©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Characterization of a Novel Keratinocyte Ubiquitin Carrier Protein (*)

(Received for publication, September 4, 1995; and in revised form, November 20, 1995)

Zhi Liu (1) (2)(§) Arthur L. Haas (2) Luis A. Diaz (1) (3) Catherine A. Conrad (2) George J. Giudice (1) (2)(¶)

From the  (1)Departments of Dermatology and (2)Biochemistry, Medical College of Wisconsin, and the (3)Veterans Affairs Medical Center, Milwaukee, Wisconsin 53226

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

A novel member of the ubiquitin carrier protein family, designated E2, has been cloned by our laboratory and expressed in a bacterial system in an active form. Ubiquitin carrier proteins, or E2s, catalyze one step in a multistep process that leads to the covalent conjugation of ubiquitin to substrate proteins. In this paper, we show that recombinant E2 catalyzes auto/multiubiquitination, the conjugation of multiple ubiquitin molecules to itself. Multiubiquitination has been shown previously to be required for targeting of a substrate protein for rapid degradation. Using a rabbit reticulocyte lysate system, E2 was shown to support the degradation of a model substrate in an ATP- and ubiquitin-dependent fashion. In contrast to a previous study which showed that selective protein degradation in one system is dependent upon multiubiquitination via the lysine 48 residue of ubiquitin, multiubiquitination, and proteolytic targeting by E2 was shown here to be independent of the lysine 48 multiubiquitin linkage. This functional characterization of E2 revealed a combination of features that distinguishes this enzyme from all previously characterized members of the ubiquitin carrier protein family. These results also suggest several possible autoregulatory models for E2 involving auto- and multiubiquitination.


INTRODUCTION

Ubiquitin, a highly conserved 76-amino acid polypeptide, is present in all eukaryotic cells either in a free state or covalently conjugated to various cellular proteins(1, 2) . Ub-protein conjugates are formed through an isopeptide linkage between the carboxyl-terminal glycine residue of ubiquitin and -amino groups of substrate proteins (3, 4) . Ub-protein conjugates typically account for about 50% of total cellular ubiquitin (5) and have been localized to various cellular compartments including the cytosol(6) , nucleus(7) , cell surface(8) , and mitochondrion(6) . Ubiquitin conjugation has been implicated in a variety of cellular functions. The best understood of these is the targeting of the substrate protein for selective degradation via a nonlysosomal pathway(9) . Other cellular processes mediated by the ubiquitin conjugation system include DNA repair(10) , cell cycle progression(11) , regulation of chromatin structure(12) , cell surface recognition(13) , and regulation of transcription factors, e.g. NFkappaB(14) .

The formation of ubiquitin-protein conjugates involves a three-step process(15, 16) . The first step is the ATP-dependent activation of ubiquitin by the 105-kDa ubiquitin activating (E1) (^1)enzyme (15, 17) involving the formation of a thiol ester linkage between the ubiquitin carboxyl terminus and a thiol group of E1. In the second step, ubiquitin is transferred to the active site cysteine residue of a ubiquitin carrier protein (or E2). In the last step, an isopeptide bond is formed between the carboxyl terminus of ubiquitin and a lysyl -amino group within a substrate protein, a reaction catalyzed either directly by the E2 enzyme or via a third enzyme designated isopeptide ligase (E3). Enzymes designated ubiquitin isopeptidases have also been described which deubiquitinate conjugates(18) , consistent with evidence for the dynamic balance governing ubiquitin adduct pools(5) .

E2s exist as a family of isozymes that exhibit variability in terms of molecular weight, physiological function, substrate specificity, and dependence on E3 in in vitro systems(2, 19, 20) . For example, in the yeast Saccharomyces cerevisiae, eight genes (designated UBC1-8) encoding distinct E2s have been described(2, 21) . The RAD6 (UBC2) protein functions in DNA repair, sporulation, and induced mutagenesis(10) . CDC34 (UBC3) is an E2 of 24 kDa that is essential for G(1)/S transition during mitosis(11) . The basic structure of E2s consists of a 153-amino acid core domain containing an active site cysteine within a highly conserved random coil segment. Additional carboxyl-terminal extension domains present on many isozymes are often acidic and generally show a high degree of sequence divergence, suggesting that they may play a role in substrate specificity during E3-independent conjugation or may contribute to the specificity of binding to cognate E3 isoforms.

