Characterization of the Testis-specific Proteasome Subunit α4s in Mammals

Background: Two subtypes of proteasome core particles (CPs) with tissue-specific β subunits have been identified in mammals. Results: Mammals have an additional proteasome α subunit, α4s, which forms the male germ-specific CP. Conclusion: The α4s-containing CP is a new subtype of CP with unique properties distinct from the constitutive CP. Significance: Our results provide a clue for understanding the role of the proteasome in mammalian testes. The 26 S proteasome is responsible for regulated proteolysis in eukaryotic cells. It is composed of one 20 S core particle (CP) flanked by one or two 19 S regulatory particles. The CP is composed of seven different α-type subunits (α1-α7) and seven different β-type subunits, three of which are catalytic. Vertebrates encode four additional catalytic β subunits that are expressed predominantly in immune tissues and produce distinct subtypes of CPs particularly well suited for the acquired immune system. In contrast, the diversity of α subunits remains poorly understood. Recently, another α subunit, referred to as α4s, was reported. However, little is known about α4s. Here we provide a detailed characterization of α4s and the α4s-containing CP. α4s is exclusively expressed in germ cells that enter the meiotic prophase and is incorporated into the CP in place of α4. A comparison of structural models revealed that the differences in the primary sequences between α4 and α4s are located on the outer surface of the CP, suggesting that α4s interacts with specific molecules via these unique regions. α4s-containing CPs account for the majority of the CPs in mouse sperm. The catalytic β subunits in the α4s-containing CP are β1, β2, and β5, and immunosubunits are not included in the α4s-containing CP. α4s-containing CPs have a set of peptidase activities almost identical to those of α4-containing CPs. Our results provide a basis for understanding the role of α4s and male germ cell-specific proteasomes in mammals.

The ubiquitin-proteasome system is one of the major routes for intracellular protein degradation in eukaryotes (1). Shortlived proteins and abnormal proteins are tagged with ubiquitin chains and are selectively degraded by the 26 S proteasome. This ATP-dependent protease is involved in a diverse array of biological events, including cell-cycle regulation, DNA repair, immune response, and protein quality control (2,3).
The molecular composition of the proteasome is well conserved across eukaryotes. The 26 S proteasome consists of a 20 S core particle (CP) 2 and one or two 19 S regulatory particles, which include 14 and 19 different subunits, respectively (4). The CP is composed of seven homologous ␣ subunits (␣1-␣7) and seven homologous ␤ subunits (␤1-␤7) that are arranged as a cylinder of four heteroheptameric rings: ␣ 1-7 ␤ 1-7 -␤ 1-7 ␣ 1-7 (5). The inner two ␤ rings form a proteolytic chamber, whereas the outer two ␣ rings serve as a gate for substrate entry into the chamber. Of these 14 CP subunits, ␤1, ␤2, and ␤5 have catalytic activities referred to as caspase-like, trypsin-like, and chymotrypsin-like activity, respectively (4). This type of CP is often called the constitutive CP (cCP). The cCP is the basic type of CP and is found from yeast to mammals. In mammals, the cCP is the predominant subtype of CP in most tissues.
Besides ␤1, ␤2, and ␤5, vertebrates have four additional catalytic ␤ subunits, called ␤1i, ␤2i, ␤5i, and ␤5t. They are incorporated into CPs in the place of their most closely related ␤ subunits, thus forming distinct subtypes of CPs. Two additional subtypes of CPs have been identified up to now. One is the immunoproteasome (iCP), which has ␤1i, ␤2i, and ␤5i as catalytic subunits and is constitutively expressed in lymphoid cells. The expression of iCPs can be induced by interferon ␥ in nonlymphoid cells. The other is the thymoproteasome (tCP), where ␤5t is incorporated in place of ␤5 or ␤5i together with ␤1i and ␤2i. The tCP is found exclusively in cortical thymic epithelium cells. Both iCPs and tCPs have peptidase activities different from those of the cCP that play important roles in their specific functions. The iCP efficiently generates antigenic peptides that bind to the groove of MHC class I molecules. The tCP plays a pivotal role in the positive selection of CD8 ϩ T cells (6 -8).
