Immunohistochemical study of a membrane skeletal molecule, protein 4.1G, in mouse seminiferous tubules

Protein 4.1 families have recently been established as potential organizers of an adherens system. In the adult mouse testis, protein 4.1G (4.1G) localized as a line pattern in both basal and adluminal compartments of the seminiferous tubules, attaching regions of germ cells and Sertoli cells. By double staining for 4.1G and F-actin, their localizations were shown to be different, indicating that 4.1G was localized in a region other than the basal and apical ectoplasmic specializations, which formed the Sertoli–Sertoli cell junction and Sertoli–spermatid junction, respectively. By electron microscopy, immunoreactive products were seen exclusively on the cell membranes of Sertoli cells, attaching to the various differentiating germ cells. The immunolocalization of cadherin was identical to that of 4.1G, supporting the idea that 4.1G may be functionally interconnected with adhesion molecules. In an experimental mouse model of cadmium treatment, in which tight and adherens junctions of seminiferous tubules were disrupted, the 4.1G immunostaining in the seminiferous tubules was dramatically decreased. These results indicate that 4.1G may have a basic adhesive function between Sertoli cells and germ cells from the side of Sertoli cells.     

Regulation of protein 4.1R, p55, and glycophorin C ternary complex in human erythrocyte membrane

Three binary protein-protein interactions, glycophorin C (GPC)-4.1R, GPC-p55, and p55-4.1R, constitute the GPC-4.1R-p55 ternary complex in the erythrocyte membrane. Little is known regarding the molecular basis for the interaction of 4.1R with either GPC or p55 and regarding the role of 4.1R in regulating the various protein-protein interactions that constitute the GPC-4.1R-p55 ternary complex. In the present study, we present evidence that sequences in the 30-kDa domain encoded by exon 8 and exon 10 of 4.1R constitute the binding interfaces for GPC and p55, respectively. We further show that 4.1R increases the affinity of p55 binding to GPC by an order of magnitude, implying that 4.1R modulates the interaction between p55 and GPC. Finally, we document that binding of calmodulin to 4.1R decreases the affinity of 4.1R interactions with both p55 and GPC in a Ca(2+)-dependent manner, implying that the GPC-4.1R-p55 ternary protein complex can undergo dynamic regulation in the erythrocyte membrane. Taken together, these findings have enabled us to identify an important role for 4.1R in regulating the GPC-4.1R-p55 ternary complex in the erythrocyte membrane.     

Localization of sodium bicarbonate cotransporter (NBC) protein and messenger ribonucleic acid in rat epididymis

An acidic environment is important for sperm maturation in the epididymis and also helps to maintain mature sperm in an immotile state during storage in this organ. Both an Na+/H+ exchanger and an H+ATPase have been implicated in this process. The H+ATPase is concentrated in specialized apical (and/or narrow) and clear cells of the epididymis, while the Na+/H+ exchanger has not yet been localized in situ. As in other proton-secreting epithelia, bicarbonate transport occurs in the epididymis, where it is implicated in luminal acidification. In this study we used an antibody raised against a fusion protein (maltose-binding protein: MBP-NBC-5) from the C-terminus of the recently cloned rat kidney Na+/HCO3- cotransporter (NBC) to localize this protein in the epididymis and vas deferens of the rat. The distribution of the respective mRNA was mapped by in situ hybridization. NBC message was strongly expressed in the initial segment and the intermediate zone of the epididymis, and the NBC-5 antibody gave a strong basolateral staining in both principal cells and apical/narrow cells in this region. Western blotting revealed a single band at about 160 kDa in the epididymis. The intensity of staining as well as mRNA levels decreased in the cauda epididymidis and in the vas deferens, where only weak staining was seen. Basolateral NBC may function in parallel with apical proton secretion to regulate luminal acidification and/or bicarbonate reabsorption in the excurrent duct system.     

Basolateral regulation of pHi in proximal tubules of avian loopless and long-looped nephrons in bicarbonate.