A subset of E2s has been shown to support multiubiquitination, a process in which successive ubiquitin molecules are linked by an isopeptide bond between a side chain amino group of one ubiquitin and the terminal carboxyl group of a second ubiquitin molecule. In vitro studies have demonstrated that E3-independent multiubiquitination by several E2 isozymes is sufficient for degradative targeting by the 26 S multicatalytic protease complex(22) . In contrast, polyubiquitination refers to the conjugation of multiple ubiquitin molecules directly to different lysine residues within a single substrate molecule. Studies show that RAD6, CDC34, and the rabbit reticulocyte E2 catalyze E3-indepedent multiubiquitination and support E3-dependent ubiquitin conjugation; however, these enzymes differ in their linkage specificity for multiubiquitination, with CDC34 and E2 using Lys-48 (20) and RAD6 utilizing Lys-6(20) . (^2)Multiubiquitination via lysine 48 of ubiquitin has also been demonstrated in the E2 of wheat germ (23) and E2 from calf thymus(24) . Formation of branched, multiubiquitin adducts have been shown to be involved in targeting of the substrate protein for selective degradation by an ATP-dependent protease complex(25) . Chau and co-workers (26, 27) have shown that proteolytic targeting can be inhibited by preventing multiubiquitination via the lysine 48 residue of ubiquitin.

A novel member of the E2 protein family, E2, has recently been cloned from human keratinocytes(28) . E2 is unique among E2s in that it contains a highly basic carboxyl-terminal extension domain. The E2 transcript was also shown to encode an antigenic polypeptide recognized by autoantibodies from pemphigus foliaceus patients. E2 is therefore the first member of the E2 enzyme family to be implicated in a disease process. The present paper reports the detailed enzymatic characterization of recombinant E2, revealing a set of functional properties that distinguishes this isozyme from all other characterized E2s. E2 is shown to exhibit auto- and multiubiquitination activities. We further show that this E2 supports the ubiquitin-dependent protein degradation pathway in the absence of Lys-48 multiubiquitination. The latter observation indicates that multiubiquitination by linkage other than Lys-48 are competent degradative intermediates, supporting a role for subpopulations of ubiquitin having different linkage specificities within the overall pathway of ATP, ubiquitin-dependent protein degradation.


MATERIALS AND METHODS

Ubiquitin, rcmBSA (reduced, carboxymethylated form of BSA), UbK48R (site-directed mutant of Ub in which the lysine residue at position 48 has been replaced by arginine), and rmUb (reductively methylated form of ubiquitin in which all free amino groups are blocked by methyl groups) were prepared as described previously(20) . Rabbit reticulocyte E1 and E2 were purified to homogeneity by a combination of affinity and high performance chromatography and then quantitated by stoichiometric activity assays(29) . Recombinant yeast RAD6 was expressed and purified as described previously(20) . Protein concentrations were determined by the Bio-Rad dye binding assay using BSA as a standard. All other proteins and reagents were purchased from Sigma unless otherwise indicated.

Preparation of E1/E2-depleted Reticulocyte Fraction II

The removal of endogenous ubiquitin, E1, and various E2 isozymes from rabbit reticulocyte lysate was accomplished using previously described procedures(29, 30) . Rabbit reticulocyte lysate was depleted of ubiquitin according to a modified procedure for fraction II(29, 30) followed by covalent affinity depletion of E1 and E2s as described previously(29) . The resulting unadsorbed fraction from the ubiquitin affinity column (depleted fraction II) was determined to be quantitatively depleted of ubiquitin, E1, and E2 isozymes by its inability to support I-ubiquitin conjugation without supplementation by exogenous E1 and E2 and inability to support I-rcmBSA degradation without further addition of ubiquitin.

Expression and Purification of Cloned E2

Cloned E2 was expressed in Escherichia coli DH5alpha harboring the pGEXEPF5-ORF2B expression construct(28) . Cells were grown at 37 °C in LB medium containing 50 µg/ml ampicillin. When the culture reached A of 0.5, isopropyl-1-thio-beta-D-galactopyranoside was added to a final concentration of 0.5 mM. After an additional 3-h incubation, the cells were harvested by centrifugation and resuspended in 0.04 volume of solution TD (50 mM Tris-Cl (pH 7.5) and 1 mM DTT). The cells were lysed by passing the suspension twice through a French press at 1000 p.s.i. The cell lysate containing the glutathione S-transferase (GST)-E2 fusion protein was clarified by centrifugation at 10^5 times g for 60 min at 4 °C. The fusion protein was purified by glutathione-agarose affinity chromatography as described previously (31) . Ten ml of the clarified cell extract was mixed with 10 ml of 50% (v/v) glutathione-agarose (Sigma) suspension (in solution TD) in a 50-ml capped tube and rocked at 4 °C for 30 min. The agarose beads were washed 5 times with 30 ml of solution TD. The washed beads were then poured into a column and washed again with 5 bed volumes of solution TD. The bound fusion protein was eluted with 10 mM glutathione (Sigma) in solution TD. The capacity of the glutathione-agarose affinity column was calculated to be approximately 1 mg of fusion protein per mg of the glutathione-agarose matrix under the conditions described.

