

saline for 20 min at 25 °C. The GST-ubiquitin fusion protein content 

was estimated to be 1 mg/ml by Coomassie Blue staining. 

E1 was prepared from rabbit liver. Bacterially expressed recombinant 

UBC4-1 and UBC4-testis proteins were also purified as described 

previously (29, 30). The E1, UBC4-1, and UBC4-testis enzymes were 

quantified by measuring the initial release of radioactive pyrophosphate 

following incubation in the presence of [- 32 P]ATP and ubiquitin 

(34). 

The purified pET-11d-based plasmids containing the mutant 

UBC4-1 and UBC4-testis genes were transformed into E. coli BL21 

(DE3) (Novagen), and induction of the recombinant proteins with 1 mM 

isopropyl--D-thiogalactopyranoside was carried out for 2hat30°C. 

The cells were pelleted and resuspended in 0.1 volume and then lysed 

by sonication in 50 mM Tris, pH 7.5, and 1 mM DTT. Cellular debris was 

removed by centrifugation at 12,000 g. The enzymatic activities of the 

mutant E2-containing bacterial lysates relative to the purified recombinant 

UBC4-1 and UBC4-testis enzymes were determined by thiol 

ester assays, as described below. 

The [ 35 S]methionine-labeled E6-AP (35) or rat p100 (36) proteins 

were synthesized separately in vitro using a coupled transcription/ 

translation kit (wheat germ extract TNT, Promega) with T7 polymerase. 

The translation reactions (150 l) were partially purified with 

DEAE-cellulose resin (Whatman DE52; 200 mg of wet resin/TNT reaction) 

using a batch elution procedure to remove ubiquitin, E1, and E2s 

homologous to rat UBC4 that were present in the break-though fraction. 

Briefly, the translation reactions and resin were incubated in 4.5 

ml of loading buffer (50 mM Tris-HCl, pH 7.5, and 1 mM DTT) for 1hat 

4 °C, washed two times, and eluted in loading buffer containing 0.5 M 

NaCl. The E3-containing eluates (2 ml) were then concentrated 10-fold 

by ultrafiltration with Centricon-30 concentrators (Amicon, Inc.), and 2 

l of each E3 preparation were utilized in each thiol ester assay. 

The testis cytosolic E3 and nuclear fractions eluting from a MonoQ 

anion-exchange column (Amersham Pharmacia Biotech) at 0.4 and 0.05 

M NaCl, respectively, were isolated as described previously (30). In 

some preparations, the nuclear fraction activity was found in the flow- 

E2 Residues Determining Substrate Specificity 18437 

through fraction instead of eluting at 0.05 M NaCl; however, it behaved 

identically to the original preparation eluting at 0.05 M NaCl. This 

nuclear fraction was concentrated 4-fold using a Centricon-10 concentrator 

(Amicon, Inc.). The testis cytosolic E3 activity was further purified 

by chromatography on a Superdex 200 gel filtration column (Amersham 

Pharmacia Inc.). 

Iodination of Proteins—The chloramine-T method was used to label 

bovine ubiquitin with Na 125 I to a specific radioactivity of 3000 cpm/ 

pmol (37) and histone H2A (Boehringer Mannheim) to a specific radioactivity 

of 375,000 cpm/g. Unincorporated 125 I was removed by passing 

the reaction products over a Sephadex G-25 column. 

Thiol Ester Assays—The relative enzymatic activities of the purified 

recombinant rat UBC4-1 and UBC4-testis proteins and of the mutant 

E2-containing bacterial lysates were determined by incubating the 

following in a total volume of 10 l: 50 mM Tris-HCl, pH 7.5, 1 mM DTT, 

2mM MgCl 2,2mM ATP, 100 nM E1, 20 units/ml inorganic pyrophosphatase, 

5 M 125 I-ubiquitin (3000 cpm/pmol), and 2.5 pmol of the 

purified E2s (5 pmol/l) or various amounts of the mutant E2 lysates. 

After incubation at 37 °C for 1 min, the reaction was stopped with 

Laemmli sample buffer without -mercaptoethanol and resolved by 

12.5% SDS-polyacrylamide gel electrophoresis at 4 °C followed by autoradiography. 

