Mammalian LIN-7 PDZ proteins associate with beta-catenin at the cell-cell junctions of epithelia and neurons.

The heterotrimeric PDZ complex containing LIN-2, LIN-7 and LIN-10 is known to be involved in the organization of epithelial and neuronal junctions in Caenorhabditis elegans and mammals. We report here that mammalian LIN-7 PDZ proteins form a complex with cadherin and beta-catenin in epithelia and neurons. The association of LIN-7 with cadherin and beta-catenin is Ca(2+) dependent and is mediated by the direct binding of LIN-7 to the C-terminal PDZ target sequence of beta-catenin, as demonstrated by means of co-immunoprecipitation experiments and in vitro binding assays with the recombinant glutathione S-transferase:LIN-7A. The presence of beta-catenin at the junction is required in order to relocate LIN-7 from the cytosol to cadherin-mediated adhesions, thus indicating that LIN-7 junctional recruitment is beta-catenin dependent and that one functional role of the binding is to localize LIN-7. Moreover, when LIN-7 is present at the beta-catenin-containing junctions, it determines the accumulation of binding partners, thus suggesting the mechanism by which beta-catenin mediates the organization of the junctional domain.

The GLT-1 and GLAST glutamate transporters are expressed on morphologically distinct astrocytes and regulated by neuronal activity in primary hippocampal cocultures.

The GLT-1 and GLAST astroglial transporters are the glutamate transporters mainly involved in maintaining physiological extracellular glutamate concentrations. Defects in neurotransmitter glutamate transport may represent an important component of glutamate-induced neurodegenerative disorders (such as amyotrophic lateral sclerosis) and CNS insults (ischemia and epilepsy). We characterized the protein expression of GLT-1 and GLAST in primary astrocyte-neuron cocultures derived from rat hippocampal tissues during neuron differentiation/maturation. GLT-1 and GLAST are expressed by morphologically distinct glial fibrillary acidic protein-positive astrocytes, and their expression correlates with the status of neuron differentiation/maturation and activity. Up-regulation of the transporters paralleled the content of the synaptophysin synaptic vesicle marker p38, and down-regulation was a consequence of glutamate-induced neuronal death or the reduction of synaptic activity. Finally, soluble factors in neuronal-conditioned media prevented the down-regulation of the GLT-1 and GLAST proteins. Although other mechanisms may participate in regulating GLT-1 and GLAST in the CNS, our data indicate that soluble factors dependent on neuronal activity play a major regulating role in hippocampal cocultures.

PDZ-mediated interactions retain the epithelial GABA transporter on the basolateral surface of polarized epithelial cells.

The PDZ target motifs located in the C-terminal end of many receptors and ion channels mediate protein-protein interactions by binding to specific PDZ-containing proteins. These interactions are involved in the localization of surface proteins on specialized membrane domains of neuronal and epithelial cells. However, the molecular mechanism responsible for this PDZ protein-dependent polarized localization is still unclear. This study first demonstrated that the epithelial gamma-aminobutyric acid (GABA) transporter (BGT-1) contains a PDZ target motif that mediates the interaction with the PDZ protein LIN-7 in Madin-Darby canine kidney (MDCK) cells, and then investigated the role of this interaction in the basolateral localization of the transporter. It was found that although the transporters from which the PDZ target motif was deleted were still targeted to the basolateral surface, they were not retained but internalized in an endosomal recycling compartment. Furthermore, an interfering BGT peptide determined the intracellular relocation of the native transporter. These data indicate that interactions with PDZ proteins determine the polarized surface localization of target proteins by means of retention and not targeting mechanisms. PDZ proteins may, therefore, act as a sort of membrane protein sorting machinery which, by recognizing retention signals (the PDZ target sequences), prevents protein internalization.

Sorting of two polytopic proteins, the gamma-aminobutyric acid and betaine transporters, in polarized epithelial cells.

The gamma-aminobutyric acid transporter (GAT-1) isoform of the gamma-aminobutyric acid and the betaine (BGT) transporters exhibit distinct apical and basolateral distributions when introduced into Madin-Darby canine kidney cells (Pietrini, G., Suh, Y. J., Edelman, L., Rudnick, G., and Caplan, M. J. (1994) J. Biol. Chem. 269, 4668-4674). We have investigated the presence of sorting signals in their COOH-terminal cytosolic domains by expression in Madin-Darby canine kidney cells of mutated and chimeric transporters. Whereas truncated GAT-1 (DeltaC-GAT) maintained the original functional activity and apical localization, either the removal (DeltaC-myc BGT) or the substitution (BGS chimera) of the cytosolic tail of BGT generated proteins that accumulated in the endoplasmic reticulum. Moreover, we have found that the cytosolic tail of BGT redirected apical proteins, the polytopic GAT-1 (GBS chimera) and the monotopic human nerve growth factor receptor, to the basolateral surface. These results suggest the presence of basolateral sorting information in the cytosolic tail of BGT. We have further shown that information necessary for the exit of BGT from the endoplasmic reticulum and for the basolateral localization of the GBS chimera is contained in a short segment, rich in basic residues, within the cytosolic tail of BGT.

