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

Abstract

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 membraneassociated 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 1-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.

Membrane-associated RING-CH 10 (MARCH10 Protein) Is a Microtubule-associated E3 Ubiquitin Ligase of the Spermatid Flagella

“…Immunoprecipitation-Immunoprecipitation of endogenous MARCH10 proteins from the testis was performed as described previously (19). In Fig.…”

“…Preparation of Recombinant Proteins and Antibodies-Preparation of the recombinant proteins and the immunization protocol for the production of rabbit antisera were performed as described previously (19). Polyclonal anti-MAR10N (808) and anti-MAR10M (810) antisera were obtained by immunizing rabbits with GST-MAR10N and GST-MAR10M, respectively.…”

“…In Vitro Ubiquitination Assay-In vitro ubiquitination assays were performed with GST-RING or GST-RINGmut (1 g) as described previously (19), with the exception that the reaction mixtures were incubated for 24 h.…”

“…Transmembrane MARCH proteins mediate the ubiquitination and subsequent down-regulation of cell-surface immune regulatory molecules, such as major histocompatibility complex class II and CD86 (17,18,21). Other proposed functions include endoplasmic reticulum-associated degradation (22), endosomal protein trafficking (23,24), mitochondrial dynamics (25,26), and spermatogenesis (19). MARCH7 (also known as Axotrophin) and MARCH10 are predicted to have no transmembrane spanning region.…”

“…The membrane-associated RING-CH (MARCH) family is a RING finger protein family of E3 Ub ligases, consisting of 11 members in mammals (17)(18)(19). Nine MARCH members (i.e.…”