Isolation of E2 (cDNA-encoded polypeptide without the amino-terminal GST moiety) was accomplished as follows. The immobilized fusion protein was incubated with a highly purified preparation of thrombin (4000 NIH units/mg of protein; Sigma) at a concentration of 30 NIH units/ml in TD buffer resulting in elution of the E2 while the GST moiety of the fusion protein remained bound to the column. Optimal digestion conditions were 1 unit of thrombin per 5 µg of fusion protein at 25 °C for 1-2 h. Longer incubations resulted in partial degradation of E2 (data not shown). To remove minor contaminants including thrombin, undigested fusion protein, and GST, the eluted fraction was subjected to further purification by anion exchange chromatography(29) . Prior to chromatography, the sample was filtered through a 0.2-µm membrane. The filtrate was chromatographed at 4 °C on a Mono Q HR 5/10 anion exchange column equilibrated with solution TD using a Pharmacia Biotech Inc. fast protein liquid chromatography system(29) . Flow rate was maintained at 1 ml/min during sample loading and gradient elution. After sample injection, the column was washed with solution TD and eluted with a linear 0-0.5 M NaCl gradient having a slope of 12.5 mM/min. Eluted fractions were assayed by both SDS-PAGE and ubiquitin thiol ester formation(29) . E2 consistently eluted as a single peak at 125 mM NaCl. A typical yield of E2 based on the thiol ester formation assay was approximately 10 nmol/liter bacterial culture. Purified E2 was stored at -80 °C. Under these conditions, E2 retained full activity for over 8 months and after two cycles of freeze thawing.

Assay of Thiol Ester Formation

Purified E2 was assayed for the ability to form a thiol ester linkage with ubiquitin, according to the procedure of Haas and Bright(29) . Briefly, E2 was incubated with I-ubiquitin (5 µM, 5-10 times 10^4 cpm/pmol) in the presence of 10 nM purified reticulocyte E1, 20 IU/ml inorganic pyrophosphatase, 2 mM ATP, 5 mM MgCl(2), 0.5 mM DTT, and 50 mM Tris-Cl (pH 7.5). After a 1-min incubation at 37 °C, the reaction was stopped by addition of an equal volume of 2 times SDS-PAGE sample buffer without 2-mercaptoethanol. Replicate-quenched samples were boiled for 1 min in the presence of 5% 2-mercaptoethanol to cleave the I-ubiquitin thiol ester(16) . All samples were then resolved by SDS-PAGE (12% acrylamide), and I-ubiquitin-containing bands were visualized by autoradiography(29) .

Quantification of E2 activity was accomplished using a modification of the above thiol ester assay. One-minute incubations containing different amounts of E2 were analyzed by electrophoresis and autoradiography. Thiol ester bands were cut from the gel and quantified by counting on an automatic counter (Micromedic 4/600 Plus, ICN Micromedic Systems Inc.). The absolute content of thiol ester was calculated based on the specific activity of I-ubiquitin(32) .

Assay of Ubiquitin Conjugation

Conjugation assays for measuring steady state levels of conjugates were performed at 37 °C for 60 min as described previously(29, 33) . The assay mixtures contained 50 mM Tris-Cl (pH 7.5), 2 mM ATP, 0.5 mM DTT, 10 mM MgCl(2), 20 IU/ml inorganic pyrophosphatase, various concentrations of I-ubiquitin (native, UbK48R, or rmUb), purified rabbit reticulocyte E1, and purified E2 (E2, E2, or CDC34). The reaction products were resolved by SDS-PAGE (12% acrylamide) and visualized by autoradiography.

A similar approach was used to assay the conjugation of ubiquitin to endogenous reticulocyte proteins. Assays contained 50 mM Tris-Cl (pH 7.5), 2 mM ATP, 10 mM MgCl(2), 0.5 mM DTT, 20 IU/ml inorganic pyrophosphatase, 5 µMI-ubiquitin (4-8 times 10^4 cpm/pmol), 10 nM E1, indicated concentrations of E2s, and the depleted fraction II (158 µg) as a source of both E3 and substrates.

