JBC Anatrace, Inc.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M700414200 on June 29, 2007

J. Biol. Chem., Vol. 282, Issue 34, 24806-24815, August 24, 2007
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
282/34/24806    most recent
M700414200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Morokuma, Y.
Right arrow Articles by Hirose, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Morokuma, Y.
Right arrow Articles by Hirose, S.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

MARCH-XI, a Novel Transmembrane Ubiquitin Ligase Implicated in Ubiquitin-dependent Protein Sorting in Developing Spermatids*Formula

Yuri Morokuma{ddagger}§, Nobuhiro Nakamura{ddagger}, Akira Kato{ddagger}, Michitaka Notoya{ddagger}, Yoko Yamamoto{ddagger}, Yasuhiro Sakai, Hidekazu Fukuda{ddagger}1, Shohei Yamashina, Yukio Hirata§, and Shigehisa Hirose{ddagger}2

From the {ddagger}Department of Biological Sciences, Tokyo Institute of Technology, Yokohama 226-8501, Japan, §Department of Clinical and Molecular Endocrinology, Tokyo Medical and Dental University Graduate School, Tokyo 113-8519, Japan, and the Department of Anatomy, Kitasato University School of Medicine, Sagamihara 228-8555, Japan

Received for publication, January 16, 2007 , and in revised form, June 11, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
A mechanism by which ubiquitinated cargo proteins are sorted into multivesicular bodies (MVBs) from plasma and trans-Golgi network (TGN) membranes is well established in yeast and mammalian somatic cells. However, the ubiquitin-dependent sorting pathway has not been clearly defined in germ cells. In this study we identified a novel member of the transmembrane RING-finger family of proteins, termed membrane-associated RING-CH (MARCH)-XI, that is expressed predominantly in developing spermatids and weakly in brain and pituitary. MARCH-XI possesses an E3 ubiquitin ligase activity that targets CD4 for ubiquitination. Immunoelectron microscopy of rat round spermatids showed that MARCH-XI is localized to TGN-derived vesicles and MVBs. Fluorescence staining of rat round spermatids and immunoprecipitation of rat testis demonstrated that MARCH-XI forms complexes with the adaptor protein complex-1 and with fucose-containing glycoproteins including ubiquitinated forms. Furthermore, the C-terminal region of MARCH-XI mediates its interaction with µ1-adaptin and Veli through a tyrosine-based motif and a PDZ binding motif, respectively. Our data suggest that MARCH-XI acts as a ubiquitin ligase with a role in ubiquitin-mediated protein sorting in the TGN-MVB transport pathway, which may be involved in mammalian spermiogenesis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Membrane trafficking is essential for eukaryotic cells to proliferate, differentiate, and perform various physiological processes such as neurotransmitter release, hormone secretion, fibroblast motility, and pathogen engulfment. It also plays certain roles in mammalian spermiogenesis, a differentiation process of postmeiotic male germ cells (spermatids) into mature elongated spermatozoa, including acrosome and flagellum formation, nuclear elongation and condensation, and mitochondrial rearrangement (1, 2). In early developing spermatids, numerous proteins and membranes necessary for acrosomal biogenesis are delivered to the immature acrosomal vesicle via the Golgi-derived vesicles (1). Some portion of glycoproteins has been found to be simultaneously sorted away from this acrosome pathway to be transported to the endosomal system (3–6). As seen in somatic cells, these events are thought to be tightly controlled by the membrane-targeting and fusion machinery such as Rab GTPase and SNARE3 proteins, which are associated with the Golgi and acrosomal membranes (7–9). Recent studies using knock-out mice have indicated that the adaptor proteins Hrb and Golgi-associated PDZ- and coiled coil motif-containing protein (GOPC) are essential for fusion and transport of the proacrosomal vesicles (10, 11). However, major questions remain regarding the mechanisms underlying the selective recognition and recruitment of cargo proteins into transporting vesicles.

Ubiquitin is known to serve as a sorting signal of transmembrane proteins for incorporation into the multivesicular body (MVB), a part of the endosomal system, from the plasma membrane and trans-Golgi network (TGN) (12, 13). Ubiquitination is catalyzed by the sequential action of three enzymes: a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). Ubiquitin ligases confer substrate specificity and direct the transfer of the activated ubiquitin moiety to substrate proteins. They are grouped into two major families based on the presence of the E3 catalytic core domain; that is, the RING-finger and HECT families. Several ubiquitin ligases have been shown to ubiquitinate plasma membrane proteins, including receptors, transporters, and ion channels, leading to their endocytic sorting to the MVB and subsequent lysosomal degradation (14). By contrast, little is known about E3 molecules responsible for ubiquitin-mediated sorting at the TGN, with the exception of two yeast proteins, Rsp5 (15) and Tul1 (16). In rat spermatids, ubiquitinated proteins have been shown to be abundant on membranes of the TGN and MVBs (17), suggesting the presence of a ubiquitin-dependent protein sorting mechanism in the TGN-MVB pathway. As yet the E3 molecules responsible for mediating these ubiquitination have not been identified.

Recently, a novel family of RING-finger ubiquitin ligases has been identified in mammals, termed membrane-associated RING-CH (MARCH) or c-MIR (cellular modulator of immune regulation) (18, 19). Ten MARCH gene products (MARCH-I to -X) have been recognized (20), and the majority shares a similar structure, an N-terminal C4HC3-type RING finger (RING-CH finger) and two or more C-terminal transmembrane spans (18). Among them, MARCH-IV, -VIII, and -IX have been shown to ubiquitinate and down-regulate transmembrane glycoproteins, such as major histocompatibility complex class I and II, B7-2, CD166, and ICAM-1 (18, 19, 21–23). Particularly, ubiquitination of major histocompatibility complex class I leads to its endocytosis and subsequent sorting to the MVB pathway for lysosomal degradation (18). Previously we have demonstrated that MARCH-II and -III are endosomal proteins bound to the SNARE protein syntaxin 6 and are involved in the regulation of endosomal trafficking (24, 25). Hence, it is conceivable that the MARCH family could function in protein sorting and transport. In this study we identified a novel member of the MARCH family, termed MARCH-XI, that is highly expressed in developing spermatids of rats. To address the role of MARCH-XI in spermiogenesis, we determined its subcellular localization and identified associated proteins. Our results suggest that MARCH-XI is likely a ubiquitin ligase mediating protein sorting in the TGN-MVB pathway.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cloning of MARCH-XI—A partial cDNA fragment of MARCH-XI was obtained from rat brain by PCR amplification with primers 5'-TGTCTAGACACTTGGTCCACCCAAC-3' and 5'-CCTCTAGAAGTGCTATGGTTTTGCCATTC-3'. The PCR product was inserted into the XbaI site of pBluescript II SK– (Stratagene), yielding pBS-Kdg5Xba. A rat testis {lambda} cDNA library (Stratagene) was screened by the standard method with the XbaI fragment of pBS-Kdg5Xba as a probe. Positive clones were plaque-purified, and the cDNA inserts were subcloned into the EcoRI site of pBluescript II SK–, yielding pBS-Kdg5#9–4. The nucleotide sequence for rat MARCH-XI has been deposited in the DDBJ/GenBankTM/EMBL databases with accession number AB048841.