The thiol ester bands were excised from the gels to 

measure the incorporated radioactivity and thereby estimate the mutant 

E2 enzymatic activities relative to those of the purified native E2s 

(5 pmol/l). Assaying dilutions of the E2-containing extracts confirmed 

linearity of the assays. 

The [ 35 S]methionine-labeled E3s E6-AP (35) and rat p100 (36), covalently 

bound to GST-ubiquitin fusion protein in thiol ester linkages 

(17, 18), were detected by incubating the enzymes in the presence of 50 

mM Tris-HCl, pH 7.5, 1 mM DTT, 2 mM MgCl 2,2mM ATP, 50 nM E1, 20 

units/ml inorganic pyrophosphatase, 500 nM E2, and 2 l of each partially 

purified and concentrated E3 preparation. The reaction mixtures 

were preincubated at 25 °C for 3 min, and then the reaction was 

initiated with 1 g of GST-ubiquitin fusion protein (33), incubated at 

25 °C for 5 min, and stopped with Laemmli sample buffer with or 

without -mercaptoethanol. The reactions were resolved at 4 °C on an 

SDS-12.5% polyacrylamide gel, which was then soaked in ENHANCE 

(NEN Life Science Products) and autoradiographed. 

Conjugation Assays—For the conjugation of ubiquitin to endogenous 

substrates present in the testis nuclear fraction, the reaction mixture 

contained the following in a final volume of 20 l: 10 l of the 0.05 M 

NaCl nuclear fraction, 50 mM Tris-HCl, pH 7.5, 1 mM DTT, 2 mM MgCl 2, 

2mM AMP-PNP, 5 M 125 I-ubiquitin (3000 cpm/pmol), 50 nM E1, and 

250 nM E2s. The ubiquitination rate for the nuclear fraction was linear 

for 1 h at 37 °C, and this assay was performed in the presence or 

absence of 0.5 g of ubiquitin aldehyde, an isopeptidase inhibitor, or 40 

M MG132 (Proscript), a proteasome inhibitor. 

For the conjugation of ubiquitin to the exogenous substrate histone 

H2A, mediated by the testis cytosolic E3, the reaction mixture contained 

the following in a final volume of 20 l: 3 l of the cytosolic E3 

Superdex 200 fraction, 50 mM Tris-HCl, pH 7.5, 1 mM DTT, 2 mM MgCl 2, 

2mM ATP, 0.5 units pyrophosphatase, 12.5 mM phosphocreatine, 2.5 

units of creatine kinase, 250 nM E1, 125 I-histone H2A (specific activity 

of 375,000 cpm/g), and varied concentrations of E2 as indicated. Reactions 

were initiated with 25 M reductively methylated ubiquitin 

(RM-Ub), prepared, and quantified as described (38). Since histone H2A 

contains a number of lysine residues, mono- to penta-RM-Ub conjugates 

were formed, and the rate of formation of these conjugates was found to 

be linear for 10 min at 30 °C, permitting their quantification. 

RESULTS 

Differential Abilities of Rat UBC4-1 and UBC4-testis to Conjugate 

Ubiquitin to a Fraction of Testis Nuclear Proteins—To 

test whether the structural differences between rat UBC4-1 

and UBC4-testis conferred different abilities to conjugate ubiquitin 

to proteins, ubiquitination assays were performed using 

testis extracts fractionated on a MonoQ anion-exchange column. 