Assay of Protein Degradation

Degradation of reduced, carboxymethylated BSA (I-rcmBSA) was quantified by generation of trichloroacetic acid-soluble radioactivity similar to that described previously(9) . Briefly, the reactions of 50 µl total volume containing 50 mM Tris-Cl (pH 7.5), 0.5 mM DTT, 10 mM MgCl(2), 2 mM ATP, 20 IU/ml creatine phosphokinase, 5 µM ubiquitin, 4 µMI-rcmBSA (4 times 10^4cpm/pmol), 10 nM E1, the indicated concentrations of E2s, and 158 µg of depleted fraction II were incubated at 37 °C for 60 min. The reactions were stopped by addition of 100 µl of 5 mg of BSA/ml as carrier followed by 150 µl of ice-cold 16% trichloroacetic acid. Following a 10-min precipitation on ice, the samples were centrifuged at 4 °C for 5 min in an Eppendorf microcentrifuge. The level of radioactivity in each 200 µl of supernatant was determined by counting using a Micromedic 4/600 Plus Automatic Gamma Counter (ICN Micromedic Systems, Inc.). Acid-soluble radioactivity present in the blank was subtracted from each reading, and the results were expressed as the net counts/min.


RESULTS

Expression and Purification of E2

The coding region of the E2 cDNA was subcloned into the prokaryotic expression vector, pGEX-2T (Pharmacia), to generate construct pGEXEPF5-ORF2B as described previously(5) . The fusion protein encoded by this construct consists of the full-length 24-kDa E2 fused to the carboxyl terminus of a 26-kDa segment of GST. The recombinant protein was purified using the glutathione-agarose affinity chromatography procedure essentially as described by Smith and Johnson(31) . As shown in Fig. 1, lanes 1 and 2, this single step affinity purification yielded an electrophoretically homogeneous preparation of the GST-E2 fusion protein. Separation of the GST and E2 moieties of the fusion protein was accomplished by thrombin digestion followed by Mono Q anion exchange fast protein liquid chromatography as described under ``Materials and Methods.'' The purity of the resulting E2 polypeptide was demonstrated by electrophoretic analysis (Fig. 1, lane 3). The apparent molecular weight of the E2 polypeptide agreed with the size predicted by sequence analysis. The Mono Q chromatography step resolved intact E2 from minor fragmentation products generated during thrombin processing (not shown). Note that E2 exhibits a more intense level of staining by the silver technique relative to that of an equal mass of GST-E2 fusion protein (Fig. 1, lanes 2 and 3). This is attributable to differences in mole amounts between the two proteins when normalized for equal mass and a greater mole fraction of lysine for E2 compared to GST, since lysine serves as the site of silver deposition.


Figure 1: Purification of recombinant E2. Purity of GST-E2 fusion protein and E2 was determined by SDS-PAGE analysis followed by silver staining. Lane 1, total protein extract (43 µg of protein) of E. coli DH5alpha harboring pGEXEPF5-ORF2B and induced with isopropyl-1-thio-beta-D-galactopyranoside; lane 2, GST-E2 fusion protein (1 µg) eluted from a glutathione-agarose affinity column; lane 3, recombinant E2 (1 µg) after thrombin digestion and FPLC Mono Q column. The marker proteins were rabbit muscle phosphorylase b (97 kDa), BSA (66 kDa), ovalbumin (43 kDa), bovine carbonic anhydrase (31 kDa), and soybean trypsin inhibitor (22 kDa).



The GST-E2 fusion protein is catalytically active in forming the corresponding I-ubiquitin thiol ester in the presence of E1(28) . Fig. 2shows that recombinant E2 processed from GST by thrombin and subsequently purified by Mono Q FPLC is also active in ubiquitin thiol ester formation (lane 5). The amount of thiol ester formed to free E2, determined by quantification of I radioactivity associated with the corresponding band in lane 5 of Fig. 2(20) , agreed with that predicted from the mass of E2 protein determined as described under ``Materials and Methods.'' The I-ubiquitin thiol ester adducts of both E2 and GST-E2 formed in incubations parallel to those of Fig. 2were quantitatively labile to reducing conditions in the presence of 2-mercaptoethanol (not shown), confirming that the associated I-ubiquitin was in thiol ester linkage(20) . We consistently noted that, at equimolar concentrations, free E2 formed more ubiquitin thiol ester than did the GST-E2 fusion protein (Fig. 2, lanes 4 and 5). The amount of thiol ester formed to the fusion protein did not increase on longer incubation and thus did not result from a lower rate of transthiolation from the E1 ternary complex (not shown). The lower level of GST-E2 thiol ester also did not result from inactivation since the predicted amount of free E2 thiol ester, based on protein determination, was observed if the fusion protein was first incubated with thrombin (not shown). These results indicate that the presence of GST at the amino terminus of E2 sterically alters the equilibrium constant rather than the rate of thiol ester formation from the E1 ternary complex.


