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Endocrinology Vol. 143, No. 10 3740-3747
Copyright © 2002 by The Endocrine Society

ARTICLE

Characterization of Rat100, a 300-Kilodalton Ubiquitin-Protein Ligase Induced in Germ Cells of the Rat Testis and Similar to the Drosophila Hyperplastic Discs Gene

Rose Oughtred, Nathalie Bedard, Olasunkanmi A. J. Adegoke, Carlos R. Morales, Jacquetta Trasler, Venkatesh Rajapurohitam and Simon S. Wing

Departments of Medicine (R.O., N.B., O.A.J.A., V.R., S.S.W.), of Anatomy and Cell Biology (C.R.M.), and of Pediatrics, Pharmacology and Human Genetics, and Montréal Children’s Hospital Research Institute (J.T.), McGill University, Montréal, Québec, Canada H3A 2B2

Address all correspondence and requests for reprints to: S. S. Wing, Polypeptide Laboratory, McGill University, Strathcona Anatomy and Dentistry Building, 3640 University Street, Room W315, Montréal, Québec, Canada H3A 2B2. E-mail: simon.wing{at}mcgill.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Conjugation of ubiquitin to proteins is activated during spermatogenesis. Ubiquitination is mediated by ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzymes (UBCs or E2s), and ubiquitin protein ligases (E3s). Since we previously showed that the activated ubiquitination is UBC4 dependent, we characterized Rat100, a UBC4-dependent E3 expressed in the testis.

Analysis of expressed sequence tag sequences and immunoblotting showed that Rat100 is actually a 300-kDa protein expressed mainly in the brain and testis and is similar to the human E3 identified by differential display (EDD) protein and the Drosophila hyperplastic discs gene, mutants of which cause a defect in spermatogenesis. Rat100 is induced during postnatal development of the rat testis, peaking at d 25. It is localized only in germ cells and is highly expressed in spermatocytes, moderately in round and slightly in elongating spermatids. In contrast to UBC4 whose removal from a testis extract abrogates much of the conjugation activity, immmunodepletion of Rat100 from the extracts had little effect. Rat100 therefore has a limited subset of substrates, some of which appear associated with the E3 as the immunoprecipitate containing Rat100 supported incorporation of 125I-ubiquitin into high molecular weight proteins. Thus, Rat100 is the homolog of human EDD and likely of Drosophila hyperplastic discs. This homology, together with our results, suggests that induction of this E3 results in ubiquitination of specific substrates, some of which are important in male germ cell development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SPERMATOGENESIS IS A CRITICAL developmental process during which a large fraction of cellular proteins are degraded as the spermatids become remodeled into their elongated mature forms. Although the mechanisms underlying this degradation are not well understood, an increasing body of evidence indicates that the ubiquitin-dependent proteolytic system is involved in this process (reviewed in Ref. 1). In addition, ubiquitin-dependent proteolysis plays a critical role in cell cycle transitions (reviewed in Refs. 2 and 3) and therefore is almost certainly involved in regulating the transitions involved in the meiotic divisions during spermatocyte development.

This proteolytic pathway appears to be responsible for most of the highly regulated and selective proteolysis in the eukaryotic cell and could therefore be implicated in the precise protein degradation that would be required for normal development. In this pathway, the 8-kDa peptide ubiquitin is covalently linked to target proteins and marks them for recognition and degradation by the 26S proteasome (reviewed in Refs. 4, 5, 6). Because the proteasome recognizes the ubiquitin and not the protein substrate, recognition of specific substrates resides at the level of conjugation. Conjugation of ubiquitin to proteins is a multistep process involving at least three types of enzymes (7). Initially, ubiquitin is activated by ubiquitin-activating enzyme (E1) that results in the formation of a high energy thiolester linkage between the carboxy terminus of ubiquitin and a cysteine residue of the E1 (8). E1 then transfers the activated ubiquitin to a cysteine residue of a ubiquitin-conjugating enzyme (UBC or E2) (reviewed in Ref. 8). Finally, the E2 interacts with a ubiquitin-protein ligase (E3) to mediate ligation of the carboxy terminus of ubiquitin to the {epsilon}-amino group of lysine residues of the target protein substrate. The E3 serves as the substrate recognition factor in this step. Successive ubiquitin molecules are usually added to the lysine residues of the previous ubiquitin to produce a polyubiquitin chain. Polyubiquitinated substrates are then recognized by the 26S proteasome that degrades the target protein.