As shown previously (30), a nuclear fraction eluting at 

0.05 M NaCl supported conjugation of ubiquitin to proteins 

essentially only with the UBC4-1 isoform (Fig. 2A). This indicated 

that these two isoforms showed differential substrate 

specificity and suggested an enhanced ability of the ubiquitous 

isoform to conjugate ubiquitin to endogenous proteins in this 

fraction. To evaluate the possibility that UBC4-testis was conjugating 

ubiquitin to proteins that are preferentially de-ubiq-

18438 

FIG. 2.Differential abilities of UBC4-1 and UBC4-testis to conjugate 

ubiquitin to a fraction of testis nuclear proteins. A, a 

nuclear testis extract was chromatographed on a MonoQ anion-exchange 

column. The fraction eluting at 0.05 M NaCl was incubated with 

E1, AMP-PNP, and 125 I-ubiquitin in the presence or absence (E2) of 

rat UBC4-1 (4-1) or UBC4-testis (4-T)for1hat37°C.Similar reactions 

were also performed in the presence of ubiquitin aldehyde (Ub Ald), an 

isopeptidase inhibitor, or MG132, a proteasome inhibitor. Ubiquitinated 

endogenous substrates were then analyzed by SDS-polyacrylamide 

gel electrophoresis and autoradiography. The high molecular 

mass ubiquitin-substrate conjugates remain within the stacking gel 

during electrophoresis. B, the preparations of UBC4-1 and UBC4-testis 

(5 pmol/l each) used in A were tested for their abilities to form thiol 

esters with 125 I -ubiquitin in the presence of E1 and ATP. Ub, ubiquitin. 

uitinated by a co-purifying isopeptidase activity, the conjugation 

assay was performed in the presence of the isopeptidase 

inhibitor ubiquitin aldehyde. Notably, the level of UBC4-testisdependent 

conjugation was not increased by the addition of this 

reagent, rendering unlikely the possibility of a co-purifying 

interfering isopeptidase. Likewise, to rule out the possibility of 

enhanced proteasomal degradation of the UBC4-testis-dependent 

conjugates, the conjugation assay was performed in the 

presence of the proteasomal inhibitor MG132. Similarly, 

MG132 did not increase the levels of UBC4-testis-dependent 

conjugates. 

Significantly, the observed difference in conjugating ability 

between UBC4-1 and UBC4-testis was not due to a difference 

in the ability of these E2s to accept ubiquitin from E1 because 

UBC4-1 and UBC4-testis formed similar amounts of ubiquitin 

thiol esters (Fig. 2B). These thiol ester assays are end-point 

assays, and the results cannot exclude the possibility of different 

affinities of these two isoforms for E1. However, more 

detailed thiol ester-based enzyme kinetic studies suggest that 

UBC4-1 and UBC4-testis show less than a 2-fold difference in 

their affinities for E1 (data not shown). 

Rat Isoforms UBC4-1 and UBC4-testis Interact with the E3s 

E6-AP and Rat p100—Since the observed difference in conjugation 

by rat UBC4-1 and UBC4-testis was found not to be due 

to significant differences in their interaction with E1, this 

suggested that the specificity might arise at the E2-E3 level. 

We therefore tested the abilities of rat UBC4-1 and UBC4testis 

to interact with some well defined E3s, E6-AP (10, 17) 

and rat p100 (18), which contain HECT domains and thereby 

form thiol ester linkages with ubiquitin by accepting ubiquitin 

from E2 thiol esters. We tested the abilities of rat UBC4-1 and 

UBC4-testis to transfer ubiquitin to E6-AP and rat p100 produced 

by in vitro translation (Fig. 3). Since these E3s are 

relatively large, a GST-ubiquitin fusion protein (molecular 

mass of 34 kDa) (18) was used to permit resolution of the 

E3-ubiquitin thiol ester linkage by gel electrophoresis. In the 

presence of E1, GST-ubiquitin, and rat UBC4-1 or UBC4-testis, 

bands 34 kDa larger than the expected translation products 

were evident. These bands were not present when the thiol 

ester reactions were treated with -mercaptoethanol or when 

the reaction was performed in the absence of rat UBC4-1 and 

UBC4-testis. Again, these results obtained with thiol ester 


FIG. 3.UBC4-1 and UBC4-testis transfer ubiquitin similarly to 

the E3s E6-AP and rat p100. The rat p100 cDNA and human E6-AP 

cDNA were transcribed and translated in vitro using wheat germ extract. 

The 35 S-labeled translation products were partially purified over 

a DE52 column to remove endogenous E2s homologous to UBC4/UBC5. 

Thiol ester assays were then performed with the E3s using rat UBC4-1 

(4-1) or UBC4-testis (4-T) and a GST-ubiquitin fusion protein (GST- 

Ub). Reactions were quenched with SDS-polyacrylamide gel electrophoresis 

sample buffer without or with -mercaptoethanol (2-Me). After 

electrophoresis, the E3-ubiquitin thiol ester adducts (indicated by arrows) 

were detected by autoradiography. 

assays do not eliminate the possibility that these E2 isoforms 

could differ somewhat in their affinities for these E3s. More 

detailed kinetic assays are not feasible in these crude preparations 

from in vitro translation, where the concentrations of the 

E3s cannot be readily determined. Nonetheless, the results 

demonstrated that these two isoforms are both capable of interacting 

with ubiquitin-protein ligases as shown by their ability 

to transfer ubiquitin to at least two different E3s, E6-AP 

and rat p100. 

