Uptake of Na(+) from the environment is an indispensable strategy for the survival of freshwater fish, as they easily lose Na(+) from the plasma to a diluted environment. Nevertheless, the location of and molecules involved in Na(+) uptake remain poorly understood. In this study, we utilized Sodium Green, a Na(+)-dependent fluorescent reagent, to provide direct evidence that Na(+) absorption takes place in a subset of the mitochondria-rich (MR) cells on the yolk sac surface of zebrafish larvae. Combined with immunohistochemistry, we revealed that the Na(+)-absorbing MR cells were exceptionally rich in vacuolar-type H(+)-ATPase (H(+)-ATPase) but moderately rich in Na(+)-K(+)-ATPase. We also addressed the function of foxi3a, a transcription factor that is specifically expressed in the H(+)-ATPase-rich MR cells. When foxi3a was depleted from zebrafish embryos by antisense morpholino oligonucleotide injection, differentiation of the MR cells was completely blocked and Na(+) influx was severely reduced, indicating that MR cells are the primary sites for Na(+) absorption. Additionally, foxi3a expression is initiated at the gastrula stage in the presumptive ectoderm; thus, we propose that foxi3a is a key gene in the control of MR cell differentiation. We also utilized a set of ion transport inhibitors to assess the molecules involved in the process and discuss the observations.
Membrane-associated RING-CH (MARCH) is a recently identified member of the mammalian E3 ubiquitin ligase family, some members of which down-regulate the expression of immune recognition molecules. Here, we have identified MARCH-II, which is ubiquitously expressed and localized to endosomal vesicles and the plasma membrane. Immunoprecipitation and in vitro binding studies established that MARCH-II directly associates with syntaxin 6. Overexpression of MARCH-II resulted in redistribution of syntaxin 6 as well as some syntaxin-6 -interacting soluble N-ethylmaleimidesensitive factor attachment protein receptors (SNAREs) into the MARCH-II-positive vesicles. In addition, the retrograde transport of TGN38 and a chimeric version of furin to trans-Golgi network (TGN) was perturbed-without affecting the endocytic degradative and biosynthetic secretory pathways-similar to effects caused by a syntaxin 6 mutant lacking the transmembrane domain. MARCH-II overexpression markedly reduced the cell surface expression of transferrin (Tf) receptor and Tf uptake and interfered with delivery of internalized Tf to perinuclear recycling endosomes. Depletion of MARCH-II by small interfering RNA perturbed the TGN localization of syntaxin 6 and TGN38/46. MARCH-II is thus likely a regulator of trafficking between the TGN and endosomes, which is a novel function for the MARCH family. INTRODUCTIONVesicular traffic between different organelles requires highly regulated docking and fusion of a transport vesicle with a specific target membrane. This process is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) localized to a specific subcellular compartment along the secretory and endocytic pathways (Jahn and Sü dhof, 1999;Chen and Scheller, 2001;Hay, 2001;Mayer, 2002). Only cognate pairs of SNAREs on transport vesicles (v-SNARE) and on target membranes (t-SNARE) form the SNARE complex through their conserved amphipathic helices (SNARE motifs) and draw two membranes into apposition, allowing membrane fusion to occur Sutton et al., 1998). Syntaxin 6 is a ubiquitously expressed SNARE that localizes to the transGolgi network (TGN) and endosomes (Bock et al., 1996(Bock et al., , 1997. It is comprised of an N-terminal helical domain (H1 domain) followed by a SNARE motif (H2 domain) and a C-terminal membrane anchor (Bock et al., 1996;Misura et al., 2002). Syntaxin 6 is thought to function in multiple membrane-trafficking events because it forms a complex with a varied set of SNAREs: syntaxin 16, Vti1a/Vti1-rp2, and VAMP3/cellubrevin or VAMP4 Steegmaier et al., 1999;Kreykenbohm et al., 2002;Mallard et al., 2002); syntaxin 7, Vti1b, and VAMP7 or VAMP8 (Wade et al., 2001); or SNAP-29/GS32 (Wong et al., 1999). Recent studies have demonstrated that syntaxin 6 plays roles in the early/recycling endosomes-to-TGN transport (Mallard et al., 2002), maturation of secretory granules (Klumperman et al., 1998;Kuliawat et al., 2004), regulation of GLUT4 trafficking (Perera et al., 2003;Shewan et al., 2003), and neutrophil ex...
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.
MARCH comprises a recently identified family of transmembrane RING-finger proteins which is implicated in diverse biological functions, such as immune regulation, protein quality control, and membrane trafficking. We previously identified MARCH-II, as a binding partner of syntaxin 6, which plays a role in endosomal protein transport. In this paper, we describe the cloning and characterization of MARCH-III which is the closest homolog of MARCH-II. It is broadly expressed at relatively high levels in spleen, colon, and lung. An immunofluorescence study of HeLa cells demonstrated that MARCH-III is present in peripheral vesicles partially colocalized with transferrin receptor. Overexpression of MARCH-III resulted in the redistribution of TGN46 and strong inhibition of transferrin uptake. Immunoprecipitation studies revealed that MARCH-III is associated with syntaxin 6 and MARCH-II. Mutational analyses revealed that the PDZ-binding motif and RING finger are essential for the subcellular localization of MARCH-III and the inhibitory effect on transferrin uptake. The location, associated molecules, and effects of overexpression suggest that MARCH-III is involved in the regulation of vesicular trafficking in endosomes.
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.
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