In isolated, nonperfused chicken proximal tubules from both loopless reptilian-type and long-looped mammalian-type nephrons, resting intracellular pH (pHi), measured with pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF), was approximately 7.1 under control HCO3- conditions [20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES)/5 mM HCO3(-)-buffered medium with pH 7.4 at 37 degrees C] and was reduced to approximately 6.8 in response to NH4Cl pulse. The rate of recovery of pHi (dpHi/dt) from this level to the resting level in proximal tubules from both nephron types was (1) significantly reduced by the removal of Na+ or both Na+ and Cl- from the bath, and (2) unaffected by the removal of Cl- from the bath or the presence of a high K+ concentration or Ba2+ in the bath. In proximal tubules from long-looped mammalian-type, but not loopless reptilian-type, nephrons, dpHi/dt was significantly reduced by the addition of either 5-(N-ethyl-N-isopropyl) amiloride (EIPA) or 4,4'-diisothiocyanostilbene-2,2'disulfonate (DIDS) to the bath. These data suggest that a Na+/H+ exchanger and most likely a Na(+)-dependent Cl-/HCO3- exchanger are involved in basolateral regulation of pHi in mammalian-type nephrons whereas none of the commonly identified basolateral acid-base transporters appear to be involved in regulation of pHi in reptilian-type nephrons.     

Immunolocalization of protein 4.1B/DAL-1 during neoplastic transformation of mouse and human intestinal epithelium

Recently, we have reported that the protein 4.1B immunolocalization occurred only in matured columnar epithelial cells of normal rat intestines. This finding suggested that protein 4.1B expression could be examined for a possible change during neoplastic transformation of the intestinal mucosa. In the present study, we first present the distribution of mouse protein 4.1B in normal intestinal epithelial cells and tumor cells using the adenomatous polyposis coli (Apc) mutant mouse model. A low level of protein 4.1B expression coincided with the phenotypic transition to carcinoma. To examine the protein 4.1B expression in human intestinal mucosa, we used another antibody against an isoform of the human protein 4.1B, DAL-1 (differentially expressed adenocarcinoma of the lung). Human DAL-1 was also expressed in matured epithelial cells in human colons, with a definite expression gradient along the crypt axis. In human colorectal cancer cells, however, DAL-1 expression was not detected. These results suggest that mouse protein 4.1B and human DAL-1 might have a striking analogy of functions, which may be integrally involved in epithelial proliferation. We propose that loss of protein 4.1B/DAL-1 expression might be a marker of intestinal tumors, indicative of a tumor suppressor function in the intestinal mucosa.     

Contraction-mediated mTOR, p70S6k, and ERK1/2 phosphorylation in aged skeletal muscle.

With age, skeletal muscle experiences substantial atrophy and weakness. Although resistance training can increase muscle size and strength, the myogenic response to exercise and the capacity for muscle hypertrophy in older humans and animals is limited. In the present study, we assessed the ability of muscle contractile activity to activate cellular pathways involved in muscle cell growth and myogenesis in adult (Y; 6 mo old) and aged (O; 30 mo old) Fischer 344 x Brown Norway rats. A single bout of rat hindlimb muscle contractile activity was elicited by high-frequency electrical stimulation (HFES) of the sciatic nerve. Plantaris (Pla) and tibialis anterior (TA) muscles were assayed for mammalian target of rapamycin (mTOR), 70-kDa ribosomal protein S6 kinase (p70(S6K)), and extracellular signal-regulated kinase (ERK) 1/2 phosphorylation and total protein either at baseline, immediately after, or 6 h after HFES. mTOR phosphorylation was elevated in Pla (1.3 +/- 0.3-fold, P < 0.05) immediately after HFES and to a lesser extent 6 h after HFES (0.6 +/- 0.1-fold, P < 0.05) in O rats. Post-HFES, p70(S6K) phosphorylation increased 1.2 +/- 0.3-fold in TA (P < 0.05) and remained elevated 6 h later (0.6 +/- 0.2-fold, P < 0.05) in O rats. ERK phosphorylation was lower in O rats immediately after exercise in both TA (11.1 +/- 2.9 vs. 2.1 +/- 0.5-fold, P < 0.05) and Pla (6.5 +/- 1.5 vs. 1.8 +/- 0.5-fold, P < 0.05) and returned to baseline by 6 h in both Y and O rats. Phosphorylation of mTOR, p70(S6K), and ERK1/2 are increased in skeletal muscle after a single bout of in situ muscle contractile activity in aged animals, and the response is less than that observed in adult animals. These observations suggest that the anabolic response to a single bout of contraction is attenuated with aging and may help explain the reduced capacity for hypertrophy in aged animals.     

Latent membrane protein 1 of Epstein-Barr virus stimulates processing of NF-kappa B2 p100 to p52

Recent studies have identified a limited number of cellular receptors that can stimulate an alternative NF-kappa B activation pathway that depends upon the inducible processing of NF-kappa B2 p100 to p52. Here it is shown that the latent membrane protein (LMP)-1 of Epstein-Barr virus can trigger this signaling pathway in both B cells and epithelial cells. LMP1-induced p100 processing, which is mediated by the proteasome and is dependent upon de novo protein synthesis, results in the nuclear translocation of p52.RelB dimers. Previous studies have established that LMP1 also stimulates the canonical NF-kappa B-signaling pathway that triggers phosphorylation and degradation of I kappa B alpha. Interestingly, LMP1 activation of these two NF-kappa B pathways is shown here to require distinct regions of the LMP1 C-terminal cytoplasmic tail. Thus, C-terminal-activating region 1 is required for maximal triggering of p100 processing but is largely dispensable for stimulation of I kappa B alpha phosphorylation. In contrast, C-terminal-activating region 2 is critical for maximal LMP1 triggering of I kappa B alpha phosphorylation and up-regulation of p100 levels but does not contribute to activation of p100 processing. Because p100 deletion mutants that constitutively produce p52 oncogenically transform fibroblasts in vitro, it is likely that stimulation of p100 processing by LMP1 will play an important role in its transforming function.     

Expression of Bcl-2 and Bax in mouse renal tubules during kidney development

Bcl-2 and Bax play an important role in apoptosis regulation, as well as in cell adhesion and migration during kidney morphogenesis, which is structurally and functionally related to mitochondria. In order to elucidate the role of Bcl-2 and Bax during kidney development, it is essential to establish the exact location of their expression in the kidney. The present study localized their expression during kidney development. Kidneys from embryonic (E) 16-, 17-, 18-day-old mouse fetuses, and postnatal (P) 1-, 3-, 5-, 7-, 14-, 21-day-old pups were embedded in Epon. Semi-thin serial sections from two E17 kidneys underwent computer assisted 3D tubule tracing. The tracing was combined with a newly developed immunohistochemical technique, which enables immunohistochemistry on glutaraldehyde fixated plastic embedded sections. Thereby, the microstructure could be described in detail, and the immunochemistry can be performed using exactly the same sections. The study showed that Bcl-2 and Bax were strongly expressed in mature proximal convoluted tubules at all time points, less strongly expressed in proximal straight tubules, and only weakly in immature proximal tubules and distal tubules. No expression was detected in ureteric bud and other earlier developing structures, such as comma bodies, S shaped bodies, glomeruli, etc. Tubules expressing Bcl-2 only were occasionally observed. The present study showed that, during kidney development, Bcl-2 and Bax are expressed differently in the proximal and distal tubules, although these two tubule segments are almost equally equipped with mitochondria. The functional significance of the different expression of Bcl-2 and Bax in proximal and distal tubules is unknown. However, the findings of the present study suggest that the mitochondrial function differs between mature proximal tubules and in the rest of the tubules. The function of Bcl-2 and Bax during tubulogenesis still needs to be investigated.     

Interaction of a peripheral protein of the erythrocyte membrane, band 4.1, with phosphatidylserine-containing liposomes and erythrocyte inside-out vesicles