Rat Isoforms UBC4-1 and UBC4-testis Support Ubiquitination 

of Histone H2A Mediated by a Testis Cytosolic E3—Since 

the assays described above do not permit ready quantitative 

determinations of reaction rates, we tested both isoforms for 

their ability to support conjugation of ubiquitin to an exogenous 

substrate, histone H2A, mediated by a testis E3. To this 

end, a testis cytosolic E3 activity (29, 30) eluting at 0.4 M NaCl 

from a MonoQ anion-exchange column was further fractionated 

using a gel filtration column. This E3 activity was found to 

support conjugation of ubiquitin to the exogenous substrate 

125 I-labeled histone H2A in the presence of rat UBC4-1 and 

UBC4-testis. The effectiveness of these two isoforms in supporting 

this E3-dependent ubiquitination of 125 I-histone H2A 

was compared using E1, an ATP-regenerating system, RM-Ub 

(38), and different concentrations of rat UBC4-1 and UBC4testis 

(Fig. 4A). RM-Ub was utilized in the assays to restrict the 

ubiquitination of histone H2A to the mono-ubiquitinated form, 

which would facilitate the quantification of conjugates. Multiubiquitinated 

forms of histone H2A were observed, indicating 

that ubiquitin is attached to several different lysine residues of 

the molecule. In contrast to the differential ubiquitin-conjugating 

abilities of rat UBC4-1 and UBC4-testis observed in the

FIG. 4.UBC4-1 and UBC4-testis support conjugation of ubiquitin 

to histone H2A mediated by a testis cytosolic E3. A, the 

effectiveness of UBC4-1 or UBC4-testis in supporting E3-dependent 

ubiquitination of 125 I-histone H2A was compared using E1, an ATPregenerating 

system, RM-Ub, and different concentrations of rat 

UBC4-1 or UBC4-testis, incubated for 10 min at 30 °C. After electrophoresis, 

the RM-Ub- 125 I-histone H2A conjugates were detected by 

autoradiography. B, the differential conjugating abilities of rat UBC4-1 

and UBC4-testis observed in the nuclear fraction are not due to a 

UBC4-testis-specific inhibitor in the 0.05 M NaCl nuclear fraction. The 

above conjugation assay with UBC4-testis was performed in the presence 

or absence of the indicated amounts of the testis nuclear fraction 

eluting at 0.05 M NaCl from a MonoQ column. 

testis nuclear fraction, both isoforms support conjugation of 

ubiquitin to histone H2A mediated by the testis cytosolic E3 to 

similar extents. Thus, the preferential conjugating activity of 

UBC4-1 as compared with UBC4-testis observed in the nuclear 

fraction demonstrates that such specificity appears to be selective 

for specific E3s or substrates. 

To ascertain that the differential conjugating ability of rat 

UBC4-1 and UBC4-testis observed in the testis nuclear fraction 

was not due to an inhibitor of the UBC4-testis activity 

present in this fraction, the histone conjugation assay was 

performed in the presence or absence of the nuclear fraction 

(Fig. 4B). UBC4-testis supported E3-mediated conjugation of 

ubiquitin to 125 I-histone H2A to similar levels with or without 


the nuclear fraction. Thus, these data, in conjunction with the 

absence of effects of ubiquitin aldehyde and MG132, are consistent 

with the differential conjugating abilities of rat UBC4-1 

and UBC4-testis in the nuclear fraction being attributable to 

differences in their interactions with specific, and as yet unidentified, 

E3s or substrates. 

Four Amino Acid Differences Are Responsible for the Abilities 

of Rat Isoforms UBC4-1 and UBC4-testis to Conjugate Ubiquitin 

to Different Subsets of Testis Nuclear Proteins—Since the 

rat UBC4-1 and UBC4-testis isoforms differ by only 11 amino 

acids (Fig. 1) (30), this provided an unprecedented opportunity 

to identify, by site-directed mutagenesis, critical residues of the 

E2 molecule responsible for the observed difference in substrate 

specificity. Site-directed mutagenesis of the UBC4-testis 

isoform was undertaken to identify regions of the molecule that 

can confer the UBC4-1-like ability to conjugate ubiquitin to the 

endogenous substrates present in the nuclear fraction. 