Plasmids—A mammalian expression vector for MARCH-XI (pcDNA3-MARCH-XI) was constructed by cloning the 1.4-kilobase PstI-EcoRI fragment of pBS-Kdg5#9–4 into pcDNA3 (Invitrogen). To construct a mammalian expression vector for the deletion mutant lacking the last amino acid residue of MARCH-XI (pcDNA3-MARCH-XI{Delta}), the cDNA fragment encoding residues 167–397 of MARCH-XI was amplified by PCR with the primers 5'-AAGATCTGCTTCCAGGGCGCGGAGCAGGGT-3' and 5'-CTCGAGTCAAGACGTCACTCTCATCACTAC-3', digested with BglII and XhoI, and then inserted into the same sites of pBS-Kdg5#9–4, yielding pBS-Kdg5{Delta}. Then, the SacII-XhoI fragment of pBS-Kdg5{Delta} was inserted into the same sites of pcDNA3-MARCH-XI, yielding pcDNA3-MARCH-XI{Delta}. To generate a glutathione S-transferase (GST)-His6-Xpress expression vector (pGex-His6-Xpress), a cDNA fragment encoding a His6-Xpress tag was amplified by PCR from pTrcHis (Invitrogen) with the primers 5'-GGCCAGATCTATGGGGGGTTCTCATCAT-3' and 5'-GGCCGGATCCTTTATCGTCATCGTC-3'. The PCR product was digested with BglII and BamHI and then inserted into the BamHI site of pGex4T (GE Healthcare). GST-His6-Mar11N was constructed by cloning the fragment encoding residues 1–146 of MARCH-XI into the BamHI-EcoRI sites of pGex-His6-Xpress. His-Mar11C and His-Mar11C{Delta} were constructed by cloning the fragments encoding residues 323–398 and 323–397 of MARCH-XI, respectively, into the XhoI-EcoRI sites of pRSET (Invitrogen). GST-RING was constructed by cloning the fragment encoding residues 147–227 of MARCH-XI into the BamHI-EcoRI sites of pGex4T. GST-GOPC was constructed by cloning of the coding region of rat GOPC into pGex4T. FLAG-CD4 was constructed by cloning of the fragment encoding residues 29–457 of rat CD4 into the EcoRI-XbaI sites of the FLAG-furin expression vector (25). 3 x FLAG-tagged µ1-adaptin was constructed by cloning of the coding region of rat µ1A-adaptin into the HindIII-XhoI sites of p3 x FLAG-CMV10 (Sigma). Myc-tagged Veli-3 was constructed by cloning of the coding region of rat Veli-3 into the EcoRI-XhoI sites of pCMV-Myc (Clontech). GST-Veli was described previously (25). All point mutations were introduced by site-direct mutagenesis using PCR.

Preparation of Recombinant Proteins—The GST-fusion and His6-tagged proteins were produced in the Escherichia coli strain BL21(DE3) pLysS (Novagen) and purified with glutathione-Sepharose 4B beads (GE Healthcare) and Talon metal affinity resins (Clontech), respectively. Purified recombinant proteins were dialyzed against saline, with the exception that His-Mar11C and His-Mar11C{Delta} were done against 2% Triton X-100 solution. For detail information, see supplemental "Experimental Procedures."

Antibodies—The following primary antibodies were purchased. Anti-ubiquitin (Ubi-1) was from Zymed Laboratories Inc., anti-{gamma}-adaptin (clone 88) and anti-TGN38 (clone 2) were from BD Transduction Laboratories, anti-ubiquitin (FK1) was from Biomol, anti-FLAG (M2), anti-His6, and anti-GST were from Sigma, and anti-Myc (9E10) and anti-HA (3F10) were from Roche Applied Science. Rabbit and rat polyclonal anti-Mar11 antisera were raised against a GST-His6-Mar11N protein. Rabbit polyclonal anti-Veli and anti-GOPC antisera were raised against GST-Veli and GST-GOPC proteins, respectively. The immunization protocols and the specificity of rabbit and rat anti-Mar11 antisera are described in the supplemental data. The anti-Veli and anti-GOPC immunoaffinity beads were prepared as follows. The appropriate antigen (2 mg) was covalently coupled to 1 ml of the HiTrap NHS-activated HP column (GE Healthcare) according to the manufacture's protocol. The antiserum (5 ml) was incubated with the affinity column for 30 min at room temperature. Then, after washing with 15 ml of 75 mM Tris-HCl, pH 8.0, bound antibodies were eluted with 0.5 M NaCl and 0.1 M glycine-HCl, pH 2.7 and subsequently dialyzed against 0.1 M Na2PO4, pH 7.0. The affinity-purified anti-Veli and anti-GOPC antibodies (500 µg of IgG each) were coupled to 500 µl of the Carbolink gel (Pierce) according to the manufacturer's protocol.

Northern Blot Analysis—Northern blot analysis was performed with 32P-labeled XbaI fragment of pBS-Kdg5Xba as described previously (24).

Cell Culture—Maintenance of cultured cells and transient transfection were described previously (24). For ubiquitination experiments, at 16–20 h post-transfection, cells were incubated for 4 h at 37 °C in culture medium containing 50 nM concanamycin A (Wako), a specific inhibitor of vacuolar H+-ATPase used to prevent the lysosomal degradation of ubiquitinated CD4 (26).

In Vitro Ubiquitination Assay—Purified GST-RING or GST-RINGmut (1 µg) was incubated with a mixture (20 µl) composed of 0.1 µg of rabbit E1 (Boston Biochem), 1 µg of purified recombinant His6-tagged E2 (24), 1 µg of ubiquitin (Sigma), and 1 mM creatine phosphokinase (Sigma) in an ATP-regeneration buffer (25 mM Tris-HCl, pH 7.5, 120 mM NaCl, 1 mM MgCl2, 0.3 mM dithiothreitol, 2 mM ATP, and 1 mM creatine phosphate) for 4 h at 30 °C. The samples were subjected to Western blot analysis with a tank-blotting apparatus (Atto) as described previously (27). Ubiquitin conjugates were detected with anti-ubiquitin (P4D1) antibody (Santa Cruz Biotechnology).

Immunoprecipitation—All operations were carried out at 4 °C, and all solutions and buffers were added with protease inhibitors (10 mM leupeptin, 1 mM pepstatin, 5 mg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride). For immunoprecipitation of FLAG-CD4, the membrane fractions were lysed with TNE buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 10% glycerol, and 1 mM EDTA). The lysates were subjected to immunoprecipitation with anti-FLAG M2 beads (Sigma) followed by Western blotting as described previously (27). For immunoprecipitation of MARCH-XI, rat testes (3 g) were homogenized in 30 ml of SPH buffer (0.25 M sucrose, 0.2 M KCl, and 10 mM HEPES-KOH, pH 7.0). The homogenate was layered onto a 1.5 M sucrose cushion (12 ml) in an SW28 tube (Beckman Coulter) and centrifuged for 2 h at 25,000 rpm on an SW28 rotor (Beckman Coulter). The membrane fraction (0.25/1.5 M sucrose interface) was diluted in phosphate-buffered saline (PBS) and centrifuged for 30 min at 40,000 rpm on an SW41 rotor. The resulting membrane pellet was lysed with 6 ml of PBS containing 0.5% Triton X-100. The lysate was incubated with 20 µlof protein G-Sepharose beads (GE Healthcare) and 2 µl of rabbit anti-Mar11 antiserum overnight. For immunoprecipitation of Veli or GOPC, the lysate was incubated for 4 h with 500 µl of the anti-Veli or anti-GOPC immunoaffinity beads. After washing 7 times with PBS containing 0.5% Triton X-100, the beads were eluted with 0.5% Triton X-100 and 0.1 M glycine-HCl, pH 2.5.

Lectin Blotting—The rat testicular membranes were lysed in 6 ml of TNE buffer containing protease inhibitors. The lysate (800 µg of protein) was subjected to immunoprecipitation with rabbit anti-Mar11 or preimmune serum. The eluted proteins were separated on a 10% SDS-polyacrylamide gel and then transferred to an Immobilon-P polyvinylidene fluoride (PVDF) membrane (Millipore). The filter was blocked with T-TBS (0.05% Tween 20, 150 mM NaCl, and 10 mM Tris-HCl, pH 7.6) containing 5% bovine serum albumin (BSA) for 2 h at room temperature and then incubated with biotin-labeled Aleuria aurantia lectin (AAL) (10 µg/ml in T-TBS; Vector Laboratories) overnight at 4 °C. The filter was incubated with goat antibiotin antibody (2.5 µg/ml; Vector Laboratories) followed by peroxidase-conjugated anti-goat IgG antibody (1:100,000; Jackson ImmunoResearch Laboratories).

