title: Membrane-associated RING-CH 10 (MARCH10 protein) is a microtubule-associated E3 ubiquitin ligase of the spermatid flagella.
abstract: Spermiogenesis is a complex and dynamic process of the metamorphosis of spermatids into spermatozoa. There is a great deal that is still unknown regarding the regulatory mechanisms for the formation of the sperm flagellum. In this study, we determined that the membrane-associated RING-CH 10 (March10) gene is predominantly expressed in rat testis. We isolated two March10 isoforms encoding MARCH10a and MARCH10b, which are generated by alternative splicing. MARCH10a is a long RING finger protein, and MARCH10b is a short RING finger-less protein. Immunohistochemical staining revealed that the MARCH10 proteins are specifically expressed in elongating and elongated spermatids, and the expression is absent in epididymal spermatozoa. MARCH10 immunoreactivity was observed in the cytoplasmic lobes as well as the principal piece and annulus of the flagella. When overexpressed in COS7 cells, MARCH10a was localized along the microtubules, whereas MARCH10b was distributed throughout the cytoplasm. An in vitro microtubule cosedimentation assay showed that MARCH10a is directly associated with microtubules. An in vitro ubiquitination assay demonstrated that the RING finger domain of MARCH10a exhibits an E3 ubiquitin ligase activity along with the E2 ubiquitin-conjugating enzyme UBE2B. Moreover, MARCH10a undergoes proteasomal degradation by autoubiquitination in transfected COS7 cells, but this activity was abolished upon microtubule disassembly. These results suggest that MARCH10 is involved in spermiogenesis by regulating the formation and maintenance of the flagella in developing spermatids.
authors.affiliation: Department of Biological Sciences, Tokyo Institute of Technology, 4259-B-19 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan.
authors.forename: Prasanna Vasudevan
authors.initials: PV
authors.lastname: Iyengar
authors.forename: Tsuyoshi
authors.initials: T
authors.lastname: Hirota
authors.forename: Shigehisa
authors.initials: S
authors.lastname: Hirose
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Nakamura
mesh_terms: D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D003593:Cytoplasm / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D007527:Isoenzymes / Q000096:biosynthesis / Q000235:genetics; D008297:Male; D008870:Microtubules / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D051381:Rats; D013082:Sperm Tail / Q000201:enzymology*; D013087:Spermatids / Q000201:enzymology*; D013091:Spermatogenesis / Q000502:physiology*; D044763:Ubiquitin-Conjugating Enzymes / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000096:biosynthesis* / Q000235:genetics; D054875:Ubiquitination / Q000502:physiology
chemical_list: D007527:Isoenzymes; C506127:Ube2b protein, rat; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases