The E2 Ubiquitin-Conjugating Enzyme UBE2J1 Is Required for Spermiogenesis in Mice

The E2 Ubiquitin-conjugating Enzyme UBE2J1 Is Required for Spermiogenesis in Mice The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Koenig, Paul-Albert, Peter K. Nicholls, Florian I. Schmidt, Masatoshi Hagiwara, Takeshi Maruyama, Galit H. Frydman, Nicki Watson, David C. Page, and Hidde L. Ploegh. “The E2 Ubiquitin-Conjugating Enzyme UBE2J1 Is Required for Spermiogenesis in Mice.” Journal of Biological Chemistry 289, no. 50 (October 15, 2014): 34490–34502. © 2014 American Society for Biochemistry and Molecular Biology, Inc. (ASBMB) As Published http://dx.doi.org/10.1074/jbc.M114.604132 Publisher American Society for Biochemistry and Molecular Biology (ASBMB) Version Author's final manuscript Citable link http://hdl.handle.net/1721.1/108038 Terms of Use Creative Commons Attribution-Noncommercial-Share Alike Detailed Terms http://creativecommons.org/licenses/by-nc-sa/4.0/ Male Ube2j1-/- mice are sterile The E2 Ubiquitin Conjugating Enzyme UBE2J1 is Required for Spermiogenesis in Mice Paul-Albert Koenig1, Peter K. Nicholls1, Florian I. Schmidt1, Masatoshi Hagiwara1, Takeshi Maruyama1, Galit H. Frydman2, Nicki Watson1, David C. Page1,3,4, Hidde L. Ploegh1,3 1 Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA 2 Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 3 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA 4 Howard Hughes Medical Institute, Cambridge, MA 02142, USA *Running title: Male Ube2j1-/- mice are sterile To whom correspondence should be addressed: Hidde Ploegh, Whitehead Institute for Biomedical Research, 9 Cambridge Center, Cambridge 02142, MA, USA, Tel.: +1-617-324-2031; Fax: +1-617-452- 3566; e-mail: [email protected] Keywords: ER quality control, endoplasmic‐reticulum‐associated protein degradation (ERAD), ubiquitin‐conjugating enzyme (E2 enzyme), gene knockout, spermatogenesis, mouse model, male infertility, spermiogenesis, ERAD tuning Background: The physiological roles of many ER lumen of the seminiferous tubule. Our findings quality control components are unknown. identify an essential function for the ubiquitin- Results: Male Ube2j1-/- mice are sterile with proteasome-system in spermiogenesis and defects in flagella and acrosome function, and define a novel, non-redundant physiological cytoplasm removal in sperm cells. function for the dislocation step of ER quality Conclusion: Spermatogenesis is a previously control. unknown process requiring ER quality control. Significance: ER quality control components Components of the endoplasmic reticulum (ER) might serve as diagnostic or therapeutic targets for quality control system recognize proteins no male infertility in the future. longer wanted in the ER and target them for degradation. During this process, misfolded ABSTRACT proteins are extracted from the ER (ER ER-resident proteins destined for degradation dislocation) and delivered to the cytosol for are dislocated into the cytosol by components of proteasomal degradation (1). Substrate dislocation the ER quality control machinery for is driven by concerted ubiquitylation and proteasomal degradation. Dislocation deubiquitylation reactions (2). Ubiquitylation substrates are ubiquitylated in the cytosol by involves the action of three enzymes, E1 ubiquitin- E2 ubiquitin conjugating/E3 ligase complexes. activating enzymes, E2 ubiquitin-conjugating UBE2J1 is one of the well-characterized E2 enzymes, and E3 ubiquitin ligases. enzymes that participate in this process. E2 ubiquitin-conjugating enzymes known to be However, the physiological function of Ube2j1 involved in ER dislocation include the yeast -/- is poorly defined. We find that Ube2j1 mice Ubc6p orthologs UBE2J1 (UBC6e, NCUBE1) and have reduced viability and fail to thrive early UBE2J2 (UBC6, NCUBE2) (3,4), tail-anchored -/- after birth. Male Ube2j1 mice are sterile due proteins in the ER membrane with their catalytic to a defect in late spermatogenesis. domain facing the cytosol. UBE2J1 is part of the Ultrastructural analysis shows that removal of SEL1L dislocation complex, which contains -/- the cytoplasm is incomplete in Ube2j1 several other factors known to be involved in ER elongating spermatids, compromising the dislocation. These include HRD1, Derlin-2, p97, release of mature elongate spermatids into the PDI, BiP, Calnexin, AUP1, UBXD8 and OS9 (5). 