Fluorescence Microscopy—Wister rats (8 weeks old) were perfused with PBS followed by ice-cold fixative (2% paraformaldehyde and 100 mM NaH2PO4, pH 7.4). Testes were extracted, bisect, immersed with the same fixative for 2 h, and then immersed with PBS containing 15% sucrose followed by PBS containing 30% sucrose overnight at 4 °C. Preparation and staining of sections and smear preparations of rat testis were described previously (28) with the exception that primary antibodies were incubated for 2 days at 4 °C. For staining of fucose glycoproteins, spermatids were incubated with 10 µg/ml biotin-labeled ALL in PBS at overnight at 4 °C followed by Alexa Fluor 488-conjugated mouse anti-biotin monoclonal antibody (2F5; 5 µg/ml; Molecular Probes) for 30 min at room temperature. Dilutions of primary and secondary antibodies were as follows: anti-ubiquitin FK1, 1:200; anti-{gamma}-adaptin, 1:500; anti-TGN38, 1:250; anti-Mar11, anti-Veli, and anti-GOPC, 1:1000; Alexa Fluor 488 or 546-conjugated anti-rabbit or mouse IgG antibody (Molecular Probes), 1:2000; Cy3-conjugated anti-rat IgG antibody (Jackson Immuno Research Laboratories), 1:2000. After the nuclei were stained with TO-PRO3 (2 µM; Molecular Probes) or Hoechst 33342 (5 µg/ml; Molecular Probes), the samples were observed with a confocal microscope (LSM510; Carl Zeiss).

Immunoelectron Microscopy—Pre-embedding immunoelectron microscopy was performed by the previous method (29) with modifications. Wister rats were perfused with ice-cold fixative (4% paraformaldehyde in PBS). Testes were extracted, cut into small pieces (~20 x 20 x 20 mm), and fixed in the same fixative for 2 h. The pieces of testes were further cut (~3 x 3 x 3 mm), fixed in the same fixative for 8 h, immersed with PBS containing 7% sucrose at 4 °C, and then frozen in optimum cutting temperature compound. Cryosections (15-µm thickness) were blocked with PBS containing 1% bovine serum albumin for 1 h at room temperature and incubated with rabbit anti-Mar11 (1:300 in PBS) overnight at 4 °C. The samples were treated with biotinylated anti-rabbit IgG antibody (Dako) followed by peroxidase-conjugated streptavidin (Dako). Signals were developed with diaminobenzidine tetrahydrochloride. The samples were post-fixed with 1% OsO4, dehydrated with ethanol, and embedded in Epon. Ultrathin sections (80-nm thickness) were contrasted with uranyl acetate for 10 min followed by lead citrate for 5 min and observed with an electron microscope (H-600, Hitachi).

Yeast Two-hybrid Assay—The MATCHMAKER two-hybrid system 3 was purchased from Clontech. The cDNA fragments encoding rat µ1A-adaptin and µ2-adaptin were inserted into the ClaI-XhoI sites of pGADT7. The fragments encoding residues 323–398 of MARCH-XI and its Y367A mutant were inserted into the EcoRI-BamHI sites of pGBKT7, yielding pGBKT7-Mar11C and pGBKT7-Y367A, respectively. These constructs were transformed into the yeast strain AH109 (MATa) with the Fast-yeast transformation kit (Geno Technology). AH109 cells were transformed with pGADT7–53 and pGBKT7-T or with pGADT7 and pGBKT7, which were used as a positive or negative control, respectively. The double transformants were grown on SD agar plates lacking Trp and Leu, with exception of the pGADT7-µ1-pGBKT7-Mar11C and positive control clones which were grown on plates lacking Trp, Leu, and His for 3–10 days at 30 °C. Colonies were picked and restreaked on Trp–/Leu–/His– plates for selection of interacting clones and assayed for beta-galactosidase activity. For beta-galactosidase assay, a single colony was grown overnight at 30 °C in 2 ml of Trp–/Leu– media. A portion of each culture was centrifuged, and the yeast pellet was resuspended to an optical density of 0.2 at 600 nm in 0.1% bovine serum albumin and 25 mM HEPES, pH 7.5. One hundred microliters of the yeast samples were used to test beta-galactosidase activity with the Beta-Glo assay system (Promega).


Figure 1
View larger version (58K):
[in this window]
[in a new window]

  		FIGURE 1.
Characterization of MARCH-XI. A, deduced amino acid sequence of rat MARCH-XI is shown. Dots, proline-rich sequences; underline, a RING-CH finger domain; boxes, putative transmembrane regions; double underline, a tyrosine-based motif; wavy line, a PDZ binding motif. B, phylogenetic analysis of rat (r) and human (h) MARCH proteins. The tree was constructed by the neighbor-joining method by using the ClustalW computer program. The scale bar represents a genetic distance of 0.2 amino acid substitutions per site. The GenBankTM accession numbers are as follows: hMARCH-I, NM_017923; hMARCH-II, NM_016496; hMARCH-III, NM_178450; hMARCH-IV, NM_020814; rMARCH-IV, XP_001074008; hMARCH-V, AB191202; hMARCH-VI/TEB4, AB011169; hMARCH-VII/axotrophin, NM_022826; hMARCH-VIII/c-Mir, BC025394; hMARCH-IX, NM_138396; rMARCH-IX, AB048842; hMARCH-X, Q8NA82; rMARCH-XI, AB048841; Kir7.1 potassium channel, NP_002233, as an unrelated outgroup. C, Northern blot analysis of 20 µg of total RNA samples from rat tissues was performed with 32P-labeled cDNA probes for MARCH-XI (top) and beta-actin (bottom). kb, kilobase(s). D, reverse transcription-PCR analysis of MARCH-XI (top) and beta-actin (bottom) was performed on total RNAs from indicated cultured cells and rat tissues. E, membrane lysates of rat testis (400 µg of protein) were immunoprecipitated (IP) with anti-Mar11 (Im; lanes 1 and 3) or preimmune serum (Pre; lanes 2 and 4). The immunoprecipitates were analyzed by Western blotting (WB) with anti-Mar11 (Im; lanes 1 and 2) or preimmune serum (Pre; lanes 3 and 4). Arrowhead and double-arrowhead indicate the bands corresponding to MARCH-XI and IgG heavy chain, respectively. F, total membranes of COS7 cells transfected with MARCH-XI were treated with PBS (lanes 1 and 2), 1 M NaCl (lanes 3 and 4), 0.1 M Na2CO3, pH 11.5 (lanes 5 and 6), or 1% Triton X-100 (lanes 7 and 8) for 30 min on ice. Samples were subjected to a high speed centrifugation at 100,000 x g, and resulting supernatants (S) and pellets (P) were analyzed by Western blotting with anti-Mar11.

