ABSTRACT
Myosin Va is associated with discrete vesicle populations in a number of cell types, but little is known of the function of myosin Vb. Yeast two-hybrid screening of a rabbit parietal cell cDNA library with dominant active Rab11a (Rab11aS20V) identified myosin Vb as an interacting protein for Rab11a, a marker for plasma membrane recycling systems. The isolated clone, corresponding to the carboxyl terminal 60 kDa of the myosin Vb tail, interacted with all members of the Rab11 family (Rab11a, Rab11b, and Rab25). GFP-myosin Vb and endogenous myosin Vb immunoreactivity codistributed with Rab11a in HeLa and Madin-Darby canine kidney (MDCK) cells. As with Rab11a in MDCK cells, the myosin Vb immunoreactivity was dispersed with nocodazole treatment and relocated to the apical corners of cells with taxol treatment. A green fluorescent protein (GFP)-myosin Vb tail chimera overexpressed in HeLa cells retarded transferrin recycling and caused accumulation of transferrin and the transferrin receptor in pericentrosomal vesicles. Expression of the myosin Vb tail chimera in polarized MDCK cells stably expressing the polymeric IgA receptor caused accumulation of basolaterally endocytosed polymeric IgA and the polymeric IgA receptor in the pericentrosomal region. The myosin Vb tail had no effects on transferrin trafficking in polarized MDCK cells. The GFP-myosin Va tail did not colocalize with Rab11a and had no effects on recycling system vesicle distribution in either HeLa or MDCK cells. The results indicate myosin Vb is associated with the plasma membrane recycling system in nonpolarized cells and the apical recycling system in polarized cells. The dominant negative effects of the myosin Vb tail chimera indicate that this unconventional myosin is required for transit out of plasma membrane recycling systems.
Subject(s)
Cell Membrane/chemistry , Cell Membrane/metabolism , Myosins/chemistry , Myosins/metabolism , Amino Acid Sequence , Animals , Biological Transport , DNA, Complementary/metabolism , Dogs , Gene Library , Green Fluorescent Proteins , HeLa Cells , Humans , Luminescent Proteins/metabolism , Molecular Sequence Data , Mutation , Protein Binding , Protein Structure, Tertiary , Rabbits , Receptors, Fc/metabolism , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino Acid , Time Factors , Transfection , Transferrin/chemistry , Transferrin/metabolism , Two-Hybrid System Techniques , rab GTP-Binding Proteins/chemistry , rab GTP-Binding Proteins/metabolismABSTRACT
Rab11a, Rab11b, and Rab25 in mammals are thought to comprise a subfamily of Rab proteins, although Rab25 has two amino acid differences in its effector domain. We have isolated and characterized the genomic sequences of murine Rab11a and Rab25 and compared them with those of previously characterized mammalian Rab genes. The Rab11a gene spans 29 kb and Rab25 spans 9 kb. The genes have TATA-less promoters, but contain GC-rich areas in their upstream 5' regions. Both genes have 5 exons, with the introns containing characteristic repeats. Rab11a has an unusually long 8. 5-kb fourth intron. The Rab11a and Rab25 genes are localized to chromosomes 9C and 3E3/F1, respectively. The overall organization of the Rab11a, Rab11b, and Rab25 genes is similar, with homologous exon-intron boundaries, and differs markedly from those of Rab3A and Rab1A. These results confirm that Rab11A, Rab11b, and Rab25 represent a closely related gene family.
Subject(s)
rab GTP-Binding Proteins/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA Primers , Mice , Molecular Sequence Data , TATA Box , rab GTP-Binding Proteins/chemistryABSTRACT
We report the characterization of an Na+/H+ exchanger (NHE) in embryonic fibroblasts (SL-29 cells) of the chicken, a terrestrial vertebrate, where Na+ conservation is important. This exchanger is electroneutral, has a single Na+ binding site, and is highly sensitive to amiloride (IC50 2 microM), dimethyl amiloride (350 nM), and ethyl-isopropyl amiloride (25 nM). It is stimulated by serum, transforming growth factor-alpha, hypertonicity, and okadaic acid. Although these features make it resemble mammalian NHE1, other characteristics suggest distinct differences. First, in contrast to mammalian NHE1 it is inhibited by cAMP and shows a biphasic response to phorbol esters and a highly variable response to increased intracellular Ca2+ concentration. Second, whereas full-length human and rat NHE1 cDNA probes recognize a 4.8-kb transcript in rat tissues, they recognize only a 3.9-kb transcript in chicken tissues. An antibody against amino acids 631-746 of human NHE1 sequence fails to recognize a protein in SL-29 cells. Rat NHE2 and NHE3 probes do not recognize any transcript in chicken fibroblasts. The SL-29 exchanger differs markedly from the previously characterized chicken intestinal apical exchanger in its amiloride sensitivity and regulation by phorbol esters. These results suggest that a modified version of mammalian NHE1 is present in chicken tissues and imply that another functionally distinct Na+/H+ exchanger is expressed in aves.
Subject(s)
Chick Embryo/metabolism , Fibroblasts/metabolism , Sodium-Hydrogen Exchangers/metabolism , Amiloride/analogs & derivatives , Amiloride/pharmacology , Animals , Blood , Cell Line , Chick Embryo/cytology , Cyclic AMP/analogs & derivatives , Cyclic AMP/pharmacology , Humans , Hypertonic Solutions/pharmacology , Kinetics , Okadaic Acid/pharmacology , Phorbol 12,13-Dibutyrate/pharmacology , RNA, Messenger/metabolism , Rats , Sodium-Hydrogen Exchangers/antagonists & inhibitors , Sodium-Hydrogen Exchangers/genetics , Thionucleotides/pharmacology , Time Factors , Transforming Growth Factor alpha/pharmacologyABSTRACT
Recent evidence suggests that apical and basolateral endocytic pathways in epithelia converge in an apically located, pericentriolar endosomal compartment termed the apical recycling endosome. In this compartment, apically and basolaterally internalized membrane constituents are thought to be sorted for recycling back to their site of origin or for transcytosis to the opposite plasma membrane domain. We report here that in the epithelial cell line Madin-Darby Canine Kidney (MDCK), antibodies to Rab11a label an apical pericentriolar endosomal compartment that is dependent on intact microtubules for its integrity. Furthermore, this compartment is accessible to a membrane-bound marker (dimeric immunoglobulin A [IgA]) internalized from either the apical or basolateral pole, functionally defining it as the apical recycling endosome. We have also examined the role of a closely related epithelial-specific Rab, Rab25, in the regulation of membrane recycling and transcytosis in MDCK cells. When cDNA encoding Rab25 was transfected into MDCK cells, the protein colocalized with Rab11a in subapical vesicles. Rab25 transfection also altered the distribution of Rab11a, causing the coalescence of immunoreactivity into multiple denser vesicular structures not associated with the centrosome. Nevertheless, nocodazole still dispersed these vesicles, and dimeric IgA internalized from either the apical or basolateral membrane was detected in endosomes labeled with antibodies to both Rab11a and Rab25. Overexpression of Rab25 decreased the rate of IgA transcytosis and of apical, but not basolateral, recycling of internalized ligand. Conversely, expression of the dominant-negative Rab25T26N did not alter either apical recycling or transcytosis. These results indicate that both Rab11a and Rab25 associate with the apical recycling system of epithelial cells and suggest that Rab25 may selectively regulate the apical recycling and/or transcytotic pathways.
Subject(s)
GTP-Binding Proteins/metabolism , Kidney/metabolism , rab GTP-Binding Proteins , Animals , Biological Transport, Active , Cell Line , Cell Polarity , Cytoskeleton/metabolism , Dogs , Endocytosis , Endosomes/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , GTP Phosphohydrolases/metabolism , GTP-Binding Proteins/genetics , GTP-Binding Proteins/immunology , Immunoglobulin A/metabolism , Immunohistochemistry , Kidney/cytology , Microtubules/metabolism , TransfectionABSTRACT
Ezrin, a membrane-cytoskeleton linker protein, is involved in the recruitment of H+/K(+)-ATPase-containing tubulovesicles to the canalicular membrane during acid secretion in the parietal cell. Ezrin exists as monomers and head-to-tail dimers in vivo, and oligomerization is presumably important for activation. In this study, we mapped regions of ezrin-ezrin interaction using the yeast two-hybrid assay. We observed that the N-terminal 283 amino acids are sufficient for interaction with the carboxyl terminal 140 amino acids. The region 333.446 inhibits this association. However, the inclusion of amino acids 283-310 appears to release the inhibition. These specific interactions may play a critical role in the formation of dimerization-competent ezrin molecules.
Subject(s)
Dimerization , Phosphoproteins/chemistry , Saccharomyces cerevisiae Proteins , Animals , Cytoskeletal Proteins , DNA-Binding Proteins , Gene Transfer Techniques , Histidine/genetics , Humans , Parietal Cells, Gastric/chemistry , Phosphoproteins/genetics , Rabbits , Recombinant Fusion Proteins , Saccharomyces cerevisiae/genetics , Sequence Homology , Transcription Factors , beta-Galactosidase/geneticsABSTRACT
Intestinal sodium transporters, such as the Na+/H+ exchanger (NHE) are important for Na+ conservation in land birds. In mammals, at least five isoforms of the exchanger, NHEs 1-5, have been cloned, with NHE-1 occurring in epithelial basolateral and nonepithelial cell membranes and NHE-3 being restricted to epithelial apical/brush border membranes. We had demonstrated earlier that chicken intestinal brush border membranes possess NHE activity that functionally resembles mammalian NHE-3. In this study, we used mammalian NHE-1 and NHE-3 probes to examine if chicken enterocytes possess these transporters. Antisera against rat NHE-3 recognized a 97 kDa protein in chicken intestinal brush border membrane, while a NHE-3 cDNA probe failed to recognize any transcript. A NHE-1 antibody failed to recognize any protein in brush border or basolateral membrane, while a NHE-1 cDNA probe recognized a 3.9 kb transcript. Thus, there is more than one NHE isoform in chicken intestine, and our results suggest a novel avian NHE family.