Since rat UBC4-1 and UBC4-testis differ with respect to 

their pI values (30), mutagenesis of UBC4-testis began with the 

four residues responsible for the dramatic change in pI. Of 

particular interest were the two residues, aspartic acid 55 and 

glutamic acid 68, located near the active-site cysteine, that 

were mutated to histidine and alanine, respectively. However, 

mutation of UBC4-testis to the UBC4-1 residues at these two 

sites did not confer UBC4-1-like ability to conjugate 125 I-ubiquitin 

to the substrates present in the testis nuclear fraction 

(Fig. 5A). Additional mutagenesis of glutamines 15 and 125, 

the two other residues responsible for the change in pI, to 

arginines resulted in a small increase in conjugating activity of 

the mutated UBC4-testis. Since mutagenesis of the two glutamine 

residues appeared to increase the conjugating activity of 

the mutated UBC4-testis, further residues nearby were mutated. 

Mutation of alanine 49 to valine conferred some increase 

in activity, and an additional mutation of serine 107 to cysteine 

conferred a UBC4-1 phenotype in conjugating ability to the 

mutated UBC4-testis. 

To determine the minimal number of substitutions required 

for the UBC4-1-like conjugating activity of the mutated UBC4testis, 

subtractive mutagenesis was then performed. Since mutagenesis 

of Gln-15, Ala-49, Ser-107, and Gln-125 improved 

conjugating activity, these four mutations were tested together 

and found to be sufficient (Fig. 5A). Further removal of any one 

of these substitutions decreased conjugating ability significantly, 

and therefore, these four substitutions are necessary 

(Fig. 5B). 

Our observation that these residues are important would 

predict that mutating the rat UBC4-1 molecule at these positions 

to the UBC4-testis residues would decrease the conjugating 

activity of UBC4-1. To test this prediction, mutagenesis of 

UBC4-1 to UBC4-testis was performed at these four residues. 

As expected, mutagenesis of each of the critical residues (Arg- 

15, Val-49, Cys-107, or Arg-125) resulted in decreased conjugating 

activity of rat UBC4-1 in the testis nuclear fraction (Fig. 

6, A and B). Interestingly, the four residues in UBC4-testis, 

which were found to be necessary and sufficient for the UBC4- 

1-like conjugating activity, are present on surfaces away from 

the active site (Fig. 7) (31). 

DISCUSSION 

We have, for the first time, identified, in the core domain 

conserved among all ubiquitin-conjugating enzymes, specific 

sites that are involved in determining substrate specificity of 

conjugation. These studies revealed a number of intriguing 

insights. First, although we anticipated, based on the 93% 

amino acid identity between the two isoforms, that only a 

limited number of residues in the core domain of E2s may 

dictate conjugation of ubiquitin to different proteins, surpris-

18440 

FIG. 5. Only four residues are necessary 

and sufficient to confer a rat 

UBC4-1 phenotype on UBC4-testis. A, 

bacterially expressed mutant UBC4-testis 

proteins were tested for their ability to 

support conjugation with the 0.05 M NaCl 

testis nuclear fraction in assays as described 

in the legend to Fig. 2. B, the 

minimal number of substitutions required 

for UBC4-1-like conjugating activity 

in the testis nuclear fraction was determined. 

Removal of some of these 

substitutions appeared to decrease conjugating 

ability, and therefore, these four 

substitutions appear necessary. C, the 

conjugating activities of the UBC4-testis 

mutants relative to the UBC4-1 isoform 

were compared by counting the radioactivity 

in the gel lanes (S.E.) and were 

normalized to the value for UBC4-1. D, 

Asp-55; E, Glu-68; Q1, Gln-15; Q2, Gln- 

125; A, Ala-49; S, Ser-107. 

ingly only four substitutions (Figs. 5 and 6) were sufficient and 

necessary to produce the change in substrate specificity. 

Second, some of these substitutions were physiochemically 

relatively conserved, indicating that subtle changes in primary 

structure can be important in determining selectivity of substrates. 