 
Dot-blot Assay—Various amounts of GST and GST fusion proteins were blotted onto Immobilon-P PVDF membranes using a vacuum. The membranes were blocked with T-TBS containing 5% nonfat milk followed by incubation with His-Mar11C or His-Mar11C{Delta} in 2% Triton X-100 overnight at 4 °C. The membrane was probed with anti-His6 or anti-GST antibody (1:3000 or 1:1000 in T-TBS) followed by anti-mouse IgG secondary antibody conjugated with alkaline phosphatase or horseradish peroxidase, respectively (1:3000; Jackson Immuno-Research Laboratories).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of a Novel Member of the MARCH Family—The sequences of a novel mammalian MARCH member were found by Blast searches of the GenBankTM expression sequence tag (EST) databases (GenBankTM accession numbers AW070319, AA868748, and AI046596). A corresponding cDNA fragment was obtained by PCR amplification from rat brain and used to screen a rat testis cDNA library. Positive clones contained a single open reading frame encoding a protein of 398 amino acid residues with a calculated molecular mass of 44.1 kDa (Fig. 1A). A phylogenetic analysis revealed that the cloned protein is the newest 11th MARCH member closely related to MARCH-IV and -IX (see Fig. 1B and supplemental Fig. S1 for sequence alignment). Therefore, we designated this novel protein "MARCH-XI." A hydropathy plot analysis predicted that MARCH-XI has a typical MARCH membrane topology with N- and C-terminal cytoplasmic tails flanking two transmembrane spans. As shown in Fig. 1A, the N-terminal tail contains proline-rich sequences (dots) and a RING-CH finger (underline), and the C terminus contains a PDZ binding sequence (wavy line; the consensus sequence E(S/T)X(V/I)) (30).

Northern blot analysis of rat tissues showed that MARCH-XI mRNA (~1.7 kilobases) is predominantly expressed in testis and weakly in brain (Fig. 1C). In addition, reverse transcription-PCR experiments revealed that MARCH-XI is also expressed in rat pituitary and the mouse pituitary corticotroph tumor cell line AtT20/D16v (Fig. 1D). To assess the protein expression of MARCH-XI, we generated rabbit and rat polyclonal antisera specific to MARCH-XI (anti-Mar11, see supplemental Fig. S2). When membrane extracts of rat testis were subjected to immunoprecipitation followed by Western blot analysis with anti-Mar11, a single band was detected at ~48 kDa corresponding to endogenous MARCH-XI protein (Fig. 1E, arrowhead). MARCH-XI was extracted from membranes by Triton X-100 treatment but not with high salt or alkaline carbonate solution (Fig. 1F), indicating that MARCH-XI is a transmembrane protein.

To examine whether the RING-CH finger of MARCH-XI possesses an E3 ubiquitin ligase activity, in vitro ubiquitination assays were performed using an established method with recombinant GST fusion proteins of the MARCH-XI RING-CH finger (GST-RING) or its mutant containing point mutations in the conserved cysteine residues (C169S, C182S, and C184S; GST-RINGmut). As shown in Fig. 2A, GST-RING but not GST-RINGmut catalyzed the formation of polyubiquitinated products in the presence of the E2 ubiquitin-conjugating enzymes UbcH5B and UbcH5C. Next, we sought to address whether the full-length MARCH-XI protein exhibits an E3 activity. A previous report has demonstrated that MARCH-IV down-regulates CD4 in a manner dependent on the lysine residues in the cytoplasmic tail of CD4 (18), suggesting that CD4 is likely an E3 substrate for MARCH-IV. The structural similarity of MARCH-XI to MARCH-IV allowed us to determine whether MARCH-XI has ability to ubiquitinate CD4. FLAG-tagged CD4 (FLAG-CD4) was coexpressed in COS7 cells with HA-tagged ubiquitin in the absence or presence of MARCH-XI or its RING-finger mutant. Immunoprecipitation of FLAG-CD4 followed by Western blotting for HA-ubiquitin revealed increased levels of FLAG-CD4 ubiquitination in the cells expressing wild-type MARCH-XI but not the RING-finger mutant (Fig. 2B). In addition, Western blotting of the anti-FLAG immunoprecipitates revealed that both wild-type and mutant MARCH-XI were co-immunoprecipitated with FLAG-CD4, indicating that MARCH-XI acts as a ubiquitin ligase through its RING-finger domain, targeting CD4 for ubiquitination. Together, these results indicate that MARCH-XI is a transmembrane ubiquitin ligase highly expressed in testis.

Stage-specific Expression in Rat Spermatids—To determine cell types that express MARCH-XI, immunofluorescence histochemistry of rat testicular sections was performed with anti-Mar11. Strong punctate staining was observed in the middle part of the seminiferous tubules, where spermatids are localized (Fig. 3 and supplemental Fig. S3). No signal was observed in spermatogonia, spermatocytes, or somatic cells (i.e. peritubular, Leydig, and Sertoli cells). Rat spermiogenesis is subdivided into 19 steps on the basis of the size and shape of the nucleus, acrosome, and cell body of spermatids (1). MARCH-XI expression became prominent in early round spermatids at step 4 (Fig. 3B), remained until step 11 (Fig. 3, B–D), decreased at steps 12–15 (Fig. 3, E and F), and diminished after step 16 (Fig. 3, A and B). Such staining was also detected with antigen-purified antibodies but not detected with preimmune sera or antisera absorbed with the antigen (supplemental Fig. S3). These results indicate that MARCH-XI is expressed in early developing spermatids.


Figure 2
View larger version (63K):
[in this window]
[in a new window]

  		FIGURE 2.
Ubiquitin ligase activity of MARCH-XI. A, reaction mixtures containing E1, E2 (UbcH2, UbcH5B, UbcH5C, or UbcH7), and ubiquitin were incubated in the presence of GST fusion proteins of the MARCH-XI RING finger (GST-RING) or its mutant (GST-RINGmut) for 4 h at 30 °C. Ubiquitinated materials were detected by Western blotting with anti-ubiquitin antibody (top). Bottom shows Coomassie Brilliant Blue staining of the E2 proteins (1 µg) used in this assay. B, plasmids encoding HA-ubiquitin (0.6 µg) and FLAG-CD4 (1 µg) were co-transfected into COS7 cells together with either an empty pcDNA3 plasmid (pcDNA3; 1.4 µg), MARCH-XI (MARCH-XI; 1.4 µg), or its RING-finger mutant (RINGmut; 1.4 µg). After treatment with concanamycin A, membrane lysates were subjected to immunoprecipitation (IP) with anti-FLAG beads followed by Western blotting with anti-HA (top left), anti-FLAG (top middle), or anti-Mar11 antibody (top right). Bottom panels show the results of Western blotting of the lysates (10 µg; 10% of the input) with anti-FLAG (left) or anti-Mar11 antibody (right). Arrowheads and asterisk indicate the bands of ubiquitinated FLAG-CD4 and of IgG heavy chain used for immunoprecipitation, respectively.

 
Subcellular Localization in Round Spermatids—To identify structures of early spermatids to which MARCH-XI associates, immunoperoxidase electron microscopy was performed with anti-Mar11 on rat testicular sections. Electron micrographs of round spermatids (steps 4–6; Fig. 4 and supplemental Fig. S4) revealed that strong immunostaining of MARCH-XI was detected in several vesicles in the Golgi medulla, a concave region near the trans-Golgi stacks (Fig. 4A). Dense staining was also found in membranes of the MVBs (Fig. 4B). No signal was observed in the Golgi or acrosomal vesicle membranes.


Figure 3
View larger version (119K):
[in this window]
[in a new window]

  		FIGURE 3.
Immunofluorescence staining of MARCH-XI in rat testis. Sections of adult rat testis were stained with rabbit anti-Mar11 antiserum (green) and TO-PRO3 (magenta) and observed with a confocal microscope. Stages II–III (A), IV–VI (B), IX (C), X and XI (D), XII (E), and XIV–XVI (F) of seminiferous tubules are shown. Arabic numbers indicate the steps of the spermatids. Bar, 20 µm.