Although the subtypes of catalytic ␤ subunits have been well studied, the diversity of ␣ subunits has remained unclear. A recent study showed that the mammalian testis has an additional ␣ subunit, referred to as ␣4s (9). However, its function remains unknown. Here, we further characterized the expression pattern of ␣4s and the composition of ␣4s-containing CPs in the mouse testis.

EXPERIMENTAL PROCEDURES
Animals-C57BL/6J mice and imprinting control region (ICR) mice were housed in pathogen-free facilities. All experimental protocols described in this study were approved by the Animal Research Committee of the Graduate School of Pharmaceutical Sciences, The University of Tokyo. For synchrony in testicular development, male pups were raised as described previously (10).
DNA Constructs-The short hairpin sequence targeting ␣4 was 5Ј-UGUUGAUGACUAUCCUUGCAUCGGC-3Ј. Oligonucleotides with a loop sequence (TTCAAGAGA) were cloned into the pMX vector (a gift from T. Kitamura, The University of Tokyo) together with the mouse U6 promoter. cDNAs of mouse ␣4, mouse ␣4s, and human ␣4 were synthesized from total RNA isolated from mouse testes or HeLa cells. cDNA of human ␣4s was acquired from the SuperScript Human Testis cDNA Library (Invitrogen). PCR was carried out using Prime-STAR Max DNA polymerase (TaKaRa). An shRNA-resistant mutant of ␣4 (referred to as ␣4*) was generated by introducing point mutations into the sequence targeted by the shRNA (A240T and A241C) without changing amino acid sequences. Amplified fragments were subcloned into either the pcDNA3.1 (Invitrogen) or pIRES vector (Clontech) and sequenced for confirmation.
Separation of Sertoli Cells and Germ Cells-Sertoli cells and germ cells were separated by enzymatic digestion as described previously, with minor modifications (15). Decapsulated testes from adult mice were incubated in DMEM (Nacalai) containing 1 mg/ml collagenase (Sigma) and 0.5 mg/ml deoxyribonuclease I for 45 min at 32°C in a shaking water bath (80 cycles/min). Leydig and interstitial cells were discarded with the supernatant after sedimentation at unit gravity. The sedimented seminiferous tubules were subsequently digested with 2.5 mg/ml trypsin (Invitrogen) for 20 min at 32°C in a shaking water bath (80 cycles/min). Cell aggregates were suspended with DMEM containing 10% FBS (MP Biochemicals), passed through a nylon mesh (100 m), and washed three times with DMEM by centrifugation (5 min, 100 ϫ g). The cells were plated on a 10-cm dish and cultured in DMEM containing 10% FBS at 37°C in humidified 5% CO 2 air. Sertoli cells attach to the dish, whereas germ cells do not. 12 h after plating, floating cells were collected as a germ cell-rich fraction. Sertoli cells were cultured further for 4 days to remove the remaining dead germ cells.
Immunoblotting and Immunoprecipitation-Various mouse tissues, prepuberal testes, Sertoli cells, and germ cells were lysed in a buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 10 mM 2-mercaptoethanol, and 1% SDS for immunoblotting. HEK293T cells, adult mouse testes, and sperm from cauda epididymis were homogenized in an ice-cold buffer containing 25 mM Tris-HCl (pH 7.5), 0.2% Nonidet P-40, 1 mM dithiothreitol, 2 mM ATP, and 5 mM MgCl 2 for immunoblotting, immunoprecipitation, and glycerol density gradient centrifugation analysis. For immunoprecipitation of proteasomes, anti-␣6 or anti-␣4s antibodies bound to protein G-Sepharose (GE Healthcare) were used. Homogenates or lysates were clarified by centrifugation at 20,000 ϫ g for 10 min, separated by SDS-PAGE, transferred to a PVDF membrane, and subjected to immunoblot analysis.