Figure 2: Ubiquitin thiol ester formation with GST-E2 and E2. Thiol ester formation of radioiodinated ubiquitin was performed at 37 °C for 1.5 min in the presence or absence of rabbit reticulocyte E1 (2.5 pmol), GST-E2 fusion protein (1 pmol), or E2 (1 pmol). The reaction products were analyzed by 12% SDS-PAGE under nonreducing conditions. Lane 1, E1 alone; lane 2, GST-E2 alone; lane 3, E2 alone; lane 4, E1 plus GST-E2; lane 5, E1 plus E2.



E2 Catalyzes Auto- and Multiubiquitination

The results of Fig. 2demonstrate that transthiolation from the E1 ternary complex to either free E2 or the GST fusion is complete within the 1.5-min incubation time employed, as is typical of the analogous loading reaction with other members of the E2 family(20) . On longer incubation with free E2, a series of higher molecular weight I-ubiquitin adducts was consistently observed on nonreducing thiol ester gels (Fig. 3A). Unlike the I-ubiquitin-E2 thiol ester formed following short incubation (Fig. 2), the higher molecular weight bands formed during prolonged incubation were stable to reducing conditions, Fig. 3B, indicating that they represented peptide conjugates to radioiodinated ubiquitin. In addition, a small fraction of the monoubiquitin adduct was also stable to reducing conditions and showed a time-dependent accumulation on prolonged incubation. Therefore, the E2-Ub1 band in Fig. 3represents a mixture of I-ubiquitin thiol ester and conjugate adducts at long incubation times but principally only thiol esters at short times. The relative molecular weights of the ladder of ubiquitin conjugates corresponded to integer multiples of the molecular weight for ubiquitin ligated to free E2. The bands present under reducing conditions in Fig. 3B were confirmed as E2-ubiquitin conjugates by comigration of identical bands when similar incubations containing unlabeled ubiquitin were analyzed on immunoblots probed with either anti-E2 or antiubiquitin antibodies (not shown). Thus, E2 catalyzes an E3-independent autoubiquitination reaction resulting in the formation of multiubiquitin homopolymer chains.


Figure 3: Time course of E2-dependent multiubiquitination. Conjugation of I-ubiquitin to E2 was allowed to proceed for the amount of time indicated at 37 °C in incubations of 25 µl containing 2.5 pmol of E1 and 1 pmol of E2. Conjugation reactions were quenched with 25 µl of sample buffer, and 20-µl aliquots were resolved by 12% SDS-PAGE under either nonreducing (A) or reducing conditions (B). Lane c shows the products of the above reaction in the absence of E2. Migration positions for E1 thiol ester and E2-Ub1 are indicated by the upper and lower arrows, respectively, shown at the left of each panel.



E2 Catalyzes the Formation of Multiubiquitin Chains with Linkage Specificity Distinct from Lysine 48

Accumulation of autoubiquitinated E2 adducts of molecular weights greater than that predicted for monoubiquitination of all 17 lysines within E2 (Fig. 3) indicates that some or all of the attached ubiquitin moieties are present as multiubiquitin chains. To demonstrate this conclusively, E2 autoubiquitination was carried out in the presence of I-rmUb which is incapable of supporting chain elongation due to the absence of primary lysyl -amines(20, 33) . Inability to form adducts above E2-Ub(2) (Fig. 4, lane 2), under conditions for which there is accumulation of these products with wild-type ubiquitin (lane 1), demonstrates that ubiquitin moieties ligated to E2 can undergo chain elongation. The appearance of two ubiquitinated-E2 adducts with I-rmUb indicates that under these incubation conditions there are at least two lysines residues within E2 which can become modified by ubiquitination, with one site being kinetically preferred. Control experiments performed with CDC34 showed no formation of higher molecular weight CDC34-ubiquitin conjugates when autoubiquitinated with I-rmUb (Fig. 4, lane 5) indicating that larger molecular weight adducts also result from chain elongation which is consistent with previous reports(20) . Longer incubation of E2 autoubiquitination products with wild type Ub results in the loss of monoubiquitinated E2. During multiubiquitin chain elongation, E2 catalyzes a linkage specificity distinct from lysine 48 since both native I-ubiquitin and I-UbK48R show similar conjugate patterns (Fig. 4, lanes 1 and 3). This is in contrast to CDC34 which utilizes lysine 48(20) , confirmed by the absence of higher molecular weight CDC34-ubiquitin adducts (Fig. 4, lanes 4 and 6).