One involved an Ala to Val conversion, whereas the 

other involved a Cys for Ser substitution. The other two substitutions, 

on the other hand, were nonconserved. The polar 

uncharged Gln residues in UBC4-testis have been mutated to 

the polar basic Arg residue. Thus, part of the interaction of 

UBC4-1 and a testis nuclear substrate or E3 could be due to 

electrostatic interactions between the molecules. However, differences 

in electrostatic attractions alone are unlikely to account 

for the specificity observed here because all four residues, 

including the two conserved substitutions, are necessary for 

conferring a UBC4-1-like phenotype on UBC4-testis. Since all 

four residues are on the surface, their substitution is unlikely 

to induce a significant conformational change on the E2 molecule. 

Instead, it may be that all four of these critical residues 

result in a surface configuration that functions to lower the free 

energy of binding of UBC4-1 to an E3 or substrate, thereby 

facilitating the UBC4-1-dependent conjugation of ubiquitin to 

specific proteins in the testis nuclear fraction. 

Third, the four critical residues in UBC4-1 are not localized, 

but spread over a broad surface of the E2 molecule away from 

the active site (Fig. 7), and so a large surface of the E2 core may 

be involved in binding an E3 or substrate. The critical residues 

are localized as follows: the N-terminal Gln-15 is in the stretch 

between the first -helix and the first -strand; the N-terminal 

Ala-49 is in the third -strand; the C-terminal Ser-107 is in the 

second -helix; and finally, the N-terminal Gln-125 is in the 

third -helix. 


Although all E2s exhibit limited sequence identity and are 

functionally different, the overall three-dimensional folding of 

E2 core domains that have been crystallized to date is remarkably 

similar (31, 40–42). The 150 amino acids of the E2 core 

domains show 25% sequence identity, and notably, most of 

the identical residues are either buried or clustered on one 

surface adjacent to the active-site cysteine (31). It has been 

suggested that the highly conserved surface region around the 

active site may be specific for ubiquitin and/or E1 binding, 

whereas the divergent surface regions may enable individual 

E2 enzymes to bind their respective substrates or E3s (31). Our 

data now provide experimental evidence to support this 

hypothesis. 

Although we have not to date been able to identify an exogenous 

substrate that requires the nuclear fraction for conjugation, 

other studies (30, 43) showing that this family of E2s 

interacts extensively with E3s would suggest that the nuclear 

fraction probably does contain an E3 activity. Recent findings 

suggest that a putative E2-binding site exists in the C terminus 

of HECT domain-containing E3s and that a variable E3 N 

terminus may be involved in binding substrates (44). Since the 

residues found to be critical for the UBC4-1-like conjugating 

activity lie on a surface away from the active site, this surface 

may be responsible for the selective interaction of UBC4-1 with 

a nuclear E3. This could permit the small E2 molecule, while 

bound to the larger E3 protein (known E3s are 95 kDa in 

size), to expose its active-site cysteine, thereby facilitating the 

transfer of ubiquitin to the E3 if it contains a HECT domain or 

directly to a substrate if the E3 is functioning primarily as a 

docking protein (Fig. 8). 

Fourth, it is possible that some E2s can functionally overlap 

with some E3s or substrates, yet be selective for other E3s or

FIG. 6. Mutation of the critical residues in UBC4-1 confirms 

that they are necessary to conjugate ubiquitin to a subset of 

testis nuclear proteins. A, bacterially expressed mutant UBC4-1 

proteins were assayed for their ability to support conjugation with the 

0.05 M NaCl testis nuclear fraction as described in the legend to Fig. 2. 

B, the conjugating activities of the UBC4-1 mutants relative to the 

UBC4-1 isoform were compared by counting the radioactivity in the gel 

lanes (S.E.) and were normalized to the value for UBC4-1. R1, Arg-15; 

R2, Arg-125; V, Val-49; C, Cys-107. 

substrates. For example, the critical residues that confer a 

UBC4-1-like phenotype on UBC4-testis likely result in a distinctive 

E2 surface configuration that is selectively recognized 

by a specific nuclear E3. However, other E3s, such as rat p100, 

E6-AP, and the testis cytosolic E3 we have identified, may 

interact principally with residues that are conserved among 

these E2s (Figs. 3 and 4). Indeed, the functional specificity of 

distinct E2s may be contingent upon specific residues in these 

molecules that facilitate or impair their interaction with different 

E3s or substrates. In support of this, it has been shown that 

the mouse homologues of yeast Rad6, mHR6A and mHR6B, 

which show 95% amino acid sequence identity (Fig. 1), may be 

functionally distinct since inactivation of the mHR6B gene, but 

not the mHR6A gene, in mice causes male sterility (45). Thus, 

mHR6A appears to complement some functions of mHR6B, but 

not all of them. The minor sequence differences between the 

two isoforms may therefore be responsible for the selectivity of 

binding to some E3s or substrates. 