We have previously shown that ubiquitination increases during the first wave of spermatogenesis in the developing testis (9). This increased ubiquitination is associated with increased rates of ubiquitination measured in testis extracts and appears dependent on the UBC4 family of E2 enzymes. Furthermore, a testis-specific UBC4 isoform is induced in spermatids at the time when conjugation is activated (10). Although our results indicate that UBC4 plays a quantitatively important role during development, other E2s also play a role in this process. Inactivation of the gene encoding the mouse E2 HR6B, a homolog of S. cerevisiae UBC2, results in incomplete spermatogenesis and male sterility (11). In addition, there are germ cell-specific isoforms of other ubiquitin system genes. In both mouse and marsupials, a Y chromosome-linked testis-specific gene highly homologous to E1 has been found (12). In Drosophila, there are two genes encoding testis specific subunits of the proteasome (13). Finally, we have recently identified germ cell-specific deubiquitinating enzymes that are primarily expressed in late elongating spermatids (14).

To date there has been very little characterization of E3s in the testis. E3 enzymes appear to support ubiquitination by two general mechanisms, either functioning as docking proteins (15) or else as catalytic intermediates in a thiolester cascade (16). E3s that serve as docking proteins bind both specific substrates and E2s, thereby permitting the transfer of ubiquitin from an E2 to a substrate (15). For example, the E3, SCFcdc4, binds the E2 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 (17, 18). These docking E3s generally contain a subunit with a RING finger motif, which appears essential for binding the E2 (19). Alternatively, E3s may function as the final intermediate in the ubiquitin thiol ester cascade. The E3, E6-AP (E6-associated protein), forms a thiol ester linkage with ubiquitin before catalyzing the ubiquitination of p53 in the presence of the viral E6 protein (16, 20). The catalytically active cysteine in E6-AP is found within its carboxy-terminal domain, and a number of putative E3s have been identified based on the presence of such HECT (homology to E6-AP carboxyl terminus) domains (21), including Rat100, Rsp5, and Nedd 4 (16, 21, 22).

Since we have previously demonstrated activation of a UBC4-dependent pathway of conjugation during spermatogenesis, it would be important to now characterize UBC4-dependent E3s that are expressed in the testis. Rat100 was previously identified as a 100-kDa protein that was highly expressed in the rat testis (23). It was subsequently observed to contain a HECT domain, suggesting that it was an E3. Indeed, it was found capable of accepting ubiquitin from several UBC4 isoforms (21, 24). To date though, only limited characterization of this E3 has been carried out. Therefore, we now report detailed characterization of it in the testis. In so doing, we also clarify confusion in the literature regarding its structure as other investigators suggested that Rat100 was actually encoded by a gene predicting a protein of 300 kDa but were unable to confirm expression of a protein of this size (25).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Identification of N-terminal Rat100 sequences similar to human E3 identified by differential display (EDD)
The human EDD sequence that was upstream from the C-terminal region encoding sequence nearly identical to Rat100 was used as query sequence in Blast searches of the GenBank mouse expressed sequence tag (EST) database. Sequences obtained were then aligned with each other and the human EDD sequence. Gaps where there was no apparent mouse sequence were filled in by amplifying by PCR the corresponding region from mouse testis cDNA using primers whose sequences were derived from sequences of the adjacent ESTs.

Preparation of tissue and cell extracts
Testis extracts used for anti-Rat100 immunoblotting or in ubiquitin conjugation assays were prepared using testes from Sprague Dawley rats (Charles River Laboratories, St. Constant, Québec, Canada). Tissues, including the liver, heart, kidney, lungs, brain, and skeletal muscles (gastrocnemius and tibialis anterior), were dissected from Sprague Dawley rats (175–200 g). The testes or tissues were sliced and homogenized using a Potter Elvehjem (Wheaton Science Products, Millville, NJ) homogenizer [Polytron (Brinkmann Instruments, Inc., Westbury, NY) tissue disrupter for heart and skeletal muscles] in 5 vol ice-cold buffer containing 0.25 M sucrose, 50 mM Tris (pH 7.5) (at 4 C), 1 mM dithiothreitol (DTT), 1 mM EDTA, and protease inhibitors 1 mM phenylmethylsulfonyl fluoride, pepstatin A (10 µg/ml), and leupeptin (10 µg/ml). The homogenates were centrifuged at 10,000 x g for 10 min and then at 100,000 x g for 60 min at 4 C. Alternatively, in some immunoblot analyses, sodium dodecyl sulfate-solubilized testis extracts were prepared using 25 mM Tris, pH 7.5 (at 4 C), 2% sodium dodecyl sulfate, and 1 mM DTT. The homogenates were then centrifuged at 10,000 x g for 15 min at room temperature. The final supernatants were used for immunoblotting studies.