Figure 4: Characterization of ubiquitin linkages formed by E2. E3-independent multiubiquitination was conducted at 37 °C for 8 min with E2 and for 30 min with CDC34. Incubations of 50 µl contained 4 pmol of liver E1, 4 pmol of either E2 or yeast CDC34, and 5.0 µMI-Ub (lanes 1 and 4), I-rmUb (lanes 2 and 5), or I-UbK48R (lanes 3 and 6). The products were resolved by 10% SDS-PAGE under reducing conditions and visualized by autoradiography. Sample volumes were adjusted to correct for slight variations in specific activities of the three radioiodinated proteins.



E2 Supports E3-dependent Ubiquitin Conjugation

Those E2 isoforms capable of catalyzing E3-independent conjugation are bifunctional and support significant rates of E3-dependent ubiquitin ligation(20, 33) . The ability of E2 to also support E3-dependent I-ubiquitin conjugation was tested using reticulocyte fraction II depleted of endogenous E1 and E2 isoforms by prior passage through a ubiquitin affinity column (see ``Materials and Methods''). Initial rates of conjugation were measured in 5-min incubations to preclude significant accumulation of the E2 auto-multiubiquitination products observed in Fig. 3B. Fig. 5shows the electrophoretic pattern of the products of these conjugation reactions. No conjugation of radioiodinated ubiquitin is observed in the presence of only pure E1 (lane 2) or E2 (lane 3), confirming that the fraction II is quantitatively depleted of both activating enzyme and the major cognate E3-dependent E2 isoform(20) . Comparison of lanes 1-3 of Fig. 5also demonstrate that the purified E1 and E2 proteins do not contain catalytically significant concentrations of other components required for ubiquitin ligation. Supplementation of the depleted fraction II with both E1 and E2 results in a significant accumulation of I-ubiquitin conjugates (Fig. 5, lane 4). Like reticulocyte E2, E2 supported conjugation of ubiquitin to endogenous proteins present in the depleted fraction II in the presence of added E1 (Fig. 5, lane 6), but not in its absence (lane 5). The size distribution of the resulting adducts indicated that they consist primarily of conjugates between ubiquitin and endogenous reticulocyte proteins, although certain of the lower molecular weight bands may correspond to E2-Ub1 and E2-Ub2 (compare lane 6 of Fig. 5to the lane corresponding to the 5-min time point in Fig. 3B). It was also noted that the ubiquitin conjugate patterns for E2 and E2 are qualitatively similar when incubated at similar concentrations and resolved by SDS-PAGE (Fig. 5, lanes 4 and 6). Densitometric measurements revealed that the conjugation rate of E2 is approximately 67% of that exhibited by an equivalent concentration of E2.


Figure 5: E3-dependent ubiquitin conjugation supported by E2. Conjugation reactions were carried out in the presence of I-ubiquitin and depleted fraction II of reticulocyte extract as described under ``Materials and Methods'' with the indicated combinations of E1 (5 pmol) and E2 (2 pmol). The reactions were incubated for 5 min at 37 °C and terminated by the addition of an equal volume of SDS-PAGE sample buffer containing beta-mercaptoethanol. The reaction products were analyzed by 12% SDS-PAGE followed by autoradiography. Lane 1, no E1 or E2; lane 2, E1 alone; lane 3, E2 alone; lane 4, E1 plus E2; lane 5, E2 alone; lane 6, E1 plus E2.



E2 Supports Ubiquitin-dependent Degradation of I-rcmBSA

Since E2 supports E3-dependent conjugation in depleted fraction II extracts, the ability of E2 also to support ubiquitin-dependent proteolysis of I-rcmBSA was assayed in parallel incubations(34) . Initial rates of I-rcmBSA degradation were measured by the formation of trichloroacetic acid-soluble radioactivity (Table 1). Degradation of I-rcmBSA was not stimulated above the basal rate of depleted fraction II alone when supplemented with saturating concentrations of ATP, ubiquitin, or both (Table 1, Experiment A). Also, degradation was not stimulated by addition of either E1 or E2 alone (Table 1, Experiment B); however, when the depleted fraction II was supplemented with ubiquitin, ATP, purified E1, and recombinant E2, a significant stimulation in degradation above the basal rate was observed (Table 1, Experiment B). The results of Experiments A and B confirm the conclusions from Fig. 5that the fraction II extract is depleted of E1 and E2 isoforms.