A role for N-myristoylation in protein targeting: NADH-cytochrome b5 reductase requires myristic acid for association with outer mitochondrial but not ER membranes.

N-myristoylation is a cotranslational modification involved in protein-protein interactions as well as in anchoring polypeptides to phospholipid bilayers; however, its role in targeting proteins to specific subcellular compartments has not been clearly defined. The mammalian myristoylated flavoenzyme NADH-cytochrome b5 reductase is integrated into ER and mitochondrial outer membranes via an anchor containing a stretch of 14 uncharged amino acids downstream to the NH2-terminal myristoylate glycine. Since previous studies suggested that the anchoring function could be adequately carried out by the 14 uncharged residues, we investigated a possible role for myristic acid in reductase targeting. The wild type (wt) and a nonmyristoylatable reductase mutant (gly2-->ala) were stably expressed in MDCK cells, and their localization was investigated by immunofluorescence, immuno-EM, and cell fractionation. By all three techniques, the wt protein localized to ER and mitochondria, while the nonmyristoylated mutant was found only on ER membranes. Pulse-chase experiments indicated that this altered steady state distribution was due to the mutant's inability to target to mitochondria, and not to its enhanced instability in that location. Both wt and mutant reductase were resistant to Na2CO3 extraction and partitioned into the detergent phase after treatment of a membrane fraction with Triton X-114, demonstrating that myristic acid is not required for tight anchoring of reductase to membranes. Our results indicate that myristoylated reductase localizes to ER and mitochondria by different mechanisms, and reveal a novel role for myristic acid in protein targeting.

The axonal gamma-aminobutyric acid transporter GAT-1 is sorted to the apical membranes of polarized epithelial cells.

Recent studies suggest that epithelial cells and neurons employ similar mechanisms to target proteins to the distinct subdomains of their polarized cell surface membranes. We have examined the sorting behavior of the neuronal gamma-aminobutyric acid (GABA) transporter GAT-1 expressed by transfection in the polarized epithelial Madin-Darby canine kidney (MDCK) cell line. We find that the GABA transporters endogenously expressed by polarized hippocampal neurons in culture are restricted to axonal plasma membranes. In transfected MDCK cells, the GABA transporter is found to be localized primarily to the apical cell surface when examined by immunocytochemistry, cell surface biotinylation, and transport assay. MDCK cells exposed to hyperosmotic stress express a close relative of GAT-1, the betaine transporter (BGT-1). We find that BGT-1 expressed by transfection in MDCK cells accumulates predominantly at the basolateral cell surface. These observations suggest that the sorting information required for axonal targeting may be similar to that which mediates apical localization in epithelia. Furthermore, it would appear that despite their high degree of homology, the BGT-1 and GAT-1 transporters manifest sorting signals which specify their targeting to distinct cell surface domains.

NADH-cytochrome b5 reductase and cytochrome b5 isoforms as models for the study of post-translational targeting to the endoplasmic reticulum.

Cytochrome b5 and NADH-cytochrome b5 reductase are integral membrane proteins with cytosolic active domains and short membrane anchors, which are inserted post-translationally into their target membranes. Both are produced as different isoforms, with different localizations, in mammalian cells. In the rat, the reductase gene generates two transcripts by an alternative promoter mechanism: a ubiquitous mRNA coding for the myristylated membrane-bound form, and an erythroid mRNA which generates both the soluble form and a nonmyristylated membrane-binding form. The available evidence indicates that the ubiquitous myristylated form binds to the cytosolic face of both outer mitochondrial membranes and ER. In contrast, two genes code for two homologous forms of cytochrome b5, one of which is found on outer mitochondrial membranes, the other on the ER. The gene specifying the ER form probably also generates an erythroid-specific mRNA by alternative splicing, which codes for soluble cytochrome b5. Possible molecular mechanisms responsible for the observed localizations of these different enzyme isoforms are discussed.

Sorting of ion transport proteins in polarized cells.

The plasma membranes of polarized epithelial cells and neurons express distinct populations of ion transport proteins in their differentiated plasma membrane domains. In order to understand the mechanisms responsible for this polarity it will be necessary to elucidate the nature both of sorting signals and of the cellular machinery which recognizes and acts upon them. In our efforts to study sorting signals we have taken advantage of two closely related families of ion transport proteins whose members are concentrated in different epithelial plasmalemmal domains. The H+,K(+)-ATPase and the Na+,K(+)-ATPase are closely related members of the E1-E2 family of ion transporting ATPases. Despite their high degree of structural and functional homology, they are concentrated on different surfaces of polarized epithelial cells and pursue distinct routes to the cell surface in cells which manifest a regulated delivery pathway. We have transfected cDNAs encoding these pumps' subunit polypeptides, as well as chimeras derived from them, in a variety of epithelial and non-epithelial cell types. Our observations suggest that these pumps encode multiple sorting signals whose relative importance and functions may depend upon the cell type in which they are expressed. Recent evidence suggests that the sorting mechanisms employed by epithelial cells may be similar to those which operate in neurons. We have examined this proposition by studying the distributions of ion pumps and neurotransmitter re-uptake co-transporters expressed endogenously and by transfection in neurons and epithelial cells, respectively. We find that one of the classes of proteins we studied obeys the correlation between neuronal and epithelial sorting while another does not.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoforms of the Na,K-ATPase are present in both axons and dendrites of hippocampal neurons in culture.

The distributions of isoforms of the Na,K-ATPase alpha subunit were determined in mature cultured hippocampal neurons and in a polarized epithelial cell line. We find that hippocampal neurons express the alpha 1 and alpha 3 isoforms in the membranes of both axons and dendrites. In contrast the alpha 1 and alpha 3 proteins are exclusively basolateral when expressed endogenously or by stable transfection in renal epithelial cells. These data suggest that epithelial cells and hippocampal neurons localize these proteins by different mechanisms. These observations contrast with those made for the vesicular stomatitis virus and the influenza glycoproteins, which are polarized in both epithelial and neuronal cells.

A single mRNA, transcribed from an alternative, erythroid-specific, promoter, codes for two non-myristylated forms of NADH-cytochrome b5 reductase.

Two forms of NADH-cytochrome b5 reductase are produced from one gene: a myristylated membrane-bound enzyme, expressed in all tissues, and a soluble, erythrocyte-specific, isoform. The two forms are identical in a large cytoplasmic domain (Mr approximately 30,000) and differ at the NH2-terminus, which, in the membrane form, is responsible for binding to the bilayer, and which contains the myristylation consensus sequence and an additional 14 uncharged amino acids. To investigate how the two differently targeted forms of the reductase are produced, we cloned a reductase transcript from reticulocytes, and studied its relationship to the previously cloned liver cDNA. The reticulocyte transcript differs from the liver transcript in the 5' non-coding portion and at the beginning of the coding portion, where the seven codons specifying the myristoylation consensus are replaced by a reticulocyte-specific sequence which codes for 13 non-charged amino acids. Analysis of genomic reductase clones indicated that the ubiquitous transcript is generated from an upstream "housekeeping" type promoter, while the reticulocyte transcript originates from a downstream, erythroid-specific, promoter. In vitro translation of the reticulocyte-specific mRNA generated two products: a minor one originating from the first AUG, and a major one starting from a downstream AUG, as indicated by mutational analysis. Both the AUGs used as initiation codons were in an unfavorable sequence context. The major, lower relative molecular mass product behaved as a soluble protein, while the NH2-terminally extended minor product interacted with microsomes in vitro. The generation of soluble reductase from a downstream AUG was confirmed in vivo, in Xenopus oocytes. Thus, differently localized products, with respect both to tissues and to subcellular compartments, are generated from the same gene by a combination of transcriptional and translational mechanisms.

Two transcripts encode rat cytochrome b5 reductase.

A cDNA expression library in lambda gt11 was screened with affinity-purified polyclonal anti-rat cytochrome b5 reductase antibodies. One positive clone out of 450,000 clones was isolated and found to be incomplete. This clone was used to rescreen the library, and a second, overlapping clone that contained the entire coding sequence was isolated. RNA gel blots showed that the two overlapping clones contained approximately 90% of the reductase mRNA sequence. Sequencing data showed (i) that rat reductase has a 93% sequence similarity with bovine and human reductase and (ii) that reductase is not synthesized as a high molecular weight precursor. Results of Southern blot analysis were consistent with the hypothesis that a single gene codes for the soluble and membrane-bound (microsomal and mitochondrial) forms of the reductase, present in erythrocytes and liver, respectively. The cloned cDNA was used to study reductase transcripts in liver and reticulocytes. Two antisense RNA probes that together covered the entire coding region and part of the noncoding region of reductase mRNA were used in RNase A protection experiments. These probes detected only one transcript in liver, suggesting that endoplasmic reticulum and mitochondrial reductase are translated from the same mRNA. In contrast, two transcripts were detected in reticulocytes, one of which mismatched the liver probe approximately 30 nucleotides downstream from the initiation codon. Since the soluble and membrane form of the reductase are known to differ at the N terminus, we suggest that this second transcript encodes soluble reductase.

Concentration of NADH-cytochrome b5 reductase in erythrocytes of normal and methemoglobinemic individuals measured with a quantitative radioimmunoblotting assay.

The activity of NADH-cytochrome b5 reductase (NADH-methemoglobin reductase) is generally reduced in red cells of patients with recessive hereditary methemoglobinemia. To determine whether this lower activity is due to reduced concentration of an enzyme with normal catalytic properties or to reduced activity of an enzyme present at normal concentration, we measured erythrocyte reductase concentrations with a quantitative radioimmunoblotting method, using affinity-purified polyclonal antibodies against rat liver microsomal reductase as probe. In five patients with the "mild" form of recessive hereditary methemoglobinemia, in which the activity of erythrocyte reductase was 4-13% of controls, concentrations of the enzyme, measured as antigen, were also reduced to 7-20% of the control values. The concentration of membrane-bound reductase antigen, measured in the ghost fraction, was similarly reduced. Thus, in these patients, the reductase deficit is caused mainly by a reduction in NADH-cytochrome b5 reductase concentration, although altered catalytic properties of the enzyme may also contribute to the reduced enzyme activity.

Three translationally regulated mRNAs are stored in the cytoplasm of clam oocytes.

In situ hybridization was used to examine the spatial distributions of three translationally controlled maternal RNAs in oocytes and two-cell embryos of the clam Spisula. 3H-labeled single-stranded RNA probes were generated from SP6 recombinant clones containing DNA inserts encoding portions of histone H3 (the DNA sequence which is presented here), cyclin A, and the small subunit of ribonucleotide reductase. Hybridization of these probes to oocytes, in which the mRNAs are translationally inactive, shows that these mRNAs are stored in the cytoplasm. There is no evidence for sequestration of any of the RNAs within the nucleus or any other discrete structure. Instead they appear to be evenly distributed throughout the cytoplasm.

Distribution of the integral membrane protein NADH-cytochrome b5 reductase in rat liver cells, studied with a quantitative radioimmunoblotting assay.

The intracellular localization of the post-translationally inserted integral membrane protein, NADH-cytochrome b5 reductase, was investigated, using a quantitative radioimmunoblotting method to determine its concentration in rat liver subcellular fractions. Subcellular fractions enriched in rough or smooth microsomes, Golgi, lysosomes, plasma membrane and mitochondrial inner or outer membranes were characterized by marker enzyme analysis and electron microscopy. Reductase levels were determined both with the NADH-cytochrome c reductase activity assay, and by radioimmunoblotting, and the results of the two methods were compared. When measured as antigen, the reductase was relatively less concentrated in microsomal subfractions, and more concentrated in fractions containing outer mitochondrial membranes, lysosomes and plasma membrane than when measured as enzyme activity. Rough and smooth microsomes had 4-5-fold lower concentrations, on a phospholipid basis than did mitochondrial outer membranes. Fractions containing Golgi, lysosomes and plasma membrane had approximately 14-, approximately 16, and approximately 9-fold lower concentrations of antigen than did mitochondrial outer membranes, respectively, and much of the antigen in these fractions could be accounted for by cross-contamination. No enzyme activity or antigen was detected in mitochondrial inner membranes. Our results indicate that the enzyme activity data do not precisely reflect the true enzyme localization, and show an extremely uneven distribution of reductase among different cellular membranes.

Rat erythrocyte NADH-cytochrome b5 reductase. Quantitation and comparison between the membrane-bound and soluble forms using an antibody against the rat liver enzyme.

The subcellular distribution of rat erythrocyte NADH-cytochrome b5 reductase was determined by radioimmunoassay, using a rabbit antibody against the cathepsin D cleaved water-soluble fragment of rat liver microsomal reductase (I-reductase), which is known to be immunologically similar to the red cell enzyme. Erythrocytes contained approximately 30 ng of reductase/mg of protein, of which 90% were recovered in the hemolysate supernatant and 2.3% in the ghost fraction. After concentration by precipitation with 70% saturated (NH4)2SO4, the NADH-cytochrome c reductase activity of the soluble enzyme could be assayed in the presence of cytochrome b5, and was found to be inhibited by anti 1-reductase antibodies. The sodium dodecyl sulfate-polyacrylamide gel electrophoretic mobilities of erythrocyte membrane-associated and soluble reductase of the liver microsomal enzyme and its cathepsin D cleaved hydrophilic fragment (I-reductase) were examined in crude fractions by blotting followed by specific and highly sensitive immunostaining. The intact microsomal enzyme and the two erythrocyte reductases all had similar mobilities and migrated behind 1-reductase. However, the ghost-associated reductase, which was not attributable to contaminating leukocyte or reticulocyte membranes, was distinguishable from the soluble form by two criteria: (i) a lower dependence on exogenous cytochrome b5 in the NADH-cytochrome c reductase assay; and (ii) a larger apparent Mr upon gel filtration in the presence of Triton X-100, presumably because of detergent binding. Considering these results, possible biogenetic relations between membrane-bound and soluble erythrocyte reductase are discussed.

Localization and biosynthesis of NADH-cytochrome b5 reductase, an iontegral membrane protein, in rat liver cells. III. Evidence for the independent insertion and turnover the enzyme in various subcellular compartments.

The biosynthesis and turnover of rat liver NADH-cytochrome b(5) reductase was studied in in vivo pulse-labeling and long-term, double-labeling experiments. Rats under thiopental anesthesia were injected into the portal vein with [(3)H]L-leucine and sacrificed at various times after the injection. NADH-cytochrome b(5) reductase was extracted from liver cell fractions by cathepsin D-catalyzed cleavage and was then immunoadsorbed onto antireductase-bearing affinity columns in the presence of excess unlabeled rat serum. After elution of the enzyme from the columns with a pH-2.2 buffer, the amount of the reductase protein in the samples was determined by radioimmunoassay, and the radioactivity in reductase was determined on SDS polyacrylamide gel reductase bands. The specific radioactivity of the reductase extracted from the homogenate as well as from rough and smooth microsomal, mitochondrial, and Golgi fractions, estimated at the end of the pulse (10 min after the injection) and at various time points thereafter, remained approximately constant over a 6-h period. These data suggest tha tth eenzyme is independently inserted into the various membranes where it is located. Moreover, the specific radioactivity of the mitochondrial reductase was lower than that of the other fractions, suggesting that it turns over at a slower rate. The lower turnover rate of the mitochondrial enzyme was confirmed by long-term, double-labeling experiments carried out according to the technique of Arias et al. (J. Biol. Chem. 244: 3303-3315.). The relevance of these findings in relation to the understanding of membrane biogenesis and turnover is discussed.

Localization and biosynthesis of NADH-cytochrome b5 reductase, an integral membrane protein, in rat liver cells. II. Evidence that a single enzyme accounts for the activity in its various subcellular locations.

NADH-cytochrome b5 reductases of rat liver microsomes, mitochondria, and heavy and light Golgi fractions (GF3 and GF 1+2) were compared by antibody inhibition and competition experiments, by peptide mapping, and by CNBr fragment analysis. The water-soluble portion of the microsomal enzyme, released by lysosomal digestion and purified by a published procedure, was used to raise antibodies in rabbits. Contaminant antimicrosome antibodies were removed from immune sera by immunoadsorption onto the purified antigen, and the F(ab')2 fragments of the pure antireductase antibody thus obtained were found to inhibit the NADH-cytochrome c reductase activity equally well in the four membrane fractions investigated, with similar dose-response relationships. Moreover, the purified water-soluble fragment of microsomal reductase, which by itself is very inefficient in reducing cytochrome c, competed for antibody binding with the membrane-bound enzymes, and therefore prevented the inhibition of their activity not only in microsomes but also in the other fractions. The reductases isolated from detergent-solubilized microsomes, mitochondria, GF3, and GF1+2 by immunoadsorption had identical mobilities in SDS polyacrylamide gels. The corresponding bands were eluted from gels, fragmented with pepsin or CNBr treatment, and the two families of peptides thus obtained were analyzed by two-dimensional mapping and SDS polyacrylamide gel electrophoresis, respectively. Both analyses failed to reveal differences among reductases of the four fractions. These findings support the hypothesis that NADH-cytochrome b5 reductase in its various subcellular locations is molecularly identical.