 
To characterize the MARCH-XI-positive vesicles, double immunofluorescence staining was performed for isolated rat round spermatids in smear samples. Confocal microscopy revealed that MARCH-XI-specific immunoreactivity was detected as a perinuclear fuzzy staining and several spherical puncta throughout the cytoplasm (Fig. 5, left and supplemental Fig. S5). The fuzzy structure appeared a cluster of small vesicles, as shown at higher magnification (Fig. 5A, right, inset) and were consistently surrounded by signals for the TGN marker protein TGN38 (31) (Fig. 5A, asterisk). In this structure MARCH-XI was colocalized with {gamma}-adaptin, a subunit of the heterotetrameric adaptor protein complex (AP)-1 associated with the vesicles in the Golgi medulla (32) (Fig. 5B, arrowhead). The colocalization was also detected in some spherical MARCH-XI puncta, most likely corresponding to the MVBs (Fig. 5B, arrow). In contrast, MARCH-XI was clearly distinguished from another medulla component GOPC (11) (Fig. 5C), which was found to be colocalized with TGN38 (Fig. 5D). Furthermore, when double staining was performed with anti-ubiquitin antibody FK1, which recognizes only polyubiquitinated proteins (33), the FK1 signal was mainly observed in the MARCH-XI-positive compartments (Fig. 5E). These results establish that, in round spermatids, MARCH-XI is localized to the TGN-derived vesicles and MVBs, both of which contain AP-1 and polyubiquitinated proteins.


Figure 4
View larger version (114K):
[in this window]
[in a new window]

  		FIGURE 4.
Immunoelectron microscopy of MARCH-XI in rat round spermatids. Round spermatids (steps 4–6) were stained with rabbit anti-Mar11 antiserum by the pre-embedding immunoperoxidase procedure. Note that heavy immunostaining for MARCH-XI was detected in vesicles of the Golgi medulla (A) and the MVB (B). N, nucleus; AG, acrosomal granule; AV, acrosomal vesicle; G, Golgi; GM, Golgi medulla; M, mitochondrion. Bars, 1 µm.

 
Association with Fucose Glycoproteins—Given the fact that during acrosomal biogenesis, fucose-containing glycoproteins are transported to the MVBs from the TGN (4, 6), we examined whether fucose glycoproteins are localized to the MARCH-XI positive compartments. Rat round spermatids were stained with anti-Mar11 and AAL that binds specifically to an {alpha}1,6-fucose residue attached to the asparagine-linked N-acetyl-D-glucosamine (34). As shown in Fig. 6A, confocal imaging revealed that overlap between MARCH-XI and AAL staining in both the TGN region (arrow) and peripheral puncta (arrowhead), raising the possibility that MARCH-XI forms complexes with them. We assessed this possibility by immunoprecipitation. The membrane extracts of rat testis were immunoprecipitated with anti-Mar11 or preimmune serum, and the immunoprecipitates were separated by electrophoresis followed by transfer to a PVDF membrane. The filter was blotted with AAL to detect fucose glycoproteins. The high molecular weight smear AAL signal was detected in the anti-Mar11 immunoprecipitates (Fig. 6B, lane 2, asterisks) but not in the control preimmune precipitates (Fig. 6B, lane 3), indicating that MARCH-XI associates with fucose glycoproteins. Noteworthy, a similar staining pattern was observed when Western blotting was performed with anti-ubiquitin antibody (Fig. 6B, lane 5, asterisk). This result together with our observation of the colocalization between MARCH-XI and ubiquitinated proteins (Fig. 5E) suggests that a certain portion of the MARCH-XI-associated fucose glycoproteins are ubiquitinated. It is possible that these glycoproteins are in vivo substrates for the MARCH-XI ubiquitin ligase.


Figure 5
View larger version (49K):
[in this window]
[in a new window]

  		FIGURE 5.
Immunofluorescence confocal microscopy of MARCH-XI in rat round spermatids. Rat round spermatids in smear preparations were stained with rabbit anti-Mar11 (A, B, and E; magenta), rat anti-Mar11 (C; magenta), or rabbit anti-GOPC antiserum (D; magenta) followed by mouse monoclonal antibodies to TGN38 (A and D; green), {gamma}-adaptin (B; green), or ubiquitin (clone FK1; E; green) or rabbit polyclonal antiserum to GOPC (C; green). Nuclei were stained with Hoechst 33342 (blue). The fluorescence signals were detected with a confocal microscope. The inset in A shows a higher magnification image of the MARCH-XI–positive structure in the Golgi medulla (asterisk). Arrowheads and arrows indicate colocalization of MARCH-XI and marker proteins in the Golgi medulla and peripheral puncta, respectively. Note that MARCH-XI is not colocalized with TGN38 (asterisk) or GOPC (double arrowheads). Bar, 2 µm.

 


Figure 6
View larger version (52K):
[in this window]
[in a new window]

  		FIGURE 6.
Association of MARCH-XI with fucose glycoproteins. A, two top and bottom left panels, rat round spermatids in smear preparations were incubated with rabbit anti-Mar11 antiserum and biotinylated AAL followed by Alexa Fluor 546-conjugated anti-rabbit IgG and Alexa Fluor 488-conjugated anti-biotin antibodies. The nucleus was stained with Hoechst 33342. The fluorescence signals for MARCH-XI (magenta), AAL (green), and the nucleus (blue) were detected with a confocal microscope. Arrow and arrowheads indicate co-staining of MARCH-XI and AAL in the Golgi medulla and peripheral puncta, respectively. Bottom right panel, as a negative control of AAL staining, the spermatids were stained with Alexa Fluor 488-conjugated anti-biotin antibodies (green) followed by Hoechst 33342 (blue). Bar, 2 µm. B, membrane lysates of rat testis were immunoprecipitated with anti-Mar11 (lanes 2, 5, and 8) or preimmune serum (lanes 3, 6, and 9). The immunoprecipitates (IP) were analyzed by lectin blotting with AAL (left panel) or by Western blotting (WB) with anti-ubiquitin (clone Ubi-1) antibody (top right) or with anti-Mar11 antiserum (bottom right panel). The lysate (32 µg; 4% of the input) was run in parallel (lanes 1, 4, and 7). The middle panel shows a prolonged exposure of the left panel. Note that signals for AAL and anti-ubiquitin are seen in the anti-Mar11 immunoprecipitates (asterisk).

 
Association with AP-1—A sequence alignment of the C-terminal cytoplasmic tails of MARCH-IV, -IX, and -XI revealed the presence of a conserved tyrosine-based motif, YXXL, which is known to be recognized by the AP µ-subunits (12, 35) (Fig. 1A, double-underline, and supplemental Fig. S1). In addition to this finding, the colocalization between MARCH-XI and AP-1 led us to determine whether the AP-1 µ-subunit µ1-adaptin can recognize the tyrosine-based motif of MARCH-XI (367YVLL370) using the yeast two-hybrid system. We constructed the pGADT7 plasmids encoding µ1-adaptin or µ2-adaptin (the AP-2 µ subunit) and the two-hybrid bait pGBKT7 plasmids encoding the last 76-amino acid residues of MARCH-XI or its mutant where Tyr367 was replaced by an alanine residue. An interaction between the MARCH-XI tail and µ1-adaptin was detected by growth on a histidine-deficient plate (Fig. 7A) and by the beta-galactosidase assay (Fig. 7B). This interaction was abolished by a Y367A mutation. In contrast, no interaction was detected between MARCH-XI and µ2-adaptin. These results indicate that the YVLL sequence of MARCH-XI is responsible for the specific interaction with µ1-adaptin. To further examine the interaction between MARCH-XI and µ1-adaptin in mammalian cultured cells, COS7 cells were transfected with expression plasmids encoding 3 x FLAG-tagged µ1-adaptin and either mock, MARCH-XI, or its mutant with the Y367A mutation. Cell lysates were immunoprecipitated with anti-Mar11 followed by Western blotting with anti-FLAG antibody. As shown in Fig. 7C, FLAG-tagged µ1-adaptin was co-immunoprecipitated with wild-type MARCH-XI (middle lane) but not with its Y367A mutant (right lane), confirming binding of µ1-adaptin to MARCH-XI. Finally, to obtain the evidence for the association between MARCH-XI and AP-1 in vivo, membrane extracts of rat testis were subjected to immunoprecipitation with anti-Mar11 followed by Western blotting with anti-{gamma}-adaptin antibody. As shown in Fig. 7D, {gamma}-adaptin was co-immunoprecipitated with anti-Mar11 (middle lane) but not with preimmune serum (right lane). Together these results suggest that MARCH-XI associates with AP-1 through binding to µ1-adaptin via its cytoplasmic tyrosine-based motif.