Immunohistochemistry-Mouse testes were fixed in 4% paraformaldehyde in PBS overnight at 4°C, dehydrated, and embedded in paraffin. Sections (5 m thick) were deparaffinized, boiled in 10 mM citrate buffer (pH 6.0), washed in Trisbuffered saline with 0.05% Tween 20 (TBST), and incubated in TBST with 1% goat serum (blocking buffer) for 1 h at room temperature. The sections were then incubated with primary antibodies in blocking buffer overnight at 4°C. The sections were washed in TBST, incubated with secondary antibodies (Alexa Fluor 488 goat anti-rabbit IgG and Alexa Fluor 594 goat anti-mouse IgG, Invitrogen) in blocking buffer. DAPI staining was performed before sections were mounted with SlowFade (Invitrogen). Images were captured on DMI6000B (Leica). Preimmune serum was used as a negative control. Stages of seminiferous tubule cross-sections were assessed by observing the population of germ cells (16).
Inhibition of Sperm Proteasomes-For inhibition of sperm proteasomes, mouse sperm was incubated in human tubal fluid medium (Millipore) containing 10 M epoxomicin (Peptide Institute) at 37°C in humidified 5% CO 2 air.

RESULTS
Identification of a New ␣-type Subunit with High Homology to ␣4 in Mammals-In eukaryotic cells, the proteasome contains seven types of ␣ subunits, called ␣1-␣7. Recent progress in whole genome sequencing has uncovered the possible existence of a new gene that encodes a protein with high homology to ␣ subunits in the mammalian genome. These genes have been annotated as PSMA8 (Gene ID 143471) and Psma8 (Gene ID 73677) in humans and mice, respectively.
A dendrogram analysis of the gene product of Psma8, which has been designated ␣4s (9), showed the highest homology to ␣4 (Fig. 1A). ␣4s shares 85% amino acid identity and 94% similarity to ␣4. Alignments of mouse ␣4 and ␣4s peptide sequences revealed that the differences were concentrated in two regions that encompass residues 178 -191 and C-terminal residues 219 -248 of ␣4 (Fig. 1B, underlined in orange and red), whereas other regions are almost identical.
To predict where these two unique regions locate in the CPs, residues comprising these regions were highlighted in the crystal structural model of the bovine CP ( Fig. 1C) (17). The unique regions of ␣4 (Fig. 1C, depicted in orange and red, corresponding to sequences underlined with the same colors in Fig. 1B) were located on the outer surface of the CP and do not come into contact with the adjacent subunits, whereas the conserved regions (blue) shaped the surfaces that faced the adjacent subunits.
We also predicted the structural models of mouse ␣4 and ␣4s by SWISS-MODEL ( Fig. 1D) (18). A large part of the surfaces generated by the unique regions of ␣4 and ␣4s is negatively charged. Because several N-and C-terminal residues of ␣4 and ␣4s were not included when the models were generated, we were unable to compare the whole C-terminal unique regions in detail, but a remarkable difference is that the C-terminal unique regions (Glu-223 for ␣4 and Lys-225 for ␣4s) have an opposite charge (Fig. 1D, arrowheads).
Taken together, differences in amino acid sequences between ␣4 and ␣4s do not seem to affect CP assembly because the difference is concentrated on the outer surface of the subunits. Rather, it is possible that incorporation of ␣4s in place of ␣4 affects interaction of the CP with other, still unidentified molecules.
␣4s Is Incorporated into the CP in the Mouse Testis-To examine where ␣4s is expressed in the mammalian body, we performed a quantitative RT-PCR analysis using various mouse tissues. This revealed that ␣4s mRNA was specifically expressed in the testis ( Fig. 2A, top panel). We next raised antibodies specific to either ␣4 or ␣4s and used them for immunoblotting of mouse tissue extracts. Although ␣4 protein was expressed in all organs examined, ␣4s protein was detected exclusively in the testis ( Fig. 2A, bottom panel).
We then asked whether ␣4s was incorporated specifically into the proteasome in the testis. Lysates from mouse testes were fractionated by glycerol gradient centrifugation, followed by measurement of proteasome activity and immunoblotting of each fraction. 26 S proteasomes and 20 S CPs were sedimented around fractions 24 and 17, respectively. The distribution of ␣4s coincided with the proteasome activity, just like that of ␣4, suggesting that ␣4s is incorporated into the CP in the testis (Fig.  2B).