Isolated populations of male germ cells (spermatocytes, round and elongating spermatids) from the testis of 91-d-old rats (one rat per cell separation) were obtained by the unit gravity sedimentation procedure (26, 27), using 2–4% BSA gradients generated with a STA-PUT apparatus (Johns Scientific, Toronto, Ontario, Canada). Fractions containing pachytene spermatocytes (average purity 84%, n = 3), round spermatids (average purity 88%, n = 3), and elongating spermatids with residual bodies (average purity 83%, n = 3) were prepared. Lysates of purified germ cell fractions were prepared by homogenization with a Dounce apparatus in 50 mM Tris (pH 7.5) at 4 C, 1 mM DTT, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 µM pepstatin A, and 1 µg/ml leupeptin. The lysates were then clarified at 10,000 x g for 10 min at 4 C.

Protein concentrations were determined using the Bio-Rad Laboratories, Inc. (Hercules, CA) protein assay kit or the BCA assay kit (Pierce Chemical Co., Rockford, IL) with BSA as the standard.

Antibodies and immunoblotting
Antiserum was prepared against nonconserved Rat100 sequences N terminal to the HECT domain. The original Rat100 cDNA (EMBL accession no. X61630) (23) was used as a template in a PCR containing as primers oligonucleotides 5'-TGATGTCTGCTCGAGGAG-3' corresponding to bases 97–112 and 5'-CGGGATCCTAGACTTTAACTCTGTGGACAG-3' complementary to bases 1184–1203 of the Rat100 coding sequence. The 1.1-kb amplified DNA fragment was then subcloned into pET15b (Novagen) to yield a plasmid expressing a 40-kDa protein with an N terminal (His)6 tag. The HisRat100 fusion protein was then purified on nickel-chelated agarose under denaturing conditions in 6 M guanidine HCl according to the manufacturer’s instructions ({Ni2+}-NTA; QIAGEN). The HisRat100 eluate was dialyzed against PBS and the dialysate was concentrated (Centriplus 10, Amicon, Beverly, MA) and used as antigen with Freund’s complete adjuvant to immunize rabbits.

Because the antiserum against Rat100 showed some cross-reactivity with other proteins, it was affinity purified using maltose binding protein (MBP)-tagged Rat100 fragment of the original HisRat100 antigen. MBP-Rat100 fragment consisted of MBP fused to the sequence corresponding to bases 655 to 1203 of the Rat100 coding sequence.

For anti-Rat100 immunoblotting, protein (100 µg) from testis or different tissue extracts was resolved on 7.5% SDS-PAGE gels and transferred to 0.45 µm polyvinylidene difluoride (PVDF) membranes (Millipore Corp., Bedford, MA). Following transfer, membranes were probed with the affinity purified antibody at 1:100 dilution. This was followed by incubation with the secondary antibody, either horseradish peroxidase-protein A (Bio-Rad Laboratories, Inc.) or 125I-protein A, and immunoblots were either visualized using the enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Baie d’Urfé, Québec, Canada), or by exposure to x-ray films, respectively. Quantification was carried out using transmittance densitometry on x-ray films from immunoblots probed with 125I-protein A. To correct for some variations in transfer between gels, protein on the blots were stained with amido black, and each lane was quantified by reflectance densitometry. The transmittance densitometry values were then normalized against the reflectance densitometry values for each lane.

In situ hybridization
Adult Sprague Dawley rats were anesthetized with pentobarbital, and the testes were fixed by perfusion through the abdominal aorta with 4% paraformaldehyde, 2% glutaraldehyde, and 3% dextran in 0.05 M phosphate buffer (pH 7.4) for 10–15 min. Following perfusion, the testes were cut into small blocks (1 mm in width, 1 mm in height, and 10 mm in length), embedded in 4% melted agar (60 C), and chopped with a Vibrotome into 60-µm-thick frontal sections. Groups of 10 sections were collected in autoclaved vials and washed three times in ribonuclease-free 0.05 M phosphate buffer to neutralize aldehyde groups.

Prehybridization and hybridization were performed as described previously (28). Briefly, the testicular sections were transferred from the phosphate buffer to prehybridization buffer containing 4x SSC (1x SSC contains 0.15 M NaCl, 0.015 M sodium citrate) and 1x Denhardt’s solution for 1 h at room temperature with gentle agitation. The sections were then immersed in hybridization buffer containing 1 ml of 8x SSC, 1 ml deionized formamide, 100 µl of Sarkosyl (2.3 mg/ml), 200 µl of 1.2 M phosphate, and 3H-labeled antisense probe (7.4 x 106 cpm). The probe was synthesized from a pBluescript plasmid (Stratagene, La Jolla, CA) containing the Rat100 sequence (23). Ssp1 digestion of this plasmid yielded a 2.6-kb fragment. In vitro transcription of this fragment was then performed using [3H]-uridine 5'-triphosphate and T3 RNA polymerase to produce the antisense strand. Unincorporated [3H]-uridine 5'-triphosphate was removed by passing the reaction products over a Sephadex G-50 column. Following hybridization overnight at 40 C, the sections were rinsed sequentially at the same temperature in 4x SSC and 0.1x SSC for 1.5 h. After the washes, the sections were postfixed in potassium ferrocyanide-reduced osmium for 15 min and quickly dehydrated in 50, 70, 90, 95, and 100% ethanol and propylene oxide and embedded in Epon. Prestained hybridized testicular sections (1 µm) were coated with Kodak (Rochester, NY) NTB-2 nuclear emulsion (29) and after 10 d exposure were developed with Kodak D-170.

Immunoprecipitation of Rat100 and conjugation assays
To determine the relative importance of Rat100 in mediating overall conjugation in testis extracts, 3 µg of affinity purified anti-Rat100 antibodies or control IgG were bound to 100 µl of a 50% slurry of protein A-Sepharose beads (Amersham Biosciences). Following washing with 20 mM Tris (pH 7.5), 0.1 M KCl, 0.1 mM CaCl2 1 mM MgCl2, and 1 mM DTT, the beads were mixed with testis extract containing 500 µg of protein. Following mixing at 4 C for 4 h, the beads were collected by centrifugation. The supernatants and pellets were then subjected to either immunoblot analysis with anti-Rat100 antibodies as above or assayed for conjugation activity. Conjugation activity in the supernatant was measured as previously described (9) by adding 5 µM 125I-ubiquitin, 2 mM 5'-adenylylimidodiphosphate (AMPPNP), and 3 µM ubiquitin aldehyde to an aliquot of the supernatant containing 25 µg of protein followed by incubation at 37 C for 10 min. (The ATP analog, AMPPNP, supports conjugation of ubiquitin to proteins, but does not support proteasome mediated hydrolysis of ubiquitinated proteins. Ubiquitin aldehyde inhibits deubiquitinating enzymes that can remove ubiquitin from proteins.) Conjugation activity in the pellet was measured by resuspending the pellet in 200 µl of 50 mM Tris (pH 7.5) and 1 mM DTT. Twenty microliters of the suspension were then added to an equal volume of the same buffer containing 100 nM E1, 500 nM UBC4, 10 µM 125I-ubiquitin, and 4 mM AMPPNP and incubated as for the reaction containing the supernatant. Reaction products were analyzed by SDS-PAGE on 10% acrylamide gels to remove unconjugated ubiquitin. Ubiquitinated proteins were detected by autoradiography of dried gels and quantitated by excising the individual lanes and counting the gel pieces in a {gamma} counter.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Rat100 has a molecular mass of 300 kDa and is expressed in the brain and testis
The original clone of Rat100 reported predicted a 100-kDa protein encoded by the distal half of a 9.5-kb mRNA transcript. These original data suggested that there was an approximately 6 kb 5' noncoding region of the mRNA. However, Callaghan et al. (25) subsequently identified EDD, a human cDNA clone that predicted a 300-kDa protein containing in the carboxyl-terminal region sequence that was 96% amino acid identical to Rat100. The overall EDD protein also showed 40% sequence identity (55% similarity) to a D. melanogaster tumor suppressor gene, hyperplastic discs. Furthermore, Callaghan et al. (25) provided evidence that the Rat100 clone contained a sequence error such that the reading frame was actually open upstream from the original proposed start site. However, they were unable to detect a 300-kDa protein by immunoblot analysis of any rat tissues. A recent search of the mouse EST databases confirms that there are many DNA sequences that are highly similar to the 5' region of the human cDNA that are not present in the original Rat100 clone (Fig. 1Go), suggesting that Rat100 may indeed be a much larger protein. When aligned, these sequences are overlapping and analogous to the EDD sequence that was missing from the original Rat100 clone. Two regions between nucleotides 3133 and 3557 (amino acids 1033–1175) and between 4681 and 4911 (amino acids 1549–1625) of EDD had no apparent homologous mouse EST. Therefore, we designed primers whose sequences were derived from the adjacent ESTs and used them in PCRs with mouse testis cDNA as template. Indeed, we were able to amplify DNA fragments whose predicted protein sequences were 97 and 98% similar to the EDD sequence. In addition, an EST that covered the 5' end of the cDNA predicted an amino acid sequence that was only 63% similar to the human EDD sequence. Because this degree of similarity was unusually low in comparison with the other ESTs that we found, we obtained the 5' end sequence directly from mouse testis RNA using the 5' RACE method (30). This fragment predicted a protein sequence identical to that of human EDD. When all these sequences were aligned together, the predicted mouse EDD/Rat100 sequence was 96% identical, 97% similar to human EDD, and 38% identical, 55% similar to the Drosophila hyperplastic discs gene. The similarity to hyperplastic discs was widely distributed throughout the length of the protein. However, five regions showed higher degrees of sequence similarity. Two of these corresponded to parts of the conserved HECT domain, whereas the third corresponded to sequence similar to the UBA domain, a motif found in many enzymes of the ubiquitin pathway (31). A PABP motif, found in the C-terminal regions of some polyA binding proteins (23) is also present in the three proteins (Fig. 1Go).



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  		Figure 1. Comparison of the sequences of Rat100 and its orthologs, human EDD and Drosophila HYD. Top, The overall sequence of Drosophila HYD is 40% identical (55% similar) in protein sequence to human EDD. Shown below the HYD bar are lines indicating regions greater than 100 amino acids in length that are at least 50% identical in sequence. The degrees of amino acid identity and similarity (in parentheses) are shown. Bottom, The original 3.2-kb cDNA clone of Rat100 was predicted to encode an approximately 100-kDa protein (23 ). However, cloning of the human cDNA EDD revealed that its C-terminal region was 96% amino acid identical (98% similar) to Rat100, but it had a large 5' extension that resulted in prediction of an approximately 300-kDa protein (25 ). Search of the mouse EST database revealed sequences (solid lines) highly similar to all regions of EDD except for two regions encoding amino acids 1033–1175 and 1549–1626. PCR amplification using mouse cDNA as template yielded DNA fragments whose sequences (dashed lines) filled these two gaps. In addition, 5' RACE analysis yielded the sequence (dashed line marked A) of the 5'end of the cDNA. Shown below each line are the degrees of amino acid similarity to human EDD. The conserved C-terminal HECT domain is indicated by the shaded box; the PABP motif found in polyA binding proteins is indicated by the solid box; a region similar to the UBA motif is indicated by the double-hatched box. (GenBank accession nos. for mouse ESTs: B, BF150508; C, BI107668; D, BF116832; E, BB239392; F, BB655206; G, BG276511; H, BB684814; I, BE986681; J, BB443775; K, BB522812; L, AA183970; M, BF159469.)

 
Therefore, to confirm that Rat100 is indeed a much larger protein than previously reported, we generated antisera to a region N-terminal of the conserved HECT domain to minimize the likelihood of any cross-reaction with other HECT domain proteins. Further possibility of cross-reaction was minimized by affinity-purifying the antibody. Indeed, this purified antibody detected a 300-kDa protein in rat tissue extracts (Fig. 2Go). Expression was seen mostly in the testis with some also in the brain. A faint band of approximately 150 kDa in size could be detected in the lung, liver, and kidney. The absence of any immunoreactivity in the 300-kDa regions suggests that this is probably a nonspecific cross-reacting band, but we cannot exclude the possibility that this is a degraded form of Rat100 in these tissues.



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  		Figure 2. Rat100 protein is a 300-kDa protein detected in the testis and brain. Protein (100 µg) from the indicated rat tissues was subjected to SDS-PAGE, and resolved proteins were transferred to PVDF membranes. The membranes were then immunoblotted with anti-Rat100 antibodies and Rat100 was detected using ECL-plus. Shown are representative samples from immunoblots performed in triplicate. Positions of molecular mass markers (kDa) are as indicated.

 
Rat100 is developmentally regulated in the testis
Because UBC4 isoforms support transfer of ubiquitin to Rat100 (24) and are subject to developmental regulation (9), we evaluated whether Rat100 expression was also modulated in a developmentally controlled manner. Testis extracts from rats of various ages were analyzed by immunoblotting using anti-Rat100 antibodies (Fig. 3Go). Low levels of Rat100 protein were detected as early as 10 d after birth, which is consistent with the previous developmental Northern that showed trace amounts of the Rat100 transcript between 1 and 14 d after birth (23). Developmental up-regulation in Rat100 protein expression was observed, with the peak levels of Rat100 being detected at d 25.