Figure 7
View larger version (57K):
[in this window]
[in a new window]

  		FIGURE 7.
Association of MARCH-XI with the adaptin subunits of AP-1. A, AH109 yeast cells were cotransformed with a plasmid encoding a GAL4 activating domain fusion protein of µ1-adaptin (µ1)or µ2-adaptin (µ2) and a plasmid encoding a GAL4 DNA binding domain fusion protein of the C-terminal region of MARCH-XI (WT) or its Y367A mutant (Y367A). The cells were grown on plates with (+His) or without (–His) histidine. The empty vectors were used as a negative control, and the vectors encoding a GAL4 activating domain fusion protein of p53 (p53) and a GAL4 DNA binding domain fusion protein of SV40 large T antigen (T) were as a positive control. B, five independent clones were selected and grown to A600 values of 0.2, and beta-galactosidase activities were measured with the Beta-Glo assay system. beta-Galactosidase activities in relative units are presented as mean values ± S.E. (bars). The asterisk indicates a significant difference between the WT-µ1- and Y367A-µ1-transformed cells (p < 0.01, Student's t test). NS, not significant. C, COS7 cells were transiently transfected with an expression plasmid for 3 x FLAG-tagged µ1-adaptin in the absence (Mock) or presence of that for MARCH-XI (WT)orits mutant with the Y367A mutation (Y367A). Cell lysates were subjected to immunoprecipitation (IP) with anti-Mar11. The anti-Mar11 immunoprecipitates (top two panels) and the lysates (20 µg; 20% of the input; two bottom panels) were analyzed by Western blotting (WB) with anti-FLAG antibody (FLAG) or anti-Mar11 (MARCH-XI). The asterisk indicates the bands of IgG heavy chain used for immunoprecipitation. D, membrane lysates of rat testis were immunoprecipitated (IP) with anti-Mar11 (middle lane) or preimmune serum (right lane). The membrane extracts (20 µg; 5% of input; left lane) and immunoprecipitates (right two lanes) were analyzed by Western blotting with anti-{gamma}-adaptin antibody (top) or anti-Mar11 antiserum (bottom).

 
Interaction with Veli PDZ Proteins—We previously demonstrated that the PDZ binding motif of MARCH-II directly binds to the PDZ protein Veli-3 (25). Because Veli is known to be expressed in rat testis (36, 37), we determined whether the PDZ binding motif of MARCH-XI (395VTSV398) is recognized by Veli by a dot-blot analysis with a recombinant His6-tagged protein containing the last 76-amino acid residues of MARCH-XI (His-Mar11C). Purified GST fusion proteins of the PDZ domain of Veli-3 (GST-Veli) were spotted on a PVDF membrane. In parallel, GST as a negative control and the PDZ domain of GOPC (GST-GOPC), a well characterized spermatid PDZ protein (11), were also spotted. The membrane was overlaid with purified His-Mar11C, and then the signals for the His6 tag were detected with anti-His6 antibody. A robust binding was detected between His-Mar11C and the Veli PDZ domain, whereas a much weaker binding affinity was observed with the GOPC PDZ domain (Fig. 8A, left panel). No binding was obvious with His-Mar11C lacking the PDZ binding motif (His-Mar11C{Delta}; Fig. 8A, middle panel). To further examine the interaction between MARCH-XI and Veli, COS7 cells were transfected with expression plasmids encoding Myc-tagged Veli-3 and either mock, MARCH-XI, or its deletion mutant lacking the last amino acid Val398 (MARCH-XI{Delta}). Cell lysates were immunoprecipitated with anti-Mar11 followed by Western blotting with anti-Myc antibody. As shown in Fig. 8B, Myc-tagged Veli-3 was co-immunoprecipitated with wild-type MARCH-XI (lane 2) but not with MARCH-XI{Delta} (lane 3), confirming the Veli-MARCH-XI interaction. Furthermore, to evaluate their association in vivo, Triton extracts of rat testis were immunoprecipitated with anti-Veli antibodies followed by Western blotting with anti-Mar11. MARCH-XI was detected in the anti-Veli immunoprecipitates (Fig. 8C, lane 2) but not in those of the control (Fig. 8C, lane 3). In contrast, MARCH-XI was not coimmunoprecipitated with GOPC (supplemental Fig. S6). Veli expression in round spermatids was confirmed by immunofluorescence confocal microscopy showing its diffuse localization throughout the cytoplasm (Fig. 8D). Moreover, subcellular fractionation analysis of rat testis revealed the membrane association of Veli (Fig. 8E). Thus, these results indicate that MARCH-XI interacts with Veli through its C-terminal PDZ binding motif.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we performed the identification and characterization of MARCH-XI, a novel transmembrane ubiquitin ligase that is predominantly expressed in round spermatids of rats. The stage-specific expression (Fig. 3) suggests that during early spermiogenesis, the specific ubiquitin system should be utilized for a certain purpose. The major event in early spermiogenesis is acrosomal formation, which is thought to be achieved by a continuous fusion of Golgi-derived vesicles to the immature acrosomal vesicle (1). In addition to this acrosomal pathway, previous cytochemical studies reported that newly synthesized fucose glycoproteins are delivered to the MVBs (4, 6), suggesting that at least two anterograde transport pathways exist from the Golgi. The membrane flow to the endosomal system should be selective because some endosomal and lysosomal proteins (i.e. mannose 6-phosphate receptors and lysosome-associated membrane proteins (LAMPs) are segregated from the acrosome (5, 32). The subcellular localization of MARCH-XI strongly suggests that MARCH-XI also follows the TGN-MVB pathway. A previous electron microscopic study demonstrated that ubiquitin conjugates are detected in the TGN area at early spermiogenic stages (steps 1–7) and increased in the MVBs at later stages (steps 8–15) (17). In addition, Eps15, a ubiquitin-binding adaptor protein with a role in endocytosis and vesicular trafficking, has been found to be associated with AP-1-positive vesicles in the Golgi medulla (10). It is, therefore, reasonable to assume that ubiquitin modification controls the selective protein sorting to the MVB pathway. We showed that MARCH-XI is colocalized with ubiquitinated proteins (Fig. 5E) and associated with fucose glycoproteins, some of which appeared to be ubiquitinated (Fig. 6B). Possibly MARCH-XI may mediate the sorting of fucose glycoproteins destined to the MVBs by ubiquitinating them. Further studies are necessary to determine the molecular identity of the in vivo E3 substrates for MARCH-XI. Our data provide evidence for a novel role of the MARCH family in the regulation of spermiogenesis.