To verify the incorporation of ␣4s into the CP and to examine the composition of ␣4s-contaning proteasomes, mouse testis lysates were immunoprecipitated using anti-␣6 and anti-␣4s antibodies. ␣6 is an invariable CP subunit, and, therefore, anti-␣6 antibody precipitates all subtypes of the CP as well as the 26 S proteasome. Indeed, the composition of the proteasome immunoprecipitates by the anti-␣6 antibody was similar to that of the testis lysate. They contained all ␣ subunits, including ␣4s, and also catalytic subunits of iCPs, ␤1i, ␤2i, and ␤5i (Fig. 2C). This is consistent with a previous report showing that the mammalian testis expresses iCP (9). When the testis lysate was immunoprecipitated with anti-␣4s antibody, ␣4 was completely absent from the precipitates (Fig. 2C). These results indicate that incorporation of ␣4 and ␣4s into the CP is mutually exclusive and that ␣4s is incorporated into the CP in place of ␣4. Also, catalytic subunits of iCPs were not observed in ␣4s-containing proteasomes, indicating that catalytic subunits of the ␣4s-containing proteasome are the constitutive catalytic subunits ␤1, ␤2, and ␤5 (Fig. 2C).
Both of the immunoprecipitates contained regulatory particle subunits (Rpt6, Rpn8, and Rpn13) and PA200, which is a CP activator highly expressed in the mammalian testis (Fig. 2C) (19). The relative amounts of these subunits to CP subunits were almost equivalent between the two precipitates, suggesting that there is no significant difference in the affinity for the regulatory particle or PA200 between the ␣4-containing CP and the ␣4s-containing CP.
␣4s Is Incorporated into the CP in Place of ␣4-To confirm incorporation of ␣4s into the CP in place of ␣4, we next examined how the expression of ␣4s affects incorporation of ␣4 into the CP using HEK293T cells, which express cCPs but do not express ␣4s. To this end, we established cells stably expressing ␣4s with a C-terminal FLAG tag.
Expression of ␣4s-FLAG slightly decreased the amount of ␣4 compared with control cells that did not express ␣4s-FLAG (Fig. 3, lanes 1 and 2). Knockdown of ␣4 by siRNA decreased CP subunits in control cells, probably because of the defect in CP biogenesis in the absence of ␣4 (Fig. 3, lane 3). In contrast, the decrease in CP subunits by ␣4 knockdown was modest in ␣4s-FLAG expressing cells, and the protein level of ␣4s-FLAG was increased markedly compared with that without treatment of ␣4 siRNA (Fig. 3, compare lane 4 to lane 2). These observations were specific to ␣4 knockdown, as revealed by the observation that ␣6 knockdown did not increase the expression of ␣4s-FLAG and that expression of ␣4s-FLAG did not increase CP levels in the absence of ␣6 (Fig. 3, lanes 5 and 6). These results indicate that ␣4s is a CP subunit that is incorporated in place of ␣4 during CP biogenesis.
Incorporation of ␣4s Does Not Alter Proteasome Activities-The two well known subtypes of CPs, the iCP and the tCP, have different catalytic ␤ subunits that confer catalytic activities different from those of the cCP (4). We asked whether ␣4s-containing CPs have altered peptidase activities. To examine catalytic activities of ␣4s-containing proteasomes, we established a HEK293T-derived cell line expressing ␣4s-FLAG. To increase the incorporation of ␣4s into the CP, a shRNA targeting ␣4 MAY 2, 2014 • VOLUME 289 • NUMBER 18

JOURNAL OF BIOLOGICAL CHEMISTRY 12367
(␣4-shRNA) was also stably expressed in the cell line (hereafter referred to as ␣4s-FLAG cell). As a control, we prepared a cell line stably expressing both ␣4-shRNA and ␣4*-FLAG (␣4*-FLAG cell). ␣4*-FLAG encodes an ␣4-FLAG protein but has synonymous mutations in its DNA sequences targeted by ␣4-shRNA so that its expression is not affected by ␣4-shRNA.
Lysates of these two cell lines as well as parental HEK293T cells (mock) were separated by glycerol gradient centrifugation. Immunoblot analysis of the fractions by ␣6 and Rpt6 showed the locations of 20 S CPs and 26 S proteasomes around fractions 15 and 25, respectively (Fig. 4A). The distribution of ␣4s-FLAG was similar to that of ␣6, and ␣4s-FLAG was hardly detected in the lighter fractions (fractions 2-12), confirming that ␣4s-FLAG was efficiently incorporated into CPs in the ␣4s-FLAG cells (Fig. 4A). We next measured three known catalytic activities of the proteasome: chymotrypsin-like, caspase-like, and trypsin-like activities. All three peptidase activities in the 20 S CP and 26 S proteasome fractions showed no significant differences between the three cell lines (Fig. 4B). Fractions 15 and 25, which correspond to the peak fractions of the 20 S CP and the 26 S proteasome, respectively, were subjected to immunoblot anal-  ysis (Fig. 4C). In both fractions, the protein level of ␣6 was comparable between the three cell lines. The protein level of ␣4 was quite low in ␣4s-FLAG cells compared with that in mock cells, indicating that peptidase activities observed in ␣4s-FLAG cells reflect those of ␣4s-containing CPs but not of cCPs (Fig.  4C).
We also assessed the protein-degrading activity of ␣4s-containing proteasomes by measuring the degradation rate of ornithine decarboxylase. Lysates of ␣4s-FLAG cells exhibited ornithine decarboxylase-degrading activity comparable with those of other cells (Fig. 4D). These results suggest that incorporation of ␣4s in place of ␣4 does not affect catalytic activities and specificities of the proteasome.
␣4s Is Specifically Expressed in Male Germ Cells-The mammalian testis consists of male germ cells, Sertoli cells, and interstitial cells. To elucidate in which cell type ␣4s is expressed, cross-sections of adult mouse testes were immunostained using anti-␣4s antibody. ␣4s was detected in the seminiferous tubules (Fig. 5, A and B, inside the broken line), whereas no signal of ␣4s was observed in interstitial cells (Fig. 5, A and B, arrowheads).
The seminiferous tubules contain germ cells and Sertoli cells. To examine whether ␣4s is expressed in either or both cells, Sertoli cells and germ cells were isolated from the seminiferous tubules, lysed, and subjected to immunoblotting, SOX9 was used as a marker for Sertoli cells (Fig. 5C) (20,21). ␣4 was expressed in both Sertoli cells and germ cells, whereas ␣4s was detected only in germ cells but not in Sertoli cells (Fig. 5C). These analyses revealed that ␣4s is specifically expressed in male germ cells.
Expression of ␣4s Occurs during Meiotic Prophase-Male germ cells undergo a complex process of differentiation called spermatogenesis, in which spermatogonia, germ stem cells, develop into mature sperm. We next examined in which types of germ cells ␣4s was expressed.
Spermatogenesis proceeds in synchronized waves along the seminiferous tubules, and every given cross-section of the tubule contains only certain cell types in a specific combination. This spermatogenic wave is divided into 12 stages (I to XII) in mice, and germ cells are arranged from the epithelial side to the luminal side of the seminiferous tubules in the order of differentiation (10,16). Immunohistochemical analysis showed that, in stage I of the seminiferous tubules, ␣4s was specifically expressed in round spermatids (RS) and elongating spermatids (ES) but not in early pachytene spermatocytes (P) positive for Sycp3, which is known to be expressed in leptotene, zygotene, and pachytene spermatocytes (Fig. 5A) (22). In stage VII, on the other hand, ␣4s was detected in middle pachytene spermatocytes, which are more differentiated than pachytene spermatocytes in stage I (Fig. 5B). ␣4s was also expressed in round spermatids (RS) but not in preleptotene spermatocytes (Pl) in stage VII (Fig. 5B). These results demonstrate that the expression of ␣4s starts in early to mid-pachytene spermatocytes and continues in spermatids (Fig. 5D).
