Journal of Biological Chemistry
Volume 286, Issue 45, 11 November 2011, Pages 39082-39090
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Cell Biology
Membrane-associated RING-CH 10 (MARCH10 Protein) Is a Microtubule-associated E3 Ubiquitin Ligase of the Spermatid Flagella*

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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.

Cytoskeleton
E3 Ubiquitin Ligase
Microtubules
Spermatogenesis
Ubiquitination
Three-dimensional SIM
Membrane-associated RING-CH
Microtubule Association
Sperm Flagella

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The nucleotide sequence(s) reported in this paper has been submitted to the DDBJ/GenBankTM/EBI Data Bank with accession number(s) AB552845 and AB552846.

*

This work was supported by Grants-in-aid for Scientific Research 21026010, 22370029, and 22770123 and the 21st Century and Global Center of Excellence Program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2 and Movies 1 and 2.