Interactions of band 4.1 with mixed phospholipid membranes [phosphatidylserine (PtdSer), phosphatidylethanolamine, phosphatidylcholine, etc.] and erythrocyte inside-out vesicles were studied. Band 4.1 showed a higher affinity to PtdSer-containing membranes. The amount of binding to PtdSer-containing liposomes was larger than that to PtdSer-lacking liposomes. The amount of binding to inside-out vesicles did not change significantly on a protease treatment of the vesicles. The amount of band 4.1 bound on inside-out vesicles decreased on PtdSer-decarboxylase treatment of the vesicles. Ca2+ acted inhibitory to the binding of band 4.1. Band 4.1 together with PtdSer-containing vesicles but not with PtdSer-lacking vesicles induced gelation of spectrin-actin copolymer solution. Ca2+ inhibited the gelation. Fluorescence energy transfer from PtdSer-containing vesicles to band 4.1 was larger than that from PtdSer-lacking vesicles. Band 4.1 caused a marked release of tempocholine from preloaded PtdSer-containing liposomes but not from PtdSer-lacking liposomes. The release was larger from liposomes containing more PtdSer. Ca2+ was inhibitory to the tempocholine release. We suggest from these results that band 4.1 provides another anchoring site for the cytoskeletal spectrin-actin network to PtdSer domains in the inner layer of erythrocyte membrane. This anchoring may be involved in functional regulation since the interaction causes the membrane permeability change that is dependent on Ca2+.     

Acute and chronic effect of dietary phosphorus restriction on protein expression in young rat renal proximal tubules

Renal proximal tubules play a vital role in phosphorus (P) homeostasis. It is well known that dietary P restriction up-regulates the activities of 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1-OHase), an enzyme that is involved in activation of vitamin D and thereby maintaining P balance. However, the mechanism involved in such regulation is not known. In the present study, we aim to identify proteins that might be involved in the renal adaptation to dietary P restriction using a proteomic approach. Renal proximal tubules were harvested from young rats fed either normal P diet or low P diet (LPD) for 1 to 7 days. Western blotting analysis of 1-OHase and signaling proteins in insulin-like growth factor I axis indicated an increase in expression of these proteins upon dietary P restriction. Using two-dimensional electrophoresis, we found that LPD reduced the total number of protein species expressed in renal proximal tubules. Differentially expressed proteins were analyzed and located using the software Melanie III, and their identities were found using matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Our results showed that beta-actin, gamma-actin, major urinary protein, phosphatidylinositol transfer protein beta isoform, and G1/S-specific cyclin D3 are up-regulated and nonspecific lipid transfer protein is down-regulated by LPD.     

A novel missense mutation in the sodium bicarbonate cotransporter (NBCe1/SLC4A4) causes proximal tubular acidosis and glaucoma through ion transport defects

In humans and terrestrial vertebrates, the kidney controls systemic pH in part by absorbing filtered bicarbonate in the proximal tubule via an electrogenic Na+/HCO3- cotransporter (NBCe1/SLC4A4). Recently, human genetics revealed that NBCe1 is the major renal contributor to this process. Homozygous point mutations in NBCe1 cause proximal renal tubular acidosis (pRTA), glaucoma, and cataracts (Igarashi, T., Inatomi, J., Sekine, T., Cha, S. H., Kanai, Y., Kunimi, M., Tsukamoto, K., Satoh, H., Shimadzu, M., Tozawa, F., Mori, T., Shiobara, M., Seki, G., and Endou, H. (1999) Nat. Genet. 23, 264-266). We have identified and functionally characterized a novel, homozygous, missense mutation (S427L) in NBCe1, also resulting in pRTA and similar eye defects without mental retardation. To understand the pathophysiology of the syndrome, we expressed wild-type (WT) NBCe1 and S427L-NBCe1 in Xenopus oocytes. Function was evaluated by measuring intracellular pH (HCO3- transport) and membrane currents using microelectrodes. HCO3- -elicited currents for S427L were approximately 10% of WT NBCe1, and CO2-induced acidification was approximately 4-fold faster. Na+ -dependent HCO3- transport (currents and acidification) was also approximately 10% of WT. Current-voltage (I-V) analysis reveals that S427L has no reversal potential in HCO3-, indicating that under physiological ion gradient conditions, NaHCO3 could not move out of cells as is needed for renal HCO3- absorption and ocular pressure homeostasis. I-V analysis without Na+ further shows that the S427L-mediated NaHCO3 efflux mode is depressed or absent. These experiments reveal that voltage- and Na+ -dependent transport by S427L-hkNBCe1 is unfavorably altered, thereby causing both insufficient HCO3- absorption by the kidney (proximal RTA) and inappropriate anterior chamber fluid transport (glaucoma).     