To confirm the temporal expression profile of ␣4s, we performed further experiments using prepubertal mouse testes. The first spermatogenic wave occurs at around a week after birth, and pachytene spermatocytes and round spermatids first appear at around P14 and P18, respectively, in mice (10). Mouse testes were homogenized and subjected to immunoblot analysis every 2 days from postnatal day 8 (P8) to P18. ␣4 was constantly expressed in testes on all days (Fig. 5D). Sycp3, which exists in two isoforms, was already expressed on P8, whereas the expression of ␣4s started on P14, suggesting that ␣4s is expressed after differentiation of germ cells into spermatocytes (Fig. 5D) (23).
These developing testes were also subjected to immunostaining (Fig. 5, E-G). No obvious signal of ␣4s was detected in the seminiferous tubules on P10, when the expression of Sycp3 was recognized (Fig. 5E). On P14, Sycp3-positive spermatocytes in some cross-sections of the seminiferous tubules (open arrowheads) expressed ␣4s, whereas others (filled arrowheads) did not (Fig. 5F), similar to adult testes, which contain both ␣4s-positive and -negative pachytene spermatocytes (Fig. 5, A  and B). On P18, round spermatids were observed that expressed ␣4s (Fig. 5G). These results are consistent with the expression profile of ␣4s observed in the adult testis. The expression of ␣4s occurs after the appearance of Sycp3, starts in the Sycp3-positive spermatocytes, and continues in spermatids. Taken together, these results demonstrate that male germ cells express ␣4s after they enter the meiotic prophase.
The ␣4s-containing CP Is the Predominant Form of the CP in Mouse Sperm-We next asked whether mature sperm expresses the ␣4s-containing CP. Glycerol gradient centrifugation analysis of sperm lysates showed that ␣4s was detected in the CP in mouse sperm (Fig. 6A). ␣4 was also detected in the CP in sperm, indicating that mouse sperm has both ␣4-containing CPs and ␣4s-containing CPs (Fig. 6A). When the protein levels of ␣4 and ␣4s were compared between whole testis lysates and sperm lysates, the amount of ␣4 relative to ␣6 was extremely small in sperm lysates compared with that in whole testis lysates (Fig. 6B). This result suggests that the expression of the ␣4-containing CP is severely attenuated in mouse sperm and replaced by the ␣4s-containing CP.
To estimate how many of the CPs are ␣4s-containing CPs in mouse sperm, sperm lysates were subjected to immunoprecipitation with either an anti-␣6 or anti-␣4s antibody, followed by immunoblot analysis of the unbound fractions. Because the anti-␣6 antibody immunoprecipitates all types of CPs, the unbound fraction contained neither ␣4s nor ␣6 (Fig. 6C). The anti-␣4s antibody immunoprecipitates ␣4s-containing CPs, whereas it does not immunoprecipitate ␣4-containg CPs. Indeed, ␣4s was  depleted by immunoprecipitation with the anti-␣4s-antibody. The anti-␣4s antibody also depleted the majority of ␣6, although a small amount of ␣6 remained in the unbound fraction (Fig. 6C). This suggests that the majority of the CP in the sperm is ␣4scontainig CPs.
To confirm this observation, the immunoprecipitates with the anti-␣6 antibody were serially diluted and compared with the amount of ␣6 in the immunoprecipitates with anti-␣4s antibody. This indicates that ϳ80% of the CPs in sperm lysates contain ␣4s (Fig. 6D). Therefore, we concluded that most of the CPs in the mouse sperm are ␣4s-containg CPs.

Ubiquitin-Proteasome Mediated Protein Degradation Occurs
in Mature Sperm-During mammalian spermatogenesis, most parts of the cytoplasm are removed from spermatids to acquire the shape of mature sperm (24,25). It is intriguing to ask whether the proteasome is still working in mature sperm cells. To examine this, mouse sperm was treated with epoxomicin, a proteasome inhibitor, and lysed for immunoblot analysis with anti-ubiquitin antibody (Fig. 6E) (26). Incubation of sperm with epoxomicin caused accumulation of ubiquitinated proteins, suggesting that degradation of ubiquitinated proteins by the proteasome is occurring in mouse sperm (Fig. 6E).