When similar experiments were conducted using recombinant E2, this isoform also was shown to stimulate I-rcmBSA degradation but only when the incubations were also supplemented with pure E1 (Table 1, Experiment C). Thus, in the in vitro system, E2 can function in targeting a substrate protein for selective degradation in an energy- and ubiquitin-dependent fashion. This targeting appears to be dependent on E3 activity since in the absence of a reticulocyte extract BSA does not function as an E2 substrate (not shown). Table 1also shows that the efficiency of E2 in supporting degradation approaches that of the cognate E2 at equimolar concentrations. The degradation rate of E2 is approximately 75% of that exhibited by E2, which agrees favorably with the difference in initial rates of E3-dependent conjugation observed between E2 and E2 in Fig. 5(lanes 4 and 6).

Ubiquitin-dependent Proteolysis Supported by E2 Does Not Exclusively Require a Lys-48 Multiubiquitin Linkage

As described above, we have shown that E2 catalyzes auto- and multiubiquitination (Fig. 3) which is not restricted to the Lys-48 residue of ubiquitin (Fig. 4) and that E2 supports ubiquitin-dependent protein degradation (Table 1). These results led to the question of whether multiubiquitination involving linkages other than Lys-48 of ubiquitin was sufficient to target substrate proteins for degradation. To address this issue, we replaced native ubiquitin with either UbK48R or rmUb in proteolytic assays otherwise identical with those of Table 1. Data summarized in Table 2demonstrate that initial rates of degradation supported by E2 are almost exclusively dependent on Lys-48 multiubiquitination since substitution with either UbK48R or rmUb yielded rates less than or equal to 2% of that observed with native ubiquitin (Experiment A). In contrast, UbK48R did not similarly block E2-supported I-rcmBSA degradation but resulted in a consistent 50% reduction compared to rates in the presence of native ubiquitin (Experiment B). The remaining stimulation of I-rcmBSA degradation by E2 supported by UbK48R must be due to multiubiquitin conjugate intermediates containing linkages other than those to Lys-48 since substitution with rmUb completely blocked proteolysis (Table 2, Experiment B).




DISCUSSION

In this paper, we report the functional characterization of a new member of the E2 enzyme family, E2, from human epidermis. Sequence comparison with all previously characterized E2s revealed that E2 exhibited the highest degree of homology with yeast UBC4, with a 60% similarity and a 38% identity (28) . UBC4 and UBC5 are central components of the ubiquitin-mediated proteolytic pathway in yeast (35) and are categorized as class I E2s (2) defined as those members of the E2 protein family that lack adjunct sequences extending from the E2 core structure. In contrast, class II E2s contain carboxyl-terminal extensions that are highly divergent and are thought to play a role in substrate specificity(12, 36) . For example, RAD6 and CDC34, which have polyacidic carboxyl-terminal tails, catalyze ubiquitination of the highly basic histone proteins(20) . E2 has a polybasic carboxyl tail with a primary structure that is unique among the E2 family. This keratinocyte enzyme may therefore have a highly restricted substrate specificity, possibly limited to certain acidic proteins. A variety of model proteins (such as histones, lysozyme, cytochrome c, myoglobin, hemoglobin, and BSA) have been assayed with E2 in the in vitro E3-independent ubiquitin conjugation assay, but none has been found to function as a substrate in this system. The identification of E2-specific substrate(s) will be an important step in characterizing the physiological role of this enzyme.

It has been demonstrated that reticulocyte E2, E2, RAD6, and CDC34 are bifunctional enzymes capable of catalyzing both E3-independent and E3-dependent ubiquitin ligation(20, 29, 33) . A recent study has shown that ubiquitination and selective degradation of some proteins is dependent upon specific association with E3(20, 33, 37) . The results presented in this report show that E2 is also bifunctional. Fig. 3and Fig. 4demonstrate that E2 can catalyze ubiquitination in the absence of an E3. Evidence that E2 also conjugates ubiquitin to substrate proteins in an E3-dependent manner came from the results of the in vitro degradation assay (Table 1). Radioiodinated rcmBSA, which is not a substrate for E2 in the absence of E3, was shown to be targeted for degradation by E2 in a ubiquitin-dependent manner using a reticulocyte lysate containing E3. We speculate that E2 may normally conjugate ubiquitin to a restricted set of specific substrate proteins in the absence of E3 and may function in an E3-dependent pathway with a more general range of substrate proteins.

