Hidekazu Fukuda

MARCH-XI, a novel transmembrane ubiquitin ligase implicated in ubiquitin-dependent protein sorting in developing spermatids

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.

Identification and properties of a novel variant of NBC4 (Na +/HCO3 - co-transporter 4) that is predominantly expressed in the choroid plexus

Secretion of HCO(3)- at the apical side of the epithelial cells of the choroid plexus is an essential step in the formation of cerebrospinal fluid. Anion conductance with a high degree of HCO(3)- permeability has been observed and suggested to be the major pathway for HCO(3)- transport across the apical membrane. Recently, it was found that NBC (Na(+)/HCO(3)- co-transporter) 4, an electrogenic member of the NBC family, was expressed in the choroid plexus. We found that a novel variant of the NBC4 [NBC4g/Slc4a5 (solute carrier family 4, sodium bicarbonate co-transporter, member 5)] is almost exclusively expressed in the apical membrane of rat choroid plexus epithelium at exceptionally high levels. RNA interference-mediated knockdown allowed the functional demonstration that NBC4g is the major player in the HCO(3)- transport across the apical membrane of the choroid plexus epithelium. When combined with a recent observation that in choroid plexus epithelial cells electrogenic NBC operates with a stoichiometry of 3:1, the results of the present study suggest that NBC4g mediates the efflux of HCO(3)- and contributes to cerebrospinal fluid production.