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  		Figure 3. Rat100 is developmentally regulated in the testis. Protein (100 µg) from testes of the indicated ages was subjected to SDS-PAGE and resolved proteins were transferred to PVDF membranes. The membranes were probed with an anti-Rat100 antibody followed by detection with 125I-labeled protein-A and then subjected to autoradiography. Shown above are representative samples and below are the results of quantification (means ± SEM) for four samples for each age by densitometric analysis. The data were subjected to one-way ANOVA and means from d 25–65 are significantly different from d 10 (P < 0.05). Molecular mass markers are as indicated.

 
Expression of Rat100 is restricted to germ cells
Although the presence of Rat100 in the testis as early as 10 d of age might suggest that it is present in somatic cells, the induction at a later age raised the possibility that it is present in germ cells and induced during spermatogenesis. To investigate what cell types express Rat100, an antisense RNA probe specific to the C-terminal of Rat100 was hybridized in situ with tissue sections from the testis and visualized radioautographically (Fig. 4Go, A and B). The antisense probe of Rat100 yielded radioautographic signals over the germ cells. Rat100 mRNA was prominent in pachytene spermatocytes and round spermatids of the seminiferous tubules (stage IV), whereas early elongating spermatids were moderately labeled and zygotene spermatocytes showed a low level of labeling (stage XII) (Fig. 4AGo). The probe also generated strong autoradiographic signals over the cytoplasm of step 7 round spermatids, but not over the Sertoli cells, spermatogonia, and late elongating spermatids (stage VII) (Fig. 4BGo). Control hybridization experiments using a sense riboprobe did not yield significant signals, indicating that the radioautographic signal generated by the antisense probe to Rat100 was specific (Fig. 4CGo). Therefore, at the mRNA level, Rat100 appears to be expressed only in germ cells of the testis. The detection of Rat100 in testis at 10 d of age (Fig. 3Go) appears to be due to a combination of its presence at high levels in pachytene spermatocytes, forms of which are present in small number at this early age (32) with some contribution from zygotene spermatocytes, which showed a low level of labeling.



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  		Figure 4. Expression of Rat100 is restricted to germ cells of the testis. A, Cross-section of rat seminiferous epithelium was hybridized in situ with a 3H-labeled Rat100 antisense probe and visualized by radioautography. Silver grains overlay uniformly pachytene spermatocytes (P) and round spermatids (R) of the seminiferous tubules (stage IV). Early elongating spermatids (E) are moderately labeled (stage XII). Magnification, x400. (S, Sertoli cells; G, spermatogonia; Z, zygotene spermatocytes). B, In situ hybridization of rat seminiferous epithelium was performed as for panel A. The probe generated strong autoradiographic signals over the cytoplasm of step 7 round spermatids (R), but not over the Sertoli cells (S), spermatogonia (G), and late elongating spermatids (E) (stage VII). Magnification, x400. C, A control probe did not yield any significant radioautographic signals. Magnification, x400. D, Protein (100 µg) from the indicated germ cell types was subjected to SDS-PAGE and resolved proteins were transferred to PVDF membranes. The membranes were then immunoblotted with antibodies to Rat100 and the presence of Rat100 was detected using ECL-plus. Shown are representative samples. Below each lane are the results of quantification of the bands (mean ± SEM) from three samples for each cell type. The mean for pachytene spermatocytes is significantly different from the elongating spermatids (P < 0.05, by one-way ANOVA).

 
Because mRNAs can be sequestered from the translational machinery in developing germ cells, the pattern of protein expression may not match RNA expression. To explore where Rat100 was expressed at the protein level, the specific cellular localization of the Rat100 protein in the testis was determined. The anti-Rat100 antibodies were not effective in immunocytochemistry. Therefore, to determine the cellular expression of the Rat100 protein, purified germ cell fractions, including either pachytene spermatocytes, round spermatids, or residual bodies/elongating spermatids were immunoblotted using anti-Rat100 antibodies. Significant levels of Rat100 were detected in pachytene spermatocytes and round spermatids, and lower levels were detected in the elongating spermatids/residual bodies (Fig. 4DGo). Rat100 protein is therefore also present in germ cells at least as early as the pachytene spermatocyte stage. The protein may also be present in zygotene spermatocytes because there is significant protein level at d 10 (Fig. 3Go) when pachytene spermatocytes are present, but in small number.

Rat100 appears to have restricted substrate specificity
Our previous studies indicated that UBC4 isoforms were responsible for the majority of ubiquitin conjugation in testis extracts and also responsible for the activation of conjugation during spermatogenesis (9). Immunodepletion of UBC4 from testis extracts removes the majority of ubiquitin conjugation activity. To determine whether the UBC4-dependent E3 Rat100 also played a quantitatively important role in the conjugation mediated by UBC4, we measured rates of ubiquitin conjugation in testis extracts before and after immunodepletion with anti-Rat100 antibodies (Fig. 5Go). The antibodies efficiently removed the 300-kDa protein from the extracts. Interestingly, in contrast with what was observed with immunodepletion of UBC4 (9), there was no significant change in rates of conjugation following removal of Rat100. Thus, Rat100 probably has a limited number of substrates, conjugation to which does not contribute significantly to overall ubiquitination in the rat testis. To explore whether some of these substrates might be associated with the enzyme, we mixed the immunoprecipitated Rat100 with E1, E2, ATP, and 125I-ubiquitin. Radiolabeled ubiquitin was indeed incorporated into proteins in reactions containing the pellets from the Rat100 immunoprecipitations, but only minimally in those in which control IgG was used instead. In addition, we have recently observed that there are other proteins that are immunoprecipitated with anti-Rat100 antibodies (data not shown).



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  		Figure 5. Rat100 has restricted substrate specificity. Extracts prepared from 30-d-old rat testes were incubated with either control IgG or affinity purified anti-Rat100 antibodies bound to protein A-Sepharose beads. Following removal of the beads with bound IgG/antibodies and any associated proteins by centrifugation, the pellet and an aliquot of the supernatant was analyzed by Western blotting with anti-Rat100 antibodies (A). The other aliquot of the supernatant was mixed with 125I-ubiquitin as described in Materials and Methods and incubated at 37 C for 10 min. The products were then resolved of free ubiquitin by SDS-PAGE and following drying of the gel, the ubiquitinated proteins were detected by autoradiography (B). Lanes of the dried gel were then excised and counted. Similar rates of incorporation were seen whether extracts were treated with IgG or anti-Rat100 antibody (IgG, 9933 cpm ± 295; anti-Rat100, 9792 cpm ± 217). C, In separate immunoprecipitations, pellets were incubated with E1, UBC4, ATP, and 125I-ubiquitin at 37 C. Reaction products were then analyzed by SDS-PAGE and detected by autoradiography.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The HECT domain containing family of E3s is presently the best defined group of E3 enzymes. Surveys of sequences in the GenBank database have identified many putative members of this family in the mammalian genome, but only a few of these such as E6-AP (21, 33) and NEDD4 (22) have received significant characterization. Although Rat100 was identified as a member of this family of E3s a number of years ago (21), there has been very little work on this enzyme published since. In this paper, we have clarified confusion regarding the nature of Rat100, and provided further characterization that reveals insights into its function and into the mechanisms of regulation of ubiquitin conjugation.

This E3 was named Rat100 after having been identified in a rat cDNA library and deduced to express a 100-kDa enzyme. Indeed, in vitro transcription and translation of this cDNA does produce a 100-kDa protein (21) with catalytic activity as shown by its ability to accept ubiquitin from E2s of the UBC4 family (24). However, Callaghan et al. (25) identified a human E3, EDD, and provided evidence that there was an error in the Rat100 sequence and that correction of this error yields an open reading frame that extends upstream and might encode a 300-kDa protein similar to their human E3. However, the exact nature of Rat100 remained unclear as these workers were unable to detect a 300-kDa protein in rat tissue lysates using anti-EDD antibodies. In this paper, we were able to confirm that Rat100 is indeed expressed as a 300-kDa protein. Furthermore, we have surveyed its expression among various tissues and have shown that it is highly expressed as a 300-kDa protein predominantly in the testis and the brain.

Within the testis, expression was cell type specific. Using RNA and protein analysis, we found that Rat100 is expressed specifically in germ cells and that within germ cells, expression is primarily in spermatocytes and round spermatids. Developmental analysis also confirmed that this E3 is precisely regulated during spermatogenesis with peak expression at d 25 of life when the germinal epithelium of the testis is populated primarily by spermatocytes and by early round spermatids. This high expression of Rat100 in the testis and its regulation in germ cells would suggest that it plays an important role during their development, beginning at the spermatocyte stage. Interestingly, in support of this, the full-length 300-kDa form of Rat100 has significant sequence similarity to the Drosophila tumor suppressor gene hyperplastic discs (25, 34). Null mutations of this Drosophila gene are lethal, but point mutants of this gene can cause hyperplasia of imaginal discs as well as male infertility, the latter being due to a lack of progression of germ cells past the primary spermatocyte stage (34).