DISCUSSION
A recent study by Qian et al. (9) first reported a newly found ␣-type subunit expressed in the mammalian male germ cell. This subunit, referred to as ␣4s, has high homology to ␣4 and was included in proteasomes purified from mammalian testes (Fig. 1) . However, the subunit composition of the ␣4s-containing proteasome has not yet been examined. In this study, we further characterized ␣4s and the ␣4s-containing proteasome in detail.
Qian et al. (9) showed that ␣4s is specifically expressed in spermatids and sperm but not in spermatocytes and spermatogonia . However, our analysis of developing testes as well as observation of adult seminiferous tubules in various stages showed that expression of ␣4s starts at an earlier stage of spermatogenesis and that spermatocytes also express ␣4s after they enter the meiotic prophase (Fig. 5).
Our biochemical analysis clarified that ␣4s is incorporated into the CP in place of ␣4 (Figs. 2 and 3). The CP is formed by dimerization of two half-CPs that have one ␣ ring and one ␤ ring, and, thus, the CP has two ␣ rings (27). Although both ␣4 and ␣4s are expressed in germ cells, CPs possessing both ␣4 and ␣4s do not seem to be present in the testis, which is revealed by immunoprecipitation by an antibody specific to ␣4s, that we generated in this study (Fig. 2C). We also showed that ␣4scontaining CPs do not include catalytic ␤ subunits of iCPs, although mammalian testes express these subunits (Fig. 2C). These findings suggest a specific mechanism by which assembly of the ␣4s-containing CP is regulated (11,27).
Comparison of the structural models of ␣4 and ␣4s showed that differences between the two subunits are located on the outer surfaces of the CP (Fig. 1). ␣4 is reported to directly interact with various molecules, such as Rab7, mitochondrial antivi-FIGURE 6. The ␣4s-containing CP is the predominant form of CP in mature sperm. A, extracts of adult mouse sperm were fractionated by glycerol gradient centrifugation. The chymotrypsin-like activity of the resultant fractions was measured in the presence of 0.025% SDS (top panel). Arrows indicate the peak locations of the 20 S CP and the 26 S proteasome. Each fraction was subjected to immunoblotting with the indicated antibodies (bottom panel). B, extracts of adult mouse testes and sperm were subjected to immunoblotting with the indicated antibodies. C, equal amounts of mouse sperm extracts (input) were immunoprecipitated with either anti-␣6 antibody or anti-␣4s antibody. The resultant unbound fractions as well as the input were subjected to immunoblotting with the indicated antibodies. D, immunoprecipitates (IP) with anti-␣6 antibody were serially diluted (20, 40, 60, 80, and 100%) and subjected to immunoblotting for ␣6. Each band was quantified by densitometry and used to generate a standard curve for estimation of the amount of ␣6. The band of ␣6 in the immunoprecipitate with anti-␣4s antibody was quantified following estimation of its amount by interpolation on the standard curve (dotted line). E, mouse sperm was incubated with dimethyl sulfoxide (DMSO) or 10 M epoxomicin for 7 h and lysed for immunoblot analysis with antibodies against ubiquitin (Ub) and Rpt6 . Data in are representative of at least three independent experiments. ral signaling protein (MAVS), c-Abl, and HIF-1␣ (28 -31). These molecules are likely to interact with residues of ␣4 that are exposed to the outer surface of the CP. Analogous to ␣4, it is possible that ␣4s interacts with some molecules distinct from the ␣4-interacting molecules in male germ cells, which might confer specific roles on the ␣4s-containing CP.
Qian et al. (9) showed that "spermatoproteasomes," which designates PA200-associated proteasomes in the testis, are required for the degradation of acetylated histones during spermatogenesis . Although they showed that PA200 is essential for this function, the physiological role of ␣4s is still totally unknown. Our immunoblot analysis using sperm lysates showed that incubation of sperm with a proteasome inhibitor caused accumulation of ubiquitinated proteins (Fig. 6E). Considering that the majority of the CPs in mouse sperm is ␣4scontaining proteasomes (Fig. 6, A-D), these results suggest that sperm has substrates that are ubiquitinated and sequentially degraded by ␣4s-containing proteasomes. Identification of such substrate proteins might give a clue to understand the role of ␣4s in the future.