1 Male Ube2j1-/- mice are sterile In the absence of β2-microglobulin, the heavy (‘pF2L2’, Addgene 13445, (7)) vector to install chain of the human class I MHC molecule HLA- LoxP sites in introns 1 and 3, allowing conditional B27 is ubiquitylated, dislocated and degraded by ablation of Ube2j1 in the presence of Cre the concerted action of UBE2J1 and the E3 ligase recombinase. A neomycin resistance cassette HRD1 (Synoviolin) (5). In contradistinction, flanked by FRT sites was inserted upstream of the UBE2J1 cooperates with the E3 ligase RMA8 and 3’ LoxP site between exon 3 and 4. The LoxP sites with Derlin-1 in the degradation of CFTRΔ508 were flanked by a ~4.6 kb 5’ and a ~2.8 kb 3’ (6). Moreover, UBE2J1 is involved in the homology arm. The linearized targeting construct degradation of other model substrates for ER was electroporated into JM8A3.N1 ES cells (strain dislocation, such as the unpaired TCRα chain, and C57BL/6N, agouti; obtained from the KOMP class I MHC heavy chains in the presence of the Repository), which were subsequently grown in HCMV-derived immunoevasin US11 (4,6). the presence of neomycin. ES cells were selected Despite accumulating data in tissue culture models with 300 µg/mL G418 (Geneticin; Gibco, Life on the function and mechanisms of ER dislocation, Technologies; Grand Island, NY) for six days. its physiological function in an organismal context G418-resistant ES cell colonies were screened for is poorly defined, due to the limited number of correct targeting by Southern blotting or long model substrates and cell lines investigated and range PCR, and correctly targeted ES cell clones due to the lack of appropriate in vivo mammalian were injected into C57BL/6J blastocysts. models. To obtain further insight into the Chimeras were bred with FLPe transgenic mice physiological role of ER dislocation, we generated (JAX strain B6.Cg-Tg(ACTFLPe)9205Dym/J; a floxed allele of Ube2j1 in mice and analyzed Jackson Laboratories, Bar Harbor, ME) to excise fl/WT Ube2j1-/- mice. the neomycin resistance cassette. Ube2j1 mice - fl/WT Although Ube2j1 has been repeatedly implicated were intercrossed to obtain FLPe Ube2j1 - fl/WT in the dislocation of various misfolded mice. FLPe Ube2j1 mice were then crossed to glycoprotein substrates, mice that lack Ube2j1 are CMV-Cre transgenic mice (JAX strain B6.C- viable and show no gross aberrations in Tg(CMV-cre)1Cgn/J; Jackson Laboratories, Bar morphology or behavior. On the assumption that Harbor, ME) to generate full-body knockout mice -/- ER quality control is essential to development and (Ube2j1 ) and to CD19-Cre mice (JAX strain survival, these results suggest remarkable B6.129P2(C)-Cd19tm1(cre)Cgn/J; Jackson redundancy in its mode of operation. The main Laboratories, Bar Harbor, ME) for specific fl/fl defect we observe in Ube2j1-/- animals, ablation in B cells (CD19-Cre Ube2j1 ). surprisingly, is male sterility due to defects in Mice were genotyped by PCR using primers flagella and acrosome function, and defective annealing to the genomic regions indicated in Fig. removal of cytoplasm from elongating spermatids. 1A (A: 5’- GCCTCTGAGGATTTCCTGTGAGG- Our results thus identify a unique, non-redundant 3’; B: 5’- ATGGAGACCCGCTACAACCT-3’; C: role for Ube2j1 in male germ cell development 5’- GCAGGATAATGCTTGGTGGTT-3’). The and define a previously unknown physiological expected amplicon sizes are 458bp for the wild- function of ER dislocation. type, 531bp for the ‘floxed’, and 538bp for the knockout allele. EXPERIMENTAL PROCEDURES Animals were housed at the Whitehead Institute Generation of Ube2j1fl/fl mice - We generated mice for Biomedical Research and maintained carrying a conditional allele of the gene encoding according to protocols approved by the UBE2J1 (also known as UBC6e or NCUBE1, Massachusetts Institute of Technology Committee gene name Ube2j1) by homologous recombination on Animal Care. (Fig. 1A). Genomic sequences were amplified by PCR from a BAC clone (Clone DB name RP24- Cell culture - MEFs were generated from E13.5 531A22; C57BL/6J, male, brain, spleen; BAC- fetuses and cultured in DME medium PAC Resources, Oakland, CA) containing the supplemented with 10% IFS, 1 mM glutamine and genomic sequence of Ube2j1 from C57BL/6J 10 mM β-mercaptoethanol (MEF medium). In the mice. Ube2j1 consists of 8 exons. We constructed experiments described here, matched pairs of a targeting vector based on the pPGKneoF2L2dta primary wild type and knock-out MEFs from 2 Male Ube2j1-/- mice are sterile littermates were used (except for experiments with affinity-purified rabbit polyclonal, 1/1000) were SV40, for which we used MEFs that had been produced by our laboratory. immortalized by serial passage). Antibodies for FACS analysis - The antibodies B cells were isolated from spleens of Ube2j1-/- or used were anti-mouse IgM PE (clone II/41; CD19-Cre Ube2j1fl/fl mice and Cre-matched eBioscience, San Diego, CA), anti-mouse B220 Ube2j1WT/WT littermates, respectively. Splenocytes Pacific Blue (clone RA3-6B2; BD Pharmingen, were enriched for B cells by depletion of non-B San Jose, CA), anti-mouse

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