Figure 8
View larger version (59K):
[in this window]
[in a new window]

  		FIGURE 8.
Interaction of MARCH-XI with Veli through the PDZ binding motif. A, dot-blot analysis. Various amounts (1, 3, 15, and 30 pmol) of purified GST, GST-Veli, and GST-GOPC were blotted on PVDF membranes. The membranes were incubated with either His-Mar11C (left) or His-Mar11C{Delta} (middle) followed by anti-His6 antibody. To confirm the presence of the GST proteins, the membrane was incubated with anti-GST antibody (right). B, COS7 cells were transiently transfected with an expression plasmid for Myc-tagged Veli-3 in the absence (lanes 1 and 4) or presence of that for MARCH-XI (lanes 2 and 5) or MARCH-XI{Delta} (lanes 3 and 6). Cell lysates were subjected to immunoprecipitation (IP) with anti-Mar11. The anti-Mar11 immunoprecipitates (lanes 1–3) and the lysates (20 µg; 4% of the input; lanes 4–6) were analyzed by Western blotting (WB) with anti-Mar11 (top) or anti-Myc antibody (bottom). The arrowhead and asterisk indicate the bands of MARCH-XI and of IgG heavy chain used for immunoprecipitation, respectively. C, membrane lysates of rat testis were immunoprecipitated with anti-Veli (lane 2) or control IgG beads (lane 3). The membrane extracts (20 µg; 1% of input; lane 1) and immunoprecipitates (lanes 2 and 3) were analyzed by Western blotting with rabbit anti-Mar11 antiserum (top), corresponding preimmune serum (middle), and anti-Veli antiserum (bottom). The arrowhead indicates the band corresponding to MARCH-XI. D, rat round spermatids were stained with rat anti-Mar11 antiserum followed by rabbit anti-Veli (top) or antigen-preabsorbed anti-Veli antiserum (bottom). Signals for Veli (left and green in right), MARCH-XI (magenta in right), and nuclei stained with Hoechst 33342 (blue in right) were detected with a confocal microscope. Bar, 2 µm. E, homogenates of rat testis were subjected to a high speed centrifugation (25,000 rpm on an SW28 rotor). The resulting supernatant (S) and membrane pellet (P) were analyzed by Western blotting with anti-Mar11 (top) and anti-Veli antiserum (bottom).

 
The Golgi medulla of round spermatids contains numerous vesicular and tubular membrane structures, including clathrincoated vesicles (4, 31, 38). MARCH-XI should be sorted to AP-1/clathrin-coated vesicles because 1) MARCH-XI is colocalized with {gamma}-adaptin near the TGN (Fig. 5B), 2) the C-terminal YVLL sequence of MARCH-XI is specifically recognized by µ1-adaptin (Fig. 7, A–C), and 3) MARCH-XI associates with {gamma}-adaptin (Fig. 7D). In early spermatids, clathrin-coated buds have been found on the TGN membrane (4, 31, 38), implying that the spermatid AP-1 mediates MARCH-XI sorting at the TGN, coupling with the formation of clathrin-coated vesicles. In yeast and mammalian somatic cells, it has been assumed that AP-1 is utilized in the TGN-to-endosome trafficking. However, the studies using AP-1-deficient cells have indicated that AP-1 more likely mediates the retrograde endosome-to-TGN trafficking (39–41). Thus, another possibility is that AP-1 is involved in retrieval of MARCH-XI back to the TGN region from the MVBs, allowing MARCH-XI to escape the lysosomal degradation. Anyway it could be argued that AP-1 would function in the TGN-MVB pathway in early spermatids. However, taking account of the fact that clathrin and Golgi-resident proteins have been detected on the acrosomal membrane (7, 8, 42), we cannot rule out the possible role of AP-1 in retrieval of mislocalized Golgi proteins.

Additional finding of this study is that MARCH-XI interacts with the PDZ protein Veli. Three Veli isoforms (Veli-1, -2, and -3) are recognized in various mammalian tissues (36). All three isoforms are likely to be expressed in rat testis because our anti-Veli antiserum detected triplex bands by Western blot and immunoprecipitation analyses (Fig. 8, C and E). It is unknown which isoform is expressed and interacts with MARCH-XI in round spermatids, whereas the dot-blot analysis demonstrated the direct binding of Veli-3 (Fig. 8A). Although no precise role has been assigned to each Veli isoform, they play crucial roles in 1) the docking and transport of synaptic vesicles to the synaptic plasma membranes of neurons (43, 44) and 2) the sorting and stable localization of basolateral transmembrane proteins in polarized epithelial cells (45–49). Additionally, we have previously demonstrated that the PDZ binding motifs of MARCH-II and -III are important for their efficient exit from the endoplasmic reticulum (24, 25). Thus, the interaction with Veli may facilitate the transport and subcellular localization of MARCH-XI. We also showed that the GOPC PDZ domain can bind to the PDZ binding motif of MARCH-XI, albeit with much lower affinity than Veli-3 (Fig. 8A). Nevertheless, it is unlikely that MARCH-XI interacts with GOPC in vivo because two proteins were not colocalized (Fig. 5C) or coimmunoprecipitated (supplemental Fig. S6).

We demonstrated that MARCH-XI mediates CD4 ubiquitination in COS7 cells (Fig. 2B) but there is no evidence for CD4 expression or ubiquitination in spermatids (50). It is evident that MARCH-XI would share overlapping E3-substrate recognition as well as the characteristic sequence motifs with MARCH-IV and -IX. Presumably these three MARCH proteins are structurally and functionally relevant with each other. In contrast to MARCH-XI, MARCH-IX has ubiquitous expression throughout human tissues (18). Interestingly, when ectopically expressed, MARCH-IX is localized to the TGN, colocalizing with AP-1 (18), and to the late endosomes and lysosomes (22). These findings raise the possibility that MARCH-IX might play a role in the regulation of intracellular ubiquitin-dependent protein sorting as somatic-cell counterpart of MARCH-XI. More detailed characterization of MARCH-IX and -XI is required to gain a better understanding of the potential role for the MARCH family in membrane trafficking. Obviously, it is anticipated that gene knock-out experiments will shed greater light on the physiological function of MARCH-XI and its impact on biological processes in brain and pituitary as well as developing spermatids.


   FOOTNOTES
 
* This work was supported by the 21st Century Center of Excellence Program and the Grant-in-Aid for Scientific Research 14104002, for Young Scientists 16710145 and 16770144, and for Scientific Research on Priority Areas "Transportsome" 18059010 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental material including Figs. S1–S6.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AB048841 and AB048842. Back

1 Present address: Department of Physiology, Kitasato University School of Medicine, Sagamihara 228-8555, Japan. Back

2 To whom correspondence should be addressed: Dept. of Biological Sciences, Tokyo Institute of Technology, 4259-B-19 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. Tel.: 81-45-924-5726; Fax: 81-45-924-5824; E-mail: shirose{at}bio.titech.ac.jp.

3 The abbreviations used are: SNARE, soluble N-ethylmaleimide factor attachment protein receptor; AAL, Aleuria aurantia lectin; AP, adaptor protein complex; GOPC, Golgi-associated PDZ- and coiled coil motif-containing protein; GST, glutathione S-transferase; MARCH, membrane-associatedRING-CH; MVB, multivesicular body; PBS, phosphate-buffered saline; PVDF, polyvinylidene fluoride; TGN, trans-Golginetwork; E1, ubiquitin-activatingenzyme; E2, aubiquitin-conjugating enzyme; E3, ubiquitin ligase; HA, hemagglutinin. Back