Identification of heat shock protein 5, calnexin and integral membrane protein 2B as Adam7-interacting membrane proteins in mouse sperm

In mammals, sperm acquire their motility and ability to fertilize eggs in the epididymis. This maturation process involves the acquisition of particular proteins from the epididymis. One such secretory protein specifically expressed in the epididymis is Adam7 (a disintegrin and metalloprotease 7). Previous studies have shown that Adam7 that resides in an intracellular compartment of epididymal cells is transferred to sperm membranes, where its levels are dependent on the expression of Adam2 and Adam3, which have critical roles in fertilization. Here, using a proteomics approach based on mass spectrometry, we identified proteins that interact with Adam7 in sperm membranes. This analysis revealed that Adam7 forms complexes with calnexin (Canx), heat shock protein 5 (Hspa5), and integral membrane protein 2B (Itm2b). Canx and Hspa5 are molecular chaperones, and Itm2b is a type II integral membrane protein implicated in neurodegeneration. The interaction of Adam7 with these proteins was confirmed by immunoprecipitation-Western blot analysis. We found that Adam7 and Itm2b are located in detergent-resistant regions known to be highly correlated with membrane lipid rafts. We further found that the association of Adam7 with Itm2b is remarkably promoted during sperm capacitation owing to a conformational change of Adam7 that occurs in concert with the capacitation process. Thus, our results suggest that Adam7 functions in fertilization through the formation of a chaperone complex and enhanced association with Itm2b during capacitation in sperm.     

Nucleic acid binding and unfolding properties of ribosomal protein S1 and the derivatives S1-F1 and m1-S1.

The nucleic acid binding and unwinding properties of wild-type Escherichia coli ribosomal protein S1 have been compared to those of a mutant form and a large trypsin-resistant fragment, both reported recently [J. Mol. Biol. 127, 41-45 (1979) and J. Biol. Chem. 254, 4309-4312 (1979). The mutant (m1-S1) contains 77% and the fragment (S1-F1) 66% of the polypeptide chain length (approximately 600 amino acid residues) of protein S1. The mutant is active in protein synthesis in vitro; the fragment, although retaining one or more of the functional domains of S1, is inactive in protein synthesis. We find that m1-S1 is is almost as effective as S1 in binding to poly(rU), phage MS2 RNA and simian virus 40 (SV40) DNA, and in unfolding poly(rU) and the helical structures present in MS2 RNA and phi X174 viral DNA. S1-F1, however, binds to poly(rU) and denatured SV40 DNA, but not to MS2 RNA. It unfolds neither poly(rU), nor the residual secondary structure of MS2 RNA or phi X174 viral DNA. Thus, there appears to be a correlation between the loss in ability of S1 to unwind RNA and the loss in its ability to function in protein synthesis.     

Voltage and cosubstrate dependence of the Na-HCO3 cotransporter kinetics in renal proximal tubule cells.

The voltage dependence of the kinetics of the sodium bicarbonate cotransporter was studied in proximal tubule cells. This electrogenic cotransporter transports one Na+, three HCO3-, and two negative charges. Cells were grown to confluence on a permeable support, mounted on a Ussing-type chamber, and permeabilized apically to small monovalent ions with amphotericin B. The steady-state, di-nitro-stilbene-di-sulfonate-sensitive current was shown to be sodium and bicarbonate dependent and therefore was taken as flux through the cotransporter. Voltage-current relations were measured as a function of Na+ and HCO3- concentrations between -160 and +160 mV under zero-trans and symmetrical conditions. The kinetics could be described by a Michaelis-Menten behavior with a Hill coefficient of 3 for HCO3- and 1 for Na+. The data were fitted to six-state ordered binding models without restrictions with respect to the rate-limiting step. All ordered models could quantitatively account for the observed current-voltage relationships and the transinhibition by high bicarbonate concentration. The models indicate that 1) the unloaded transporter carries a positive charge; 2) the binding of cytosolic bicarbonate to the transporter "senses" 12% of the electric field in the membrane, whereas its translocation across the membrane "senses" 88% of the field; 3) the binding of Na+ to the cotransporter is voltage independent.     