HECT domain E3s are characterized by the conserved approximately 360-residue carboxy-terminal HECT domain that contains the cysteine residue that accepts ubiquitin from the E2 as well as a region shown to be essential for binding to the E2 (35). The sequences amino terminal to the HECT domain are divergent and thus appear to play important roles in mediating the distinct functions of the various members of this family either through recognition of specific substrates (33, 36) or through localization to specific compartments of the cell. This structure-function arrangement also seems to apply to the UBP family of deubiquitinating enzymes which are also characterized by relatively conserved carboxy regions and more divergent amino-terminal regions (37). In the case of the two isoforms of UBP-testis, which have different amino-terminal regions but identical carboxy-terminal regions, the divergent amino termini were indeed shown to be responsible for subcellular localization and for influencing substrate specificity (14). Intriguingly, Rat100 along with its apparent fly and human orthologs has one of the largest nonconserved N-terminal regions (~2500 amino acids) of any member of this family. Thus, Rat100 may be involved in the ubiquitination of a number of substrates and/or may be localized to several different molecular complexes or structures in the cell. The multiple phenotypes of mutants of the apparent Drosophila ortholog would be consistent with this (34). However, these substrates are unlikely to be quantitatively abundant as removal of Rat100 does not affect significantly the overall rate of conjugation in testis extracts. Thus, other E3s are likely to play quantitatively more important roles in UBC4 mediated conjugation. The N-terminal regions may serve to localize the E3 to specific subcellular locations. However, the abundance of the protein in the supernatant following high speed centrifugation of extracts suggests that a significant fraction is present in the cytosol.

Regulation of degradation of proteins through the ubiquitin-dependent pathway has focused largely on regulation of substrate availability. For example, the degradation of a number of substrates of the ubiquitin system, particularly those whose ubiquitination is mediated by F-box containing E3 complexes, is initiated by phosphorylation of the substrate protein. Such modification renders the protein available for recognition by the E3. However, regulation of degradation of this pathway can also be mediated through regulation of the activities of the enzymatic components of the pathway. We have previously shown that conjugation of ubiquitin to proteins is induced during spermatogenesis and that this activation of overall rates of conjugation appears to be largely due to the UBC4 family of ubiquitin conjugating enzymes (9). Indeed, various isoforms of UBC4 as well as total UBC4 protein immunoreactivity are induced during spermatogenesis and could explain at least in part the increased rate of conjugation. However, the work in this paper also indicates that some of the activation of conjugation can arise from the induction of specific E3s that interact with UBC4. Our studies indicate that Rat100 could be one of these E3s because it is induced in spermatocytes and round spermatids, cells which also express and show induction of various isoforms of UBC4 (10, 38). Although we have observed induction of both E2s and E3s during spermatogenesis, we have not observed any regulation of E1 expression. E1 protein levels remain stable in the testis during the first wave of spermatogenesis and levels appear similar in extracts of isolated spermatocytes, round spermatids and elongating spermatids (Bedard, N., and S. S. Wing, unpublished data). Such relatively constitutive expression from a single essential E1 gene (39) would be consistent with its nonspecific function of supplying activated ubiquitin to the much larger family of E2s. In contrast, the more precise regulation of E2s and E3s, which together determine substrate specificity of conjugation, would permit regulation of rates of ubiquitination and thereby degradation of specific substrates. In the case of Rat100, its induction in spermatocytes along with the lack of progression beyond this stage in mutants of its apparent Drosophila homolog suggest that it ubiquitinates specific proteins critical to this transition which includes meiotic recombination and division. Further studies that identify its substrates as well as the effects of inactivation of its gene will critically evaluate this possibility.


   Acknowledgments
 
We thank D. Muller and J. Huibregtse for plasmids containing Rat100 sequences.


   Footnotes
 
This work was supported by the Canadian Institutes of Health Research Grants MT12121 (to S.S.W.) and MT11362 (to J.T.). R.O. is the recipient of the Eileen Peters McGill Major Fellowship and the Molson Foundation Fellowship. O.A.J.A. is supported by a fellowship from the Canadian Diabetes Association. S.S.W. and J.T. are recipients of chercheur boursier awards from the Fonds de la Recherche en Santé du Québec. J.T. is also a recipient of a Scientist award from the Canadian Institutes of Health Research.

Abbreviations: AMPPNP, 5'-Adenylylimidodiphosphate; DTT, dithiothreitol; E1, ubiquitin-activating enzyme; E2 or UBC, ubiquitin-conjugating enzymes; E3, ubiquitin protein ligases; E6-AP, E6-associated protein; ECL, enhanced chemiluminescence; EDD, E3 identified by differential display; EST, expressed sequence tag; HECT, homology to E6-AP carboxyl terminus; MBP, maltose binding protein; PVDF, polyvinylidene difluoride.

Received March 4, 2002.

Accepted for publication June 3, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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