   ACKNOWLEDGMENTS
 
We thank Toru Tateno, Osamu Katsumata, Erika Shirako, Korefumi Nakamura, Takeshi Aihara, Yugo Ikeda, and Setsuko Sato for technical and secretarial assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Clermont, Y., Oko, R., and Hermo, L. (1993) in Cell and Molecular Biology of the Testis (Desjardins, C., and Ewing, L. L., eds) pp. 332–376, Oxford University Press, New York
  2. Ramalho-Santos, J., Schatten, G., and Moreno, R. D. (2002) Biol. Reprod. 67, 1043–1051[Abstract/Free Full Text]
  3. Clermont, Y., and Tang, X. M. (1985) Anat. Rec. 213, 33–43[CrossRef][Medline] [Order article via Infotrieve]
  4. Martínez-Menárguez, J. A., Aviles, M., Madrid, J. F., Castells, M. T., and Ballesta, J. (1993) Eur. J. Cell Biol. 61, 21–33[Medline] [Order article via Infotrieve]
  5. Martínez-Menárguez, J. A., Geuze, H. J., and Ballesta, J. (1996) J. Histochem. Cytochem. 44, 313–320[Abstract]
  6. Tang, X. M., Lalli, M. F., and Clermont, Y. (1982) Am. J. Anat. 163, 283–294[CrossRef][Medline] [Order article via Infotrieve]
  7. Moreno, R. D., Ramalho-Santos, J., Chan, E. K., Wessel, G. M., and Schatten, G. (2000) Dev. Biol. 219, 334–349[CrossRef][Medline] [Order article via Infotrieve]
  8. Ramalho-Santos, J., and Moreno, R. D. (2001) Biol. Res. 34, 147–152[Medline] [Order article via Infotrieve]
  9. Ramalho-Santos, J., Moreno, R. D., Wessel, G. M., Chan, E. K., and Schatten, G. (2001) Exp. Cell Res. 267, 45–60[CrossRef][Medline] [Order article via Infotrieve]
  10. Kang-Decker, N., Mantchev, G. T., Juneja, S. C., McNiven, M. A., and van Deursen, J. M. A. (2001) Science 294, 1531–1533[Abstract/Free Full Text]
  11. Yao, R., Ito, C., Natsume, Y., Sugitani, Y., Yamanaka, H., Kuretake, S., Yanagida, K., Sato, A., Toshimori, K., and Noda, T. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 11211–11216[Abstract/Free Full Text]
  12. Bonifacino, J. S., and Traub, L. M. (2003) Annu. Rev. Biochem. 72, 395–447[CrossRef][Medline] [Order article via Infotrieve]
  13. Hicke, L., and Dunn, R. (2003) Annu. Rev. Cell Dev. Biol. 19, 141–172[CrossRef][Medline] [Order article via Infotrieve]
  14. Staub, O., and Rotin, D. (2006) Physiol. Rev. 86, 669–707[Abstract/Free Full Text]
  15. Helliwell, S. B., Losko, S., and Kaiser, C. A. (2001) J. Cell Biol. 153, 649–662[Abstract/Free Full Text]
  16. Reggiori, F., and Pelham, H. R. B. (2002) Nat. Cell Biol. 4, 117–123[CrossRef][Medline] [Order article via Infotrieve]
  17. Haraguchi, C. M., Mabuchi, T., Hirata, S., Shoda, T., Hoshi, K., and Yokota, S. (2004) J. Histochem. Cytochem. 52, 1393–1403[Abstract/Free Full Text]
  18. Bartee, E., Mansouri, M., Hovey Nerenberg, B. T., Gouveia, K., and Früh, K. (2004) J. Virol. 78, 1109–1120[Abstract/Free Full Text]
  19. Goto, E., Ishido, S., Sato, Y., Ohgimoto, S., Ohgimoto, K., Nagano-Fujii, M., and Hotta, H. (2003) J. Biol. Chem. 278, 14657–14668[Abstract/Free Full Text]
  20. Lehner, P. J., Hoer, S., Dodd, R., and Duncan, L. M. (2005) Immunol. Rev. 207, 112–125[CrossRef][Medline] [Order article via Infotrieve]
  21. Bartee, E., McCormack, A., and Früh, K. (2006) PLoS Pathog. 2, 975–988
  22. Hoer, S., Smith, L., and Lehner, P. J. (2007) FEBS Lett. 581, 45–51[CrossRef][Medline] [Order article via Infotrieve]
  23. Ohmura-Hoshino, M., Matsuki, Y., Aoki, M., Goto, E., Mito, M., Uematsu, M., Kakiuchi, T., Hotta, H., and Ishido, S. (2006) J. Immunol. 177, 341–354[Abstract/Free Full Text]
  24. Fukuda, H., Nakamura, N., and Hirose, S. (2006) J. Biochem. (Tokyo) 139, 137–145[Abstract/Free Full Text]
  25. Nakamura, N., Fukuda, H., Kato, A., and Hirose, S. (2005) Mol. Biol. Cell 16, 1696–1710[Abstract/Free Full Text]
  26. Mansouri, M., Bartee, E., Gouveia, K., Hovey Nerenberg, B. T., Barrett, J., Thomas, L., Thomas, G., McFadden, G., and Früh, K. (2003) J. Virol. 77, 1427–1440[CrossRef][Medline] [Order article via Infotrieve]
  27. Nakamura, N., Kimura, Y., Tokuda, M., Honda, S., and Hirose, S. (2006) EMBO Rep. 7, 1019–1022[CrossRef][Medline] [Order article via Infotrieve]
  28. Kato, A., Nagata, Y., and Todokoro, K. (2004) Dev. Biol. 269, 196–205[CrossRef][Medline] [Order article via Infotrieve]
  29. Sakai, Y., and Yamashina, S. (1990) Dev. Growth Differ. 32, 389–395[CrossRef]
  30. Songyang, Z., Fanning, A. S., Fu, C., Xu, J., Marfatia, S. M., Chishti, A. H., Crompton, A., Chan, A. C., Anderson, J. M., and Cantley, L. C. (1997) Science 275, 73–77[Abstract/Free Full Text]
  31. Martínez-Menárguez, J. A., Geuze, H. J., and Ballesta, J. (1996) Eur. J. Cell Biol. 71, 137–143[Medline] [Order article via Infotrieve]
  32. Moreno, R. D. (2003) Mol. Reprod. Dev. 66, 202–209[CrossRef][Medline] [Order article via Infotrieve]
  33. Fujimuro, M., Sawada, H., and Yokosawa, H. (1994) FEBS Lett. 349, 173–180[CrossRef][Medline] [Order article via Infotrieve]
  34. Yamashita, K., Kochibe, N., Ohkura, T., Ueda, I., and Kobata, A. (1985) J. Biol. Chem. 260, 4688–4693[Abstract/Free Full Text]
  35. Ohno, H., Fournier, M. C., Bosshart, H., Rhee, I., Miyatake, S., Saito, T., Galluser, A., Kirchhausen, T., and Bonifacino, J. S. (1995) Science 269, 1872–1875[Abstract/Free Full Text]
  36. Butz, S., Okamoto, M., and Sudhof, T. C. (1998) Cell 94, 773–782[CrossRef][Medline] [Order article via Infotrieve]
  37. Tseng, T. C., Marfatia, S. M., Bryant, P. J., Pack, S., Zhuang, Z., O'Brien, J. E., Lin, L., Hanada, T., and Chishti, A. H. (2001) Biochim. Biophys. Acta 1518, 249–259[Medline] [Order article via Infotrieve]
  38. Griffiths, G., Warren, G., Stuhlfauth, I., and Jockusch, B. M. (1981) Eur. J. Cell Biol. 26, 52–60[Medline] [Order article via Infotrieve]
  39. Meyer, C., Eskelinen, E. L., Guruprasad, M. R., von Figura, K., and Schu, P. (2001) J. Cell Sci. 114, 4469–4476[Medline] [Order article via Infotrieve]
  40. Meyer, C., Zizioli, D., Lausmann, S., Eskelinen, E. L., Hamann, J., Saftig, P., von Figura, K., and Schu, P. (2000) EMBO J. 19, 2193–2203[CrossRef][Medline] [Order article via Infotrieve]
  41. Valdivia, R. H., Baggott, D., Chuang, J. S., and Schekman, R. W. (2002) Dev. Cell 2, 283–294[CrossRef][Medline] [Order article via Infotrieve]
  42. Moreno, R. D., Ramalho-Santos, J., Sutovsky, P., Chan, E. K., and Schatten,