Recently, three-dimensional structure determination of 

yeast Ubc7 (41) revealed that its tertiary folding is similar to 

other class I enzymes that have been crystallized, with the 

exception of two regions where extra residues are present in 

Ubc7. Based on amino acid sequence alignment between 13 

yeast E2 enzymes, Cook et al. (41) suggested that there are four 

potential regions where extra residues could be inserted into 

the common core domain of various E2s and that these may 

represent hypervariable regions that confer specificity for bind- 


FIG. 7.The critical residues essential for UBC4-1-like conjugation 

are dispersed on surfaces away from the active-site cysteine. 

Shown is the crystal structure of yeast Ubc4 (31). Rat UBC4-1 

shows 80% amino acid identity to yeast Ubc4, and rat UBC4-testis 

shows 76% identity. Shown in red on Ubc4 are the locations of the 

corresponding residues of UBC4-testis required for UBC4-1-like conjugating 

activity in the testis nuclear fraction. These substitutions are 

located on surfaces away from the active-site cysteine (yellow). 

FIG. 8.A model for the interaction of E2s with their respective 

E3s. The critical residues on E2 molecules that facilitate their interaction 

with E3s or substrates (Sub) may be dispersed on surfaces of the E2 

core away from the active site, and these surfaces may be responsible 

for the selective interaction of E2s with different E3s. This would 

permit the E2 molecule, while bound to a larger E3 protein, to expose its 

active-site cysteine, thereby facilitating the transfer of ubiquitin (Ub)to 

an E3 if it contains a HECT domain (A) or directly to a substrate if the 

E3 is functioning primarily as a docking protein (B). 

ing substrates or E3s. Notably, these four hypervariable regions 

are located on one broad surface surrounding the activesite 

cysteine. It has been hypothesized that the two insertions 

in the Ubc7 core domain (Fig. 1) may be critical for its role in 

targeting specific substrates (e.g. Mat2, Sec61p, and YscY) (8, 

46, 47) for ubiquitin-dependent degradation. Although this hypothesis 

of a hypervariable surface contributing to substrate 

and/or E3 specificity may prove correct, functional evidence is 

still lacking. In contrast, functional evidence presented here 

demonstrates that surfaces away from the ubiquitin-accepting 

cysteine, and thus away from the surface containing the hypervariable 

regions, are critical for determining the substrate 

specificity of the rat UBC4 isoforms. This involvement of a 

large surface of the E2 (Figs. 7 and 8) in determining substrate 

selectivity is an attractive concept as it can explain how small 

E2 molecules can encode such a diverse range of specificities.

18442 

Significantly, these results represent the first detailed mutagenesis 

of an E2 molecule related to substrate specificity. 

Unlike most studies of structure-function relationships that 

use mutagenesis to create artificial mutants with distinct properties, 

our studies have been based on naturally occurring 

isoforms. Thus, the different biochemical phenotypes based on 

these four critical residues are likely to be biologically important. 

The existence of such highly similar isoforms in the same 

tissue may not be redundant, but rather may permit fine regulation 

of conjugation of ubiquitin to specific substrates. The 

precise induction of UBC4-testis at the round spermatid stage 

of spermatogenesis would also argue for a specific function of 

this isoform (30). Inactivation of this gene in the mouse is 

currently underway and will likely yield further insights into 

the determinants of function present in the core domains of 

class I E2s. 

Acknowledgments—We thank J. Huibregtse for the pGEX-ubiquitin 

plasmid, D. Muller for the cDNA encoding rat p100, P. Howley for the 

E6-AP construct, A. Ciechanover for ubiquitin aldehyde, Proscript for 

MG132, and A. Haas for the protocol for purifying E1 from rabbit liver. 

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