MP38 contains the membrane-embedded domain of the lens fiber gap junction protein MP70

A 70-kDa lens membrane polypeptide (MP70) is a specific component of the fiber gap junctions. The C-terminal portion of MP70 is removed by age-related proteolytic processing, leaving an N-terminal 38-kDa polypeptide (MP38) in the membrane. Membrane association and topology of MP70 and of its processed form MP38 have been studied by hydrophobic labeling with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine and phenyl isothio[14C]cyanate. Membrane-embedded segments have been identified. They are localized in the N-terminal 30-kDa portion of MP70 and MP38. The C-terminal 40-kDa portion of MP70 appears to be exposed entirely at the cytoplasmic side of the junctional membranes. Hence, potentially poreforming peptide segments in the MP70 molecule are conserved upon age-related processing to MP38.     

Molecular portrait of lens gap junction protein MP70

A 70-kDa membrane protein (MP70) is a component of the lens fiber gap junctions. Its membrane topology and its N-terminal sequence are similar to those of the connexin family of proteins. Some features of MP70 containing fiber gap junctions are, however, distinct from gap junctions in other mammalian tissues: (i) Lens connexons form crystalline arrays only after cleavage of junctional proteins in vitro. These hexagonal arrays have a periodicity of 13.6 nm which is significantly larger than the 8- 9-nm spacing of liver and heart gap junctions. (ii) Lens fiber gap junctions dissociate in low concentrations of nonionic detergent and this provides an avenue to purify MP70 directly from a membrane mixture. Isolated MP70 in the form of 17 S structures has an appearance consistent with connexon pairs. (iii) The C-terminal half of MP70 is cleaved in situ by a lens endogenous calcium-dependent protease. The processed from MP38 remains in the membrane and is abundant in the central region of the lens. A testable hypothesis for MP70 function is presented.     

Reconstitution of channels from preparations enriched in lens gap junction protein MP70

Summary Detergent-solubilized ovine lens membrane proteins, enriched in the 70-kDa gap junction component (MP70), were reconstituted into planar lipid bilayers and analyzed for channel activities. Three distinct activities were found. Those showing conductance steps of 290 pS (symmetrical 150-mM KCl solutions) had properties similar to those reported earlier for MIP26 (Ehring, G.R., Zampighi, G., Horwitz, J., Bok, D., Hall, J.E. 1990. J. Gen. Physiol. 96:631–664.) of which minor amounts were normally present in the detergent-solubilized preparations. Two novel channel activities had unitary conductances of 90 and 45 pS, were halothane sensitive and did not discriminate between sodium and potassium ions. The 90-pS channel was asymmetrically voltage dependent, and its properties would be consistent with the expected properties of junctional hemichannels.This work was supported by grants from the Health Research Council of New Zealand, from the New Zealand Lottery Grants Board and from the New Zealand Vice Chancellor's Committee Fund.     

A lens intercellular junction protein, MP26, is a phosphoprotein

The major protein present in the plasma membrane of the bovine lens fiber cell (MP26), thought to be a component of intercellular junctions, was phosphorylated in an in vivo labeling procedure. After fragments of decapsulated fetal bovine lenses were incubated with [32P]orthophosphate, membranes were isolated and analyzed by SDS PAGE and autoradiography. A number of lens membrane proteins were routinely phosphorylated under these conditions. These proteins included species at Mr 17,000 and 26,000 as well as a series at both 34,000 and 55,000. The label at Mr 26,000 appeared to be associated with MP26, since (a) boiling the membrane sample in SDS led to both an aggregation of MP26 and a loss of label at Mr 26,000, (b) the label at 26,000 was resistant to both urea and nonionic detergents, and (c) two-dimensional gels showed that a phosphorylated Mr 24,000 fragment was derived from MP26 with V8 protease. Studies with proteases also provided for a localization of most label within approximately 20 to 40 residues from the COOH-terminus of MP26. Published work indicates that the phosphorylated portion of MP26 resides on the cytoplasmic side of the membrane, and that this region of MP26 contains a number of serine residues. The same region of MP26 was labeled when isolated lens membranes were reacted with a cAMP-dependent protein kinase prepared from the bovine lens. After the in vivo labeling of lens fragments, phosphoamino acid analysis of MP26 demonstrated primarily labeled serines, with 5-10% threonines and no tyrosines. Treatments that lowered the intracellular calcium levels in the in vivo system led to a selective reduction of MP26 phosphorylation. In addition, forskolin and cAMP stimulated the phosphorylation of MP26 and other proteins in concentrated lens homogenates. These findings are of interest because MP26 appears to serve as a protein of cell-to-cell channels in the lens, perhaps as a lens gap junction protein.     

Cx23, a connexin with only four extracellular-loop cysteines, forms functional gap junction channels and hemichannels

Gap junction channels may be comprised of either connexin or pannexin proteins (innexins and pannexins). Membrane topologies of both families are similar, but sequence similarity is lacking. Recently, connexin-like sequences have been identified in mammalian and zebrafish genomes that have only four conserved cysteines in the extracellular domains (Cx23), a feature of the pannexins. Phylogenetic analyses of the non-canonical "C4" connexins reveal that these sequences are indeed connexins. Functional assays reveal that the Cx23 gap junctions are capable of sharing neurobiotin, and further, that Cx23 connexins form hemichannels in vitro.     

Mouse whey acidic protein is a novel member of the family of 'four-disulfide core' proteins.

Unlike in other mammalian species, the major whey protein in mouse is not alpha-lactalbumin, but a cysteine rich, acidic protein with a molecular weight of 14.0 kDa. We have deduced the amino acid sequence of this mouse acidic of whey protein from the nucleotide sequence of cloned cDNA. The positions of the half cysteines suggest that mouse whey acidic protein (WAP) is a two domain protein, very similar in structure to the plant lectin wheat germ agglutinin and the hypothalamic carrier protein neurophysin.     

Maize mesocotyl plasmodesmata proteins cross-react with connexin gap junction protein antibodies.

Polypeptide present in various cell fractions obtained from homogenized maize mesocotyls were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotted, and screened for cross-reactivity with antibodies against three synthetic polypeptides spanning different regions of the rat heart gap junctional protein connexin43 and the whole mouse liver gap junctional protein connexin32. An antibody raised against a cytoplasmic loop region of connexin43 cross-reacted strongly with a cell wall-associated polypeptide (possibly a doublet) of 26 kilodaltons. Indirect immunogold labeling of thin sections of mesocotyl tissue with this antibody labeled the plasmodesmata of cortical cells along the entire length of the plasmodesmata, including the neck region and the cytoplasmic annulus. Sections labeled with control preimmune serum were essentially free of colloidal gold. An antibody against connexin32 cross-reacted with a 27-kilodalton polypeptide that was present in the cell wall and membrane fractions. Indirect immunogold labeling of thin sections with this antibody labeled the plasmodesmata mainly in the neck region. It is suggested that maize mesocotyl plasmodesmata contain at least two different proteins that have homologous domains with connexin proteins.     

Characterization of the gap junction protein,connexin45

Connexin45 is a gap junction protein which forms channels with unique characteristics. RNA blots demonstrated that connexin45 is expressed in a number of cell lines including WB, SK Hepl, BHK, A7r5, CLEM, and BWEM cells. Connexin45 was further studied in BWEM cells using specific affinity-purified antibodies directed against a synthetic peptide representing amino acids 285–298 of its sequence. Immunofluorescence experiments demonstrated that the BWEM cells expressed both connexin43 and connexin45 and that these connexins colocalized. Connexin45 polypeptide, immunoprecipitated from BWEM cells metabolically labeled with [³⁵S]-methionine, consisted of a predominant 48 kD polypeptide. Connexin45 and connexin43 contained radioactive phosphate when immunoprecipitated from BWEM cells metabolically labeled with [³²P]-orthophosphoric acid. This phosphate label was removed from connexin45 by alkaline phosphatase digestion. Treatment of BWEM cells with the tumor promoting agent 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited intercellular passage of microinjected Lucifer yellow. While TPA treatment induced phosphorylation of connexin43 in these cells, it reduced the expression of connexin45. Furthermore, the connexin45 expressed after TPA treatment was not phosphorylated. These results suggest that treatments which alter protein phosphorylation may regulate connexin43 and connexin45 in BWEM cells by different mechanisms.These studies were supported by National Institutes of Health grants HL45466 and EY08368. J.G.L. is supported by a fellowship from the Lucille P. Markey Foundation. E.C.B. is an Established Investigator of the American Heart Association.     

The permeability of reconstituted liposomes containing the purified lens fiber cell integral membrane proteins MP20, MP26 and MP70

Summary A number of lens fiber cell integral membrane proteins have been localized to junctional regions where they have been proposed to play a role in either mediating or controlling cell-to-cell communication. We have examined the effect of three lens fiber cell membrane proteins, MP20, MP26 and MP70, on the permeability properties of unilamellar phospholipid liposomes. This approach has been previously used to examine the channel-forming properties of MP26. Liposome permeability was determined by measuring the effect of Co²⁺ on the quenching of the fluorescence of N-4-nitrobenzo-2-oxa-1,3 diazole phosphatidyl ethanolamine (NBD-PE)-containing liposomes as described previously by Scaglione and Rintoul (Invest. Ophthalmol. Vis. Sci. 30:961–966, 1989). The effect of all three proteins on liposome permeability was similar. Permeability was dependent on the protein/phospholipid ratio and was not significantly affected by agents known to modify gap junctional permeability in vivo. Glycophorin A, a non-channel-forming integral membrane protein derived from erythrocytes, was also shown to increase the permeability of unilamellar phospholipid liposomes. The ability of a non-channel membrane protein to increase Co²⁺ quenching of NBD-PE-containing liposomes (presumably in a nonspecific manner) indicates that reports describing the permeability of lens membrane protein-containing liposomes should be interpreted with caution in terms of their relationship to cell-to-cell communication.We would like to thank Dr. Rita Meyer for technical assistance with the freeze-fracture electron microscopy, Drs. Wolfgang Baumann and Barbara Malewicz for the purification of bovine lens lipids, and Dr. Gary Nelsestuen for the use of both the fluorescence and photon correlation spectrophotometers as well as for many helpful discussions. This research was supported by NIH grant EY 05684.     

Mouse oncogene protein 24p3 is a member of the lipocalin protein family.

Rigorous new methods of protein sequence analysis have been applied to the lipocalins, a diverse family of ligand binding proteins. Using three conserved sequence motifs to search for similar patterns in a large sequence database, the size and composition of this protein family have been defined in an automatic and objective way. It has allowed the identification of an existing sequence, mouse 24p3 protein, as a lipocalin and the possible rejection of other putative members from this protein family. On the basis of this newly discovered homology, a possible function for mouse 24p3 protein is proposed.     

Phosphorylation of MP26, a lens junction protein,is enhanced by activators of protein kinase C

Summary MP26, a protein thought to form gap junctional channels in the lens, and other lens proteins were phosphorylated under conditions that activate protein kinase C. Phosphorylation was detected both in lens fiber cell fragments in an in vivo labeling procedure with³²P-phosphate and in cell homogenates with³²P-ATP. In these experiments, both calcium and 12-O-tetradecanoylphorbol 13-acetate (TPA) were necessary for maximal phosphorylation of MP26. Calcium stimulated the phosphorylation of MP26 approximately fourfold and TPA with calcium led to a sevenfold increase. If TPA was present, 1 m calcium was sufficient for maximal labeling. Phosphoamino acid analysis demonstrated approximately 85% phosphoserine, 15% phosphothreonine, and no phosphotyrosine when MP26 was phosphorylated in lens homogenates in the presence of TPA and calcium and then electrophoretically purified. Phosphorylation occurred near the cytoplasmic, C-terminal of MP26. The possible involvement of other kinases was also examined. The Walsh inhibitor, which affects cAMP-dependent protein kinases, had no influence on the TPA-mediated increase in phosphorylation. In studies with isolated membranes and added kinases, MP26 was also found to not be a substrate for calcium/calmodulindependent protein kinase II. Thus, protein kinase C may have phosphorylated MP26 in a direct manner.     

Four novel members of the connexin family of gap junction proteins. Molecular cloning, expression, and chromosome mapping.

We have used low stringency hybridization and polymerase chain reaction (PCR) amplification with degenerate oligonucleotides to identify four new members of the rat connexin gene family. On the basis of their predicted molecular mass, these proteins have been designated connexin (Cx) 40 (Cx40), Cx37, Cx33, and Cx31.1. The new connexins exhibit all of the conserved structural features of the connexin family, including highly similar extracellular and transmembrane domains but divergent major cytoplasmic domains. On the basis of primary sequence similarity, the connexin family may be divided into two classes. Cx40, Cx37, and Cx33 are similar to the previously characterized Cx43 and Cx46. Cx31.1 is similar to Cx26, Cx31, and Cx32. Cx37 and Cx40 mRNAs are expressed in a wide variety of adult organs and tissues, with particular abundance in lung. However, their relative levels are different in many organs and thus their distribution is not completely coincident. Cx33 and Cx31.1 genes exhibit a much more restricted pattern of expression; mRNAs are detected only in testes and skin, respectively. Chromosomal mapping studies indicate that Cx26 and Cx46 are tightly linked on chromosome 14, and Cx37 and Cx31.1 are linked on chromosome 4, while the rest of the connexin genes are dispersed.     

Voltage-dependent gap junction channels are formed by connexin32, the major gap junction protein of rat liver.

We report here experiments undertaken in pairs of hepatocytes that demonstrate a marked voltage sensivity of junctional conductance and, thus, contradict earlier findings reported by this laboratory (Spray, D.C., R.D.ginzberg, E.A., E. A. Morales, Z. Gatmaitan and I.M. Arias, 1986, J. Cell Biol. 101:135-144; Spray C.D. R.L. White, A.C. Campos de Carvalho, and M.V.L. Bennett. 1984. Biophys. J. 45:219-230) and by others (Dahl, G., T. Moller, D. Paul, R. Voellmy, and R. Werner. 1987. Science [Wash. DC] 236:1290-1293; Riverdin, E.C., and R. Weingart. 1988. Am. J. Physiol. 254:C226-C234). Expression in exogenous systems, lipid bilayers in which fragments of isolated gap junction membranes were incorporated (Young, J.D.-E., Z. Cohn, and N.B. Gilula. 1987. Cell. 48:733-743.) and noncommunicating cells transfected with connexin32 cDNA (Eghbali, B., J.A. Kessler, and D.C. Spray. 1990. Proc. Natl. Acad. Sci. USA. 87:1328-1331), support these findings and indicate that the voltage-dependent channel is composed of connexin32, the major gap junction protein of rat liver (Paul, D. 1986. J. Cell Biol. 103:123-134).     

Structural studies of lens fiber junction protein MP26 by cyanogen bromide cleavage

The lens fiber-cell plasma membrane MP26 from chick, bovine, and human lenses yielded identical cyanogen bromide peptide maps, confirming the essential conservation of structure in the junction protein of vertebrate lens fiber cells. Immunoblot analyses of the cyanogen bromide peptide maps of human lens MP26 and of its age-dependent proteolytic product MP22 confirmed that MP22 is a derivative of MP26. The findings in this study are the first consistent with the positioning of the methionine residues in lens MP26 as predicted by its cDNA-derived sequence.     

Uncoating ATPase is a member of the 70 kilodalton family of stress proteins

The synthetic peptide, VGIDLGTTYSC, derived from the heat shock-induced genes human hsp70, Drosophila hsp70, S. cerevisiae YG100, and E. coli dnaK, elicited antibodies that recognized two constitutive proteins in bovine extracts. One of these proteins, 71 kd, has previously been identified as uncoating ATPase, an enzyme that releases clathrin from coated vesicles. This immunological data complemented the result that uncoating ATPase was indistinguishable from the constitutive mammalian 71 kd stress protein by either partial proteolytic mapping or two-dimensional gel analysis. In addition, affinity-purified uncoating ATPase antibodies recognize proteins in yeast identified as the gene products of the heat shock or heat shock cognate genes YG100 and YG102. The results show that uncoating ATPase is a member of the 70 kd heat shock protein family.     

Phosphorylation of connexin 50 by protein kinase A enhances gap junction and hemichannel function

Phosphorylation of connexins is an important mechanism regulating gap junction channels. However, the role(s) of connexin (Cx) phosphorylation in vivo are largely unknown. Here, we showed by mass spectrometry that Ser-395 in the C terminus of chicken Cx50 was phosphorylated in the lens. Ser-395 is located within a PKA consensus site. Analyses of Cx50 phosphorylation by two-dimensional thin layer chromatography tryptic phosphopeptide profiles suggested that Ser-395 was targeted by PKA in vivo. PKA activation increased both gap junction dye coupling and hemichannel dye uptake in a manner not involving increases in total Cx50 expression or relocation to the cell surface or gap junctional plaques. Single channel recordings indicated PKA enhanced transitions between the closed and ～200-pS open state while simultaneously reducing transitions between this open state and a ～65-pS subconductance state. The mutation of Ser-395 to alanine significantly attenuated PKA-induced increases in dye coupling and uptake by Cx50. However, channel records indicated that phosphorylation at this site was unnecessary for enhanced transitions between the closed and ～200-pS conductance state. Together, these results suggest that Cx50 is phosphorylated in vivo by PKA at Ser-395 and that this event, although unnecessary for PKA-induced alterations in channel conductance, promotes increased dye permeability of Cx50 channels, which plays an important role in metabolic coupling and transport in lens fibers.     

The human connexin gene family of gap junction proteins: distinct chromosomal locations but similar structures.

Connexins are protein subunits that constitute gap junction channels. Two members of this gene family, connexin43 (Cx43) and connexin32 (Cx32), are abundantly expressed in the heart and liver, respectively. Human genomic DNA analysis revealed the presence of two loci for Cx43: an expressed gene and a processed pseudogene. The expressed gene (GJA1) was mapped to human chromosome 6 and the pseudogene (GJA1P) to chromosome 5. To determine whether Cx32 was linked to Cx43, somatic cell hybrids were analyzed by polymerase chain reaction and hybridization, resulting in the assignment of the gene for Cx32 (GJB1) to the X chromosome at Xp11----q22. Comparison of the structures of connexin genes suggests that members of this multigene family arose from a single precursor, but evolved to distinct chromosomal locations.     

Investigation of the reciprocal relationship between the expression of two gap junction connexin proteins, connexin46 and connexin43

Connexins are the transmembrane proteins that form gap junctions between adjacent cells. The function of the diverse connexin molecules is related to their tissue-specific expression and highly dynamic turnover. Although multiple connexins have been previously reported to compensate for each other's functions, little is known about how connexins influence their own expression or intracellular regulation. Of the three vertebrate lens connexins, two connexins, connexin43 (Cx43) and connexin46 (Cx46), show reciprocal expression and subsequent function in the lens and in lens cell culture. In this study, we investigate the reciprocal relationship between the expression of Cx43 and Cx46. Forced depletion of Cx43, by tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate, is associated with an up-regulation of Cx46 at both the protein and message level in human lens epithelial cells. An siRNA-mediated down-regulation of Cx43 results in an increase in the level of Cx46 protein, suggesting endogenous Cx43 is involved in the regulation of endogenous Cx46 turnover. Overexpression of Cx46, in turn, induces the depletion of Cx43 in rabbit lens epithelial cells. Cx46-induced Cx43 degradation is likely mediated by the ubiquitin-proteasome pathway, as (i) treatment with proteasome inhibitors restores the Cx43 protein level and (ii) there is an increase in Cx43 ubiquitin conjugation in Cx46-overexpressing cells. We also present data that shows that the C-terminal intracellular tail domain of Cx46 is essential to induce degradation of Cx43. Therefore, our study shows that Cx43 and Cx46 have novel functions in regulating each other's expression and turnover in a reciprocal manner in addition to their conventional roles as gap junction proteins in lens cells.