Several E2s, including RAD6, CDC34, rabbit E2, and bovine E2 have been shown to support the formation of multiubiquitin chains(20, 25) . In the present study, we demonstrate that human E2 supports both auto- and multiubiquitination (Fig. 3) via sequential addition of ubiquitin to the growing multiubiquitin chain. Although rigorous kinetic studies have not yet been done, it appears that the rates of mono- and diubiquitinations are much slower than those of the subsequent elongation of ubiquitin chains to form higher order conjugates. Auto-multiubiquitination has also been documented in yeast CDC34(20) . Unlike CDC34, multiubiquitination by E2 does not appear to be restricted to the ubiquitin lysine 48 linkage since substitution of ubiquitin with UbK48R did not significantly alter the resulting conjugation pattern (Fig. 4). Parallel work with Lys to Arg ubiquitin mutants has been used subsequently to identify novel linkage specificities catalyzed by several E2 isozymes(38) .

E2-supported targeting of a substrate protein for proteolysis is dependent on the formation of branched ubiquitin chains since substitution of ubiquitin with reductively methylated ubiquitin resulted in inhibition of proteolysis. These results agree with those of a previous study involving the characterization of the Ub-dependent degradation of a test protein beta-galactosidase(26) . Proteolytic targeting by reticulocyte lysate E2 isoforms was further shown to be dependent on multiubiquitination via the lysine 48 linkage(26, 27) . In contrast, however, E2 was shown here to support selective proteolysis in the absence of lysine 48-mediated multiubiquitination, indicating that E2 and the E2 isoforms present in reticulocyte lysate may represent members of two distinct subgroups supporting ubiquitin-dependent proteolysis but differing in E3-dependent linkage specificity. Substitution of ubiquitin with UbK48R did result in a 50% decrease in E2-dependent proteolysis of the model substrate as shown in Table 2. This difference could be accounted for by a partial inhibition of E2 activity by UbK48R or by the possibility that both lysine 48 and other ubiquitin residues may be used equally by E2 for targeting protein degradation.

The 225-residue E2 protein contains 17 lysine residues (28) , 9 of which are clustered near the polybasic carboxyl terminus (within the last 30 residues). Which of these lysine residue(s) in E2 are used for multiubiquitination remains unknown at present. Preliminary evidence indicates that E2 in which the polybasic carboxyl terminus has been deleted is still capable of thiol ester formation and auto-multiubiquitination. (^3)In contrast, deletion of the carboxyl-terminal 81 residues of CDC34 prevents auto-multiubiquitination(39) . Therefore, the site of E2 autoubiquitination may reside within the catalytic core of the protein, although at this time we cannot exclude secondary effects of carboxyl-terminal extension deletion. It is thus important to confirm the preferential auto-ubiquitination site on E2 since this assignment may offer useful information related to the question of how E2 catalyzes both E3-dependent and E3-independent ubiquitin conjugation. This site may be involved in the recognition of specific substrate proteins (E3-independent pathway) and in the interaction with specific E3(s) which, in turn, determine substrate specificity (E3-dependent pathway).

The demonstration that E2 catalyzes auto- and multiubiquitination and targets substrate proteins for selective degradation suggests several possible autoregulatory models for E2. In one model, E2 could down-regulate its own activity by targeting itself for degradation using the ubiquitin-dependent pathway. Alternatively, the ubiquitin conjugation activity of E2 may be determined by the conjugation state of the enzyme (native, mono-, or multiubiquitinated). But, in either case, the possibility that autoubiquitination of E2in vivo may be coupled to an additional signal, e.g. phosphorylation or interaction with other cellular proteins, cannot be ruled out. The enzymatic properties of E2 documented in this report provide a useful model system to address these important issues regarding autoregulation of this keratinocyte E2.


FOOTNOTES

*
This work was supported in part by United States Public Health Service Grants R01-AR32599 (to L. A. D.), R01-AR32081 (to L. A. D.), R01-GM34009 (to A. L. H.), and R29-AR40410 (to G. J. G.), and National Institutes of Health Training Grant T32-AR07577 and a Veterans Affairs merit review grant (to L. A. D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a Dermatology Foundation Career Development Award sponsored by SmithKline Beecham Pharmaceuticals.

To whom correspondence should be addressed: Dept. of Dermatology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Tel.: 414-456-4087; Fax: 414-266-8673.

(^1)
The abbreviations used are: E1, ubiquitin activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; EPF, endemic pemphigus foliaceus; GST, glutathione S-transferase; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; FPLC, fast protein liquid chromatography; Ub, ubiquitin; BSA, bovine serum albumin; rcmBSA, reduced, carboxymethylated form of BSA; rmUb, reductively methylated form of Ub.

(^2)
O. V. Baboshina and A. L. Haas, manuscript in preparation.

(^3)
Z. Liu, C. A. Conrad, A. L. Haas, and G. J. Giudice, unpublished observation.


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