A Connexin50 Mutant,CX50fs,That Causes Cataracts Is Unstable,but Is Rescued by a Proteasomal Inhibitor

The mechanisms by which mutant connexins lead to disease are diverse, including those of connexin50 (CX50) encoded by the GJA8 gene. We investigated the cellular and functional behavior of CX50fs, a mutant CX50 that has a frameshift after amino acid 255 and causes recessive congenital cataracts. Cellular levels of CX50fs were much lower than those of wild type CX50 in stably transfected HeLa cells. Whereas CX50 localized at distinct gap junction plaques and supported extensive intercellular transfer of Neurobiotin, CX50fs gap junctions were rare, and their support of Neurobiotin transfer was reduced by >90%. After inhibition of new protein synthesis with cycloheximide, CX50fs disappeared much more rapidly than CX50, suggesting increased degradation of the mutant. Treatment of cells with epoxomicin (a proteasomal inhibitor) led to a dramatic increase in CX50fs levels and in the abundance of gap junctions. Epoxomicin treatment also rescued intercellular transfer of Neurobiotin to levels similar to those in cells expressing the wild type protein. Treatment with eeyarestatin I (an inhibitor of p97-dependent protein degradation) resulted in many abundant slowly migrating CX50 and CX50fs bands consistent with polyubiquitination of the proteins. These results demonstrate that the CX50fs mutant is rapidly degraded by endoplasmic reticulum-associated degradation in mammalian cells. This accelerated degradation reduces the abundance of gap junctions and the extent of intercellular communication, potentially explaining the pathogenesis of cataracts linked to this mutant. The efficacy of epoxomicin in restoring function suggests that protease inhibition might have therapeutic value for this and other diseases caused by mutants with similar defects.     

Functional characterization of a GJA1 frameshift mutation causing oculodentodigital dysplasia and palmoplantar keratoderma

A frameshift mutation generated from a dinucleotide deletion (780-781del) in the GJA1 gene encoding Cx43 results in a frameshift yielding 46 aberrant amino acids after residue 259 and a shortened protein of 305 residues compared with the 382 in wild-type Cx43. This frameshift mutant (fs260) causes oculodentodigital dysplasia (ODDD) that includes the added condition of palmoplantar keratoderma. When expressed in a variety of cell lines, the fs260 mutant was typically localized to the endoplasmic reticulum and other intracellular compartments. The fs260 mutant, but not the G138R ODDD-linked Cx43 mutant or a Cx43 mutant truncated at residue 259 (T259), reduced the number of apparent gap junction plaques formed from endogenous Cx43 in normal rat kidney cells or keratinocytes. Interestingly, mutation of a putative FF endoplasmic reticulum retention motif encoded within the 46 aberrant amino acid domain failed to restore efficient assembly of the fs260 mutant into gap junctions. Dual whole cell patch-clamp recording revealed that fs260-expressing N2A cells exerted severely reduced electrical coupling in comparison to wild-type Cx43 or the T259 mutant, whereas single patch capacitance recordings showed that fs260 could also dominantly inhibit the function of wild-type Cx43. Co-expression studies further revealed that the dominant negative effect of fs260 on wild-type Cx43 was dose-dependent, and at a predicted 1:1 expression ratio the fs260 mutant reduced wild-type Cx43-mediated gap junctional conductance by over 60%. These results suggest that the 46 aberrant amino acid residues associated with the frameshift mutant are, at least in part, responsible for the manifestation of palmoplantar keratoderma symptoms.     

Different consequences of cataract-associated mutations at adjacent positions in the first extracellular boundary of connexin50

Gap junction channels, which are made of connexins, are critical for intercellular communication, a function that may be disrupted in a variety of diseases. We studied the consequences of two cataract-associated mutations at adjacent positions at the first extracellular boundary in human connexin50 (Cx50), W45S and G46V. Both of these mutants formed gap junctional plaques when they were expressed in HeLa cells, suggesting that they trafficked to the plasma membrane properly. However, their functional properties differed. Dual two-microelectrode voltage-clamp studies showed that W45S did not form functional intercellular channels in paired Xenopus oocytes or hemichannel currents in single oocytes. When W45S was coexpressed with wild-type Cx50, the mutant acted as a dominant negative inhibitor of wild-type function. In contrast, G46V formed both functional gap junctional channels and hemichannels. G46V exhibited greatly enhanced currents compared with wild-type Cx50 in the presence of physiological calcium concentrations. This increase in hemichannel activity persisted when G46V was coexpressed with wild-type lens connexins, consistent with a dominant gain of hemichannel function for G46V. These data suggest that although these two mutations are in adjacent amino acids, they have very different effects on connexin function and cause disease by different mechanisms: W45S inhibits gap junctional channel function; G46V reduces cell viability by forming open hemichannels.     

SCAM analysis of Panx1 suggests a peculiar pore structure

Vertebrates express two families of gap junction proteins: the well-characterized connexins and the pannexins. In contrast to connexins, pannexins do not appear to form gap junction channels but instead function as unpaired membrane channels. Pannexins have no sequence homology to connexins but are distantly related to the invertebrate gap junction proteins, innexins. Despite the sequence diversity, pannexins and connexins form channels with similar permeability properties and exhibit similar membrane topology, with two extracellular loops, four transmembrane (TM) segments, and cytoplasmic localization of amino and carboxy termini. To test whether the similarities extend to the pore structure of the channels, pannexin 1 (Panx1) was subjected to analysis with the substituted cysteine accessibility method (SCAM). The thiol reagents maleimidobutyryl-biocytin and 2-trimethylammonioethyl-methanethiosulfonate reacted with several cysteines positioned in the external portion of the first TM segment (TM1) and the first extracellular loop. These data suggest that portions of TM1 and the first extracellular loop line the outer part of the pore of Panx1 channels. In this aspect, the pore structures of Panx1 and connexin channels are similar. However, although the inner part of the pore is lined by amino-terminal amino acids in connexin channels, thiol modification was detected in carboxyterminal amino acids in Panx1 channels by SCAM analysis. Thus, it appears that the inner portion of the pores of Panx1 and connexin channels may be distinct.     

Isoprenylation mediates direct protein-protein interactions between hepatitis large delta antigen and hepatitis B virus surface antigen.

Hepatitis delta antigen (HDAg) consists of two protein species of 195 and 214 amino acids, respectively, which are identical in sequence except that the large HDAg has additional 19 amino acids at its C terminus and is prenylated. Previous studies have shown that the large HDAg and the surface antigen of hepatitis B virus (HBsAg) together can form empty hepatitis delta virus (HDV) particles. To understand the molecular mechanism of HDV virion morphogenesis, we investigated the possible direct protein-protein interaction between HDAg and HBsAg. We constructed recombinant baculoviruses expressing the major form of HBsAg and various mutant HDAgs and used these proteins for far-Western protein binding assays. We demonstrated that HBsAg interacted specifically with the large HDAg but not with the small HDAg. Using mutant HDAgs which have defective or aberrant prenylation, we showed that this interaction required isoprenylates on the cysteine residue of the C terminus of the large HDAg. Isoprenylation alone, without the remainder of the C-terminal amino acids of the large HDAg, was insufficient to mediate interaction with HBsAg. This study demonstrates a novel role of prenylates in HDV virion assembly.     

Cytoplasmic Amino Acids within the Membrane Interface Region Influence Connexin Oligomerization

Gap junction channels composed of connexins connect cells, allowing intercellular communication. Their cellular assembly involves a unique quality-control pathway. Some connexins [including connexin43 (Cx43) and Cx46] oligomerize in the trans-Golgi network following export of stabilized monomers from the endoplasmic reticulum (ER). In contrast, other connexins (e.g., Cx32) oligomerize early in the secretory pathway. Amino acids near the cytoplasmic aspect of the third transmembrane domain have previously been shown to determine this difference in assembly sites. Here, we characterized the oligomerization of two connexins expressed prominently in the vasculature, Cx37 and Cx40, using constructs containing a C-terminal dilysine-based ER retention/retrieval signal (HKKSL) or treatment with brefeldin A to block ER vesicle trafficking. Both methods led to intracellular retention of connexins, since the cells lacked gap junction plaques. Retention of Cx40 in the ER prevented it from oligomerizing, comparable to Cx43. By contrast, ER-retained Cx37 was partially oligomerized. Replacement of two amino acids near the third transmembrane domain of Cx43 (L152 and R153) with the corresponding amino acids from Cx37 (M152 and G153) resulted in early oligomerization in the ER. Thus, residues that allow Cx37 to oligomerize early in the secretory pathway could restrict its interactions with coexpressed Cx40 or Cx43 by favoring homomeric oligomerization, providing a structural basis for cells to produce gap junction channels with different connexin composition.     

心肌细胞缝隙连接重塑与心律失常

缝隙连接是相邻心肌细胞间电、化学偶联的通道,亦是心室肌成为功能性合胞体的重要结构.心肌有缝隙连接蛋白(connexin,CX) 40、43与45的表达,心室肌主要表达CX43.CX43形成的缝隙连接大部分呈点状分布于闰盘部位,心肌细胞膜侧面分布极少.心肌缺血-再灌注、肥厚、衰竭、高胆同醇与糖尿病条件下,心肌细胞缝隙连接...     

The N Terminus of Connexin37 Contains an ��-Helix That Is Required for Channel Function

The cytoplasmic N-terminal domain of connexins has been implicated in multiple aspects of gap junction function, including connexin trafficking/assembly and channel gating. A synthetic peptide corresponding to the first 23 amino acids of human connexin37 was prepared, and circular dichroism and nuclear magnetic resonance studies showed that this N-terminal peptide was predominantly α-helical between glycine 5 and glutamate 16. The importance of this structure for localization of the protein at appositional membranes and channel function was tested by expression of site-directed mutants of connexin37 in which amino acids leucine 10 and glutamine 15 were replaced with prolines or alanines. Wild type connexin37 and both substitution mutants localized to appositional membranes between transfected HeLa cells. The proline mutant did not allow intercellular transfer of microinjected neurobiotin; the alanine mutant allowed transfer, but less extensively than wild type connexin37. When expressed alone in Xenopus oocytes, wild type connexin37 produced hemichannel currents, but neither of the double substitution mutants produced detectable currents. The proline mutant (but not the alanine mutant) inhibited co-expressed wild type connexin37. Taken together, our data suggest that the α-helical structure of the connexin37 N terminus may be dispensable for protein localization, but it is required for channel and hemichannel function.Gap junction channels allow intercellular passage of ions and small molecules up to 1000 Da. They are oligomeric assemblies of members of a family of related proteins called connexins (CX)² (reviewed in Ref. 1). Six connexin monomers assemble to form a hemichannel or connexon (Fig. 1, top panel), which, in turn, forms a complete gap junction channel by docking with a hemichannel from an adjacent cell. Based on sequence similarities, connexins have been separated into subfamilies designated by Greek characters (2, 3). The majority of connexins are members of the α- and β-subfamilies. Connexin polypeptides span the plasma membrane four times and have three cytoplasmic regions: the N terminus (NT), a cytoplasmic loop between the second and third transmembrane domains, and the C terminus (Fig. 1, middle panel). Structural studies of gap junctions have revealed that each hemichannel contains a ring of 24 transmembrane spanning helices (4, 5). Most topological models suggest that the NT of α-subfamily connexins contains 23 amino acids (illustrated for connexin37, CX37, in Fig. 1, bottom panel) and that of β-subfamily connexins contains 22 amino acids.Open in a separate windowFIGURE 1.Diagrams depicting the relationships between a gap junction hemichannel (top), the connexin polypeptide (middle), and the amino acid sequence of the CX37 N-terminal domain (bottom). Thick vertical lines represent the boundaries of the plasma membrane; the intracellular and extracellular spaces are indicated. The transmembrane (M1–M4), extracellular (E1 and E2), and cytoplasmic (NT, N terminus; CL, cytoplasmic loop; and CT, C terminus) domains within a connexin are indicated.The importance of the connexin NT has been emphasized by the identification of a number of connexin mutants that cause amino acid substitutions within this region and are linked to diseases including sensorineural deafness (CX26, CX30, and CX31), Charcot-Marie-Tooth disease (CX32), oculodentodigital dysplasia (CX43), and congenital cataracts (CX46 and CX50). Among the disease-linked mutants that have been studied, some show impaired protein trafficking to the cell surface, whereas others traffic properly, but show loss or alterations of channel function (6–16). Heterologous expression of site-directed mutants and chimeric connexins has demonstrated the influence of NT amino acids upon channel properties, including transjunctional voltage (V_j)-dependent gating, unitary conductance, permeability, and sensitivity to regulation by polyamines (17–22). Lagree et al. (23) have provided evidence that the NT influences the compatibility of connexin hetero-oligomerization.The structure of the NT domain of a β-group connexin, Cx26, has been investigated through circular dichroism (CD) and nuclear magnetic resonance (NMR) of a synthetic peptide corresponding to part of the predicted CX26NT (24, 25). Based on their data, Purnick et al. (24) proposed a model for the NT of CX26 with an α-helix extending from position 1 to 10 and a critical bend at positions 11 and 12 that was suggested to act as a “hinge” allowing the first 10 amino acids to swing into the pore and block the channel. Oshima et al. (5) have published structural studies of a “permeability” mutant (M34A) of CX26 (26) showing a density within the pore of the channel that they suggested might represent a bundle of N termini acting as a “plug” to close the channel.We have been studying CX37, an α-group connexin that is expressed in endothelial cells (27), which may be important for development of atherosclerotic disease (28) and that can form large conductance channels and hemichannels (27, 29). We have shown that as much as half the length of the CX37NT can be deleted without affecting formation of gap junction plaques, but a full-length N terminus is required for hemichannel gating and intercellular communication (30). These observations suggested that the CX37NT may have a structure that is required for function. Therefore, the present experiments were designed to determine the structure of the NT of CX37 and the importance of that structure for protein localization and formation of functional channels and hemichannels. Differences between our data and those previously reported in studies of CX26 suggest that the structure of the NT in α-group connexins may differ from that in β-group connexins.     

Affinity purification of a rat-brain junctional protein, connexin 43.

Immunocytochemical investigations have previously shown that antibodies specific for mammal connexins labeled in situ rat and mouse brain gap junctions. However brain gap-junction proteins have neither been identified with certainty, nor purified. By immunoblotting, anti-peptide antibodies directed against rat heart connexin 43 (CX43) detect a major protein of 41 kDa in rat brain homogenates. The specificity of these antibodies made it possible to establish an affinity-chromatography purification procedure of the 41-kDa protein. Purified antibodies specific for the sequence SAEQNRMGQ (residues 314-322) of rat heart CX43 were covalently bound to a protein-A-Sepharose-CL-4B matrix. Rat brain homogenates were recycled through the immunomatrix and the material specifically bound to the matrix was then competitively eluted with the peptide SAEQNRMGQY. Analysis by SDS/PAGE of eluates demonstrated that they contain a 41-kDa protein associated with low amounts of high-molecular-mass proteins. By immunoblotting, these proteins were shown to be specifically recognized by antibodies directed against residues 5-17, 55-56, and 314-322 of rat heart CX43. The NH2-terminal partial sequence for the 41-kDa protein was determined by microsequencing and shown to be similar to alpha 1 connexins. This is the first successful purification of a junctional protein from brain tissue and provides direct evidence that the 41-kDa protein is a CX43 gene product.     

Membrane insertion of gap junction connexins: polytopic channel forming membrane proteins

Connexins, the proteins that form gap junction channels, are polytopic plasma membrane (PM) proteins that traverse the plasma membrane bilayer four times. The insertion of five different connexins into the membrane of the ER was studied by synthesizing connexins in translation- competent cell lysates supplemented with pancreatic ER-derived microsomes, and by expressing connexins in vivo in several eucaryotic cell types. In addition, the subcellular distribution of the connexins was determined. In vitro-synthesis in the presence of microsomes resulted in the signal recognition particle-dependent membrane insertion of the connexins. The membrane insertion of all connexins was accompanied by an efficient proteolytic processing that was dependent on the microsome concentration. Endogenous unprocessed connexins were detectable in the microsomes used, indicating that the pancreatic microsomes serve as a competent recipient in vivo for unprocessed full length connexins. Although oriented with their amino terminus in the cytoplasm, the analysis of the cleavage reaction indicated that an unprecedented processing by signal peptidase resulted in the removal of an amino-terminal portion of the connexins. Variable amounts of similar connexin cleavage products were also identified in the ER membranes of connexin overexpressing cells. The amount generated correlated with the level of protein expression. These results demonstrate that the connexins contain a cryptic signal peptidase cleavage site that can be processed by this enzyme in vitro and in vivo in association with their membrane insertion. Consequently, a specific factor or condition must be required to prevent this aberrant processing of connexins under normal conditions in the cell.     

The connexin 46 mutant (V44M) impairs gap junction function causing congenital cataract

Connexin 46 (Cx46) is important for gap junction channels formation which plays crucial role in the preservation of lens homeostasis and transparency. Previously, we have identified a missense mutation (p.V44M) of Cx46 in a congenital cataract family. This study aims at dissecting the potential pathogenesis of the causative mutant of cataract. Plasmids carrying wild-type (wt) and mutant (V44M) of Cx46 were constructed and expressed in Hela cells respectively. Western blotting and fluorescence microscopy were applied to analyse the expression and subcellular localization of recombinant proteins, respectively. Scrape loading dye transfer experiment was performed to detect the transfer capability of gap junction channels among cells expressed V44M mutant. The results demonstrated that in transfected Hela cells, both wt-Cx46 and Cx46 V44M were localized abundantly in the plasma membrane. No significant difference was found between the protein expressions of the two types of Cx46. The fluorescent localization assay revealed the plaque formation, significantly reduced in the cells expressing Cx46 V44M. Immunoblotting analysis demonstrated that formation of Triton X-100 insoluble complex decreased obviously in mutant Cx46. Additionally, the scrape-loading dye-transfer experiment showed a lower dye diffusion distance of Cx46 V44M cells, which indicates that the gap junction intercellular communication activity was aberrant. Human Cx46 V44M mutant causing cataracts result in abnormally decreased formation of gap junction plaques and impaired gap junction channel function.     

Transforming p21 ras protein: flexibility in the major variable region linking the catalytic and membrane-anchoring domains.

The mammalian p21 ras proteins contain a 20-amino acid region that is highly divergent, in contrast to the strong sequence conservation that is common to other regions of these proteins. This major variable region is located near the C terminus just upstream from a conserved cysteine residue that is required for post-translational processing, membrane localization and transforming activity of the proteins. We have now used the viral oncogene (v-rasH) of Harvey sarcoma virus to study the major variable region by deleting or duplicating parts of the gene. Reducing this region to five amino acids or increasing it to 50 amino acids has relatively little effect on the capacity of the gene to induce morphological transformation of NIH 3T3 cells. Assays of GTP binding, GTPase and autophosphorylating activities of such mutant v-rasH-encoded proteins synthesized in bacteria indicated that the sequences that encode these biochemical activities are located upstream from the major variable region. In the context of transformation, we propose that the region of sequence heterogeneity serves principally to connect the N-terminal catalytic domain with amino acids at the C terminus that are required to anchor the protein in the membrane.     

The zinc finger domain of the archaeal minichromosome maintenance protein is required for helicase activity

The minichromosome maintenance (MCM) proteins, a family of six conserved polypeptides found in all eukaryotes, are essential for DNA replication. The archaeon Methanobacterium thermoautotrophicum Delta H contains a single homologue of MCM with biochemical properties similar to those of the eukaryotic enzyme. The amino acid sequence of the archaeal protein contains a putative zinc-binding domain of the CX(2)CX(n)CX(2)C (C(4)) type. In this study, the roles of the zinc finger domain in MCM function were examined using recombinant wild-type and mutant proteins expressed and purified from Escherichia coli. The protein with a mutation in the zinc motif forms a dodecameric complex similar to the wild-type enzyme. The mutant enzyme, however, is impaired in DNA-dependent ATPase activity and single-stranded DNA binding, and it does not possess helicase activity. These results illustrate the importance of the zinc-binding domain for archaeal MCM function and suggest a role for zinc binding in the eukaryotic MCM complex as well, since four out of the six eukaryotic MCM proteins contain a similar zinc-binding motif.     

Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues

Rat heart and other organs contain mRNA coding for connexin43, a polypeptide homologous to a gap junction protein from liver (connexin32). To provide direct evidence that connexin43 is a cardiac gap junction protein, we raised rabbit antisera directed against synthetic oligopeptides corresponding to two unique regions of its sequence, amino acids 119-142 and 252-271. Both antisera stained the intercalated disc in myocardium by immunofluorescence but did not react with frozen sections of liver. Immunocytochemistry showed anti-connexin43 staining of the cytoplasmic surface of gap junctions in isolated rat heart membranes but no reactivity with isolated liver gap junctions. Both antisera reacted with a 43-kD polypeptide in isolated rat heart membranes but did not react with rat liver gap junctions by Western blot analysis. In contrast, an antiserum to the conserved, possibly extracellular, sequence of amino acids 164-189 in connexin32 reacted with both liver and heart gap junction proteins on Western blots. These findings support a topological model of connexins with unique cytoplasmic domains but conserved transmembrane and extracellular regions. The connexin43-specific antisera were used by Western blots and immunofluorescence to examine the distribution of connexin43. They demonstrated reactivity consistent with gap junctions between ovarian granulosa cells, smooth muscle cells in uterus and other tissues, fibroblasts in cornea and other tissues, lens and corneal epithelial cells, and renal tubular epithelial cells. Staining with the anti-connexin43 antisera was never observed to colocalize with antibodies to other gap junctional proteins (connexin32 or MP70) in the same junctional plaques. Because of limitations in the resolution of the immunofluorescence, however, we were not able to determine whether individual cells ever simultaneously express more than one connexin type.     

Molecular cloning and characterization of a new member of the gap junction gene family, connexin-31.

A new member of the connexin gene family has been identified and designated rat connexin-31 (Cx31) based on its predicted molecular mass of 30,960 daltons. Cx31 is 270 amino acids long and is coded for by a single copy gene. It is expressed as a 1.7-kilobase mRNA that is detected in placenta, Harderian gland, skin, and eye. Cx31 is highly conserved and can be detected in species as distantly related to rat as Xenopus laevis. It exhibits extensive sequence similarity to the previously identified connexins, 58, 50, and 40% amino acid identity to Cx26, Cx32, and Cx43, respectively. When conservation of predicted phosphorylation sites is used to adjust the alignment of Cx31 to other connexins, a unique alignment of three predicted protein kinase C phosphorylation sites near the carboxyl terminus of Cx31 with three sites at the carboxyl terminus of Cx43 is revealed.     

CCN3 (NOV) interacts with connexin43 in C6 glioma cells: possible mechanism of connexin-mediated growth suppression

Many tumor cells exhibit aberrant gap junctional intercellular communication, which can be restored by transfection with connexin genes. We have previously discovered that overexpression of connexin43 (Cx43) in C6 glioma cells not only reduces proliferation but also leads to production of soluble growth-inhibitory factors. We identified that several members of the CCN (Cyr61/connective tissue growth factor/nephroblastoma-overexpressed) family are up-regulated following Cx43 expression, including CCN3 (NOV). We now report evidence for an association between CCN3 and Cx43. Western blot analysis demonstrated that the 48-kDa full-length CCN3 protein was present in the lysate and conditioned medium of growth-suppressed C6-Cx43 cells, as well as primary astrocytes, but not in C6 parental and human glioma cells. Immunocytochemical examination of CCN3 revealed diffuse localization in parental C6 cells, whereas transfection of C6 cells with Cx43 (C6-Cx43) or with a modified Cx43 tagged to green fluorescent protein on its C terminus (Cx43-GFP) resulted in punctate staining, suggesting that CCN3 co-localizes with Cx43 in plaques at the plasma membrane. In cells expressing a C-terminal truncation of Cx43 (Cx43Delta244-382), this co-localization was lost. Glutathione S-transferase pull-down assay and co-immunoprecipitation demonstrated that CCN3 was able to physically interact with Cx43. In contrast, CCN3 was not found to associate with Cx43Delta244-382. Similar experiments revealed that CCN3 did not co-localize or associate with other connexins, including Cx40 or Cx32. Taken together, these data support an interaction of CCN3 with the C terminus of Cx43, which could play an important role in mediating growth control induced by specific gap junction proteins.     

Connexins in mammalian heart function

In heart, the propagation of electrical activity is mediated by intercellular channels, referred to as junctional channels, aggregated into gap junctions and localised between myocytes. These channels consist of structurally related transmembrane proteins, the connexins, three of which (CX43, CX40 and CX45) have been shown to be associated with the myocytes of mammalian heart; a fourth, CX37, was detected exclusively in endothelial cells. In this paper, we review the recent data dealing with the topographical heterogeneity of expression of these connexins in the different cardiac tissues and the unique conductance properties of the channels they form, and attempt to assess the role played by each connexin and the consequences of their multiplicity in the propagation of action potentials.     

Mutation of a conserved threonine in the third transmembrane helix of alpha- and beta-connexins creates a dominant-negative closed gap junction channel

Single site mutations in connexins have provided insights about the influence specific amino acids have on gap junction synthesis, assembly, trafficking, and functionality. We have discovered a single point mutation that eliminates functionality without interfering with gap junction formation. The mutation occurs at a threonine residue located near the cytoplasmic end of the third transmembrane helix. This threonine is strictly conserved among members of the alpha- and beta-connexin subgroups but not the gamma-subgroup. In HeLa cells, connexin43 and connexin26 mutants are synthesized, traffic to the plasma membrane, and make gap junctions with the same overall appearance as wild type. We have isolated connexin26T135A gap junctions both from HeLa cells and baculovirus-infected insect Sf9 cells. By using cryoelectron microscopy and correlation averaging, difference images revealed a small but significant size change within the pore region and a slight rearrangement of the subunits between mutant and wild-type connexons expressed in Sf9 cells. Purified, detergent-solubilized mutant connexons contain both hexameric and partially disassembled structures, although wild-type connexons are almost all hexameric, suggesting that the three-dimensional mutant connexon is unstable. Mammalian cells expressing gap junction plaques composed of either connexin43T154A or connexin26T135A showed an absence of dye coupling. When expressed in Xenopus oocytes, these mutants, as well as a cysteine substitution mutant of connexin50 (connexin50T157C), failed to produce electrical coupling in homotypic and heteromeric pairings with wild type in a dominant-negative effect. This mutant may be useful as a tool for knocking down or knocking out connexin function in vitro or in vivo.     

A stretch of positively charged amino acids at the N terminus of Hansenula polymorpha Pex3p is involved in incorporation of the protein into the peroxisomal membrane

Pex3p is a peroxisomal membrane protein that is essential for peroxisome biogenesis. Here, we show that a conserved stretch of positively charged amino acids (Arg(11)-X-Lys-Lys-Lys(15)) in the N terminus of Hansenula polymorpha Pex3p is involved in incorporation of the protein into its target membrane. Despite the strong conservation, this sequence shows a high degree of redundancy. Substitution of either Arg(11), Lys(13), Lys(14), or Lys(15) with uncharged or negatively charged amino acids did not interfere with Pex3p location and function. However, a mutant Pex3p, carrying negatively charged amino acids at position 13 and 15 (K13E/K15E), caused moderate but significant defects in peroxisome assembly and matrix protein import. Additional changes in the N terminus of Pex3p, e.g. replacing three or four of the positively charged amino acids with negatively charged ones, led to a typical pex3 phenotype, i.e. accumulation of peroxisomal matrix proteins in the cytosol and absence of peroxisomal remnants. Also, in these cases, the mutant Pex3p levels were reduced. Remarkably, mutant Pex3p proteins were mislocalized to mitochondria or the cytosol, depending on the nature of the mutation. Furthermore, in case of reduced amounts of Pex3p, the levels of other peroxisomal membrane proteins, e.g. Pex10p and Pex14p, were also diminished, suggesting that Pex3p maybe involved in the recruitment or stabilization of these proteins (in the membrane).     

Redundancy of signal and anchor functions in the NH2-terminal uncharged region of influenza virus neuraminidase, a class II membrane glycoprotein

Class II membrane glycoproteins share a common topology of the NH2 terminus inside and the COOH terminus outside the cell. Their transport to the cell surface is initiated by the function of a single hydrophobic domain near the NH2 terminus. This functional domain serves both as an uncleaved signal sequence and as a transmembrane anchor. We examined the signal and anchor functions of influenza virus neuraminidase, a prototype class II membrane glycoprotein, by deletion analysis of its long, uncharged amino-terminal region. The results presented here show that the entire stretch of 29 uncharged amino acids (7 to 35) is not required for either a signal sequence or an anchor sequence function. On the basis of translocation and membrane stability data for different mutants, we suggest that the first 20 amino acid residues (7 to 27) are likely to provide the hydrophobic core for these functions and that within this putative subdomain some sequences are more efficient than the other sequences in providing a translocation function. Finally, it appears that neuraminidase and its mutant proteins are translocated with the proper orientation, regardless of the characteristics of the flanking sequences.