Interaction of Pik1p and Sjl proteins in membrane trafficking

Phosphatidylinositol (PtdIns) phosphates are involved in signal transduction, cytoskeletal organization, and membrane traffic. PtdIns 4-phosphate [PtdIns(4)P], produced in yeast by PtdIns 4-kinase (Pik1p), appears to regulate Golgi secretory function. PtdIns(4)P is also produced by dephosphorylation of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], catalyzed by one of the three yeast Sjl proteins, homologs of the mammalian synaptic vesicle-associated PtdIns(4,5)P2 5-phosphatase, synaptojanin. To determine whether Pik1p and Sjl proteins operate in the same pathway or regulate the same process, we used a genetic approach. Mutation in the PIK1 gene displays synthetic genetic interactions with deletions of individual SJL genes. Deletion of SJL3 gene is synthetically lethal with pik1ts, and deletions of SJL1 or SJL2 genes in pik1ts cells exacerbate the temperature sensitivity, neomycin sensitivity, and defect in invertase secretion. A diminished level of PtdIns(4)P and increased level of PtdIns(4,5)P2 in pik1(ts)sjl1delta and pik1(ts)sjl2delta cells, compared with pik1ts cells, indicate that PtdIns(4)P is specifically required for secretion. Collectively, our results suggest that Pik1p and the Sjl proteins coordinately function to regulate the dynamic phosphorylation-dephosphorylation of the polar heads of phosphoinositides, and this process appears to be important for membrane trafficking pathways.     

Protein p0071, an armadillo plaque protein of adherens junctions, is predominantly expressed in distal renal tubules

Protein p0071 is a member of the p120-subfamily of armadillo proteins and is well known as a junctional plaque component involved in cell–cell adhesion, especially in adherens junctions. By systematic immunohistochemical analysis of mouse and human kidney tissues, p0071 was prominently detected in distinct kidney tubules. Upon double-labeling immunolocalization experiments with segment-specific markers, p0071 was predominantly localized in distal straight and convoluted tubules and to a lesser extent in proximal tubules, in the ascending thin limb of loop of Henle and in the collecting ducts. In capillaries of the kidney, p0071 co-localized with VE-cadherin an endothelium-specific cadherin. Protein p0071 was also detected in both, renal cell carcinomas derived from distal tubules and in maturing nephrons of early mouse developmental stages. Immunoblotting of total extracts of cultured cells of renal origin showed that p0071 was detected in all human and murine cells analyzed. Upon immunolocalization, p0071 was observed in adherens junctions but also in distinct cytoplasmic structures at the cell periphery of cultured cells. Possible structural and functional roles of p0071 are suggested by its preferential occurrence in distinct tubule segments, and its potential use as a cytodiagnostic cell type marker in renal pathology is discussed.     

Palmitoylation of MPP1 (membrane-palmitoylated protein 1)/p55 is crucial for lateral membrane organization in erythroid cells

S-Acylation of proteins is a ubiquitous post-translational modification and a common signal for membrane association. The major palmitoylated protein in erythrocytes is MPP1, a member of the MAGUK family and an important component of the ternary complex that attaches the spectrin-based skeleton to the plasma membrane. Here we show that DHHC17 is the only acyltransferase present in red blood cells (RBC). Moreover, we give evidence that protein palmitoylation is essential for membrane organization and is crucial for proper RBC morphology, and that the effect is specific for MPP1. Our observations are based on the clinical cases of two related patients whose RBC had no palmitoylation activity, caused by a lack of DHHC17 in the membrane, which resulted in a strong decrease of the amount of detergent-resistant membrane (DRM) material. We confirmed that this loss of detergent-resistant membrane was due to the lack of palmitoylation by treatment of healthy RBC with 2-bromopalmitic acid (2-BrP, common palmitoylation inhibitor). Concomitantly, fluorescence lifetime imaging microscopy (FLIM) analyses of an order-sensing dye revealed a reduction of membrane order after chemical inhibition of palmitoylation in erythrocytes. These data point to a pathophysiological relationship between the loss of MPP1-directed palmitoylation activity and perturbed lateral membrane organization.