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J G Conboy

Publications and source records attributed to J G Conboy.

33 records · Page 2Linked to original sources
PubMed1995

Mechanochemistry of protein 4.1's spectrin-actin-binding domain: ternary complex interactions, membrane binding, network integration, structural strengthening.

Mechanical strength of the red cell membrane is dependent on ternary interactions among the skeletal proteins, spectrin, actin, and protein 4.1. Protein 4.1's spectrin-actin-binding (SAB) domain is specified by an alternatively spliced exon encoding 21 amino acid (aa) and a constitutive exon encoding 59 aa. A series of truncated SAB peptides were engineered to define the sequences involved in spectrin-actin interactions, and also membrane strength. Analysis of in vitro supramolecular assemblies showed that gelation activity of SAB peptides correlates with their ability to recruit a critical amount of spectrin into the complex to cross-link actin filaments. Also, several SAB peptides appeared to exhibit a weak, cooperative actin-binding activity which mapped to the first 26 residues of the constitutive 59 aa. Fluorescence-imaged microdeformation was used to show SAB peptide integration into the elastic skeletal network of spectrin, actin, and protein 4.1. In situ membrane-binding and membrane-strengthening abilities of the SAB peptides correlated with their in vitro gelation activity. The findings imply that sites for strong spectrin binding include both the alternative 21-aa cassette and a conserved region near the middle of the 59 aa. However, it is shown that only weak SAB affinity is necessary for physiologically relevant action. Alternatively spliced exons can thus translate into strong modulation of specific protein interactions, economizing protein function in the cell without, in and of themselves, imparting unique function.

Actins↗
PubMed1994

Mouse erythroid cells express multiple putative RNA helicase genes exhibiting high sequence conservation from yeast to mammals.

RNA secondary structure is a critical determinant of RNA function in ribosome assembly, pre-mRNA splicing, mRNA translation and RNA stability. The 'DEAD/H' family of putative RNA helicases may help regulate these processes by utilizing intrinsic RNA-dependent ATPase activity to catalyze conformational changes in RNA secondary structure. To investigate the repertoire of DEAD/H box proteins expressed in mammals, we used PCR techniques to clone from mouse erythroleukemia (MEL) cells three new DEAD box cDNAs with high similarity to known yeast (Saccharomyces cerevisiae) genes. mDEAD2 and mDEAD3 (mouse DEAD box proteins) are > 95% identical to mouse PL10 but exhibit differential tissue-specific expression patterns; mDEAD2 and mDEAD3 are also approx. 70% identical (at the aa level) to yeast DED1 and DBP1 proteins. Members of this DEAD box subclass contain C-terminal domains with high content of Arg, Ser, Gly and Phe, reminiscent of the RS domain in several Drosophila and mammalian splicing factors. mDEAD5 belongs to a second class related to translation initiation factors from yeast (TIF1/TIF2) and mammals (eIF-4A); this class contains a novel conserved peptide motif not found in other DEAD box proteins. Northern blotting shows that mDEAD5 is differentially expressed in testis vs. somatic tissues. Thus, mouse erythroid cells produce two highly conserved families of putative RNA helicases likely to play important roles in RNA metabolism and gene expression.

Amino Acid Sequence↗
PubMed1993

Mechanochemistry of the alternatively spliced spectrin-actin binding domain in membrane skeletal protein 4.1.

Protein 4.1's interaction with the erythroid skeletal proteins spectrin and actin and its essential role in regulating membrane strength are both attributable to expression of an alternatively spliced 63-nucleotide exon. The corresponding 21-amino acid (21-aa) cassette is within the previously identified spectrin-actin binding domain (10 kDa molecular mass) of erythroid protein 4.1. This cassette is absent, however, in several isoforms that are generated by tissue- and development-specific RNA splicing. Four isoforms of the 10-kDa domain were constructed for comparative assessment of functions particularly relevant to red cells. In vitro translated isoforms containing the 21-aa cassette, denoted 10k21 and 10k19,21, were able to bind spectrin, stabilize spectrin-actin complexes, and associate with red cell membrane. Isoforms replacing or lacking the 21-aa cassette, 10k19 and 10k0, did not function in these assays. A bacterially expressed fusion protein with glutathione-S-transferase, designated GST-10k21, congealed spectrin-actin into a network in vitro as found with purified protein 4.1. Additionally, incorporation of GST-10k21 into mechanically weak, 4.1-deficient membranes increased mechanical strength of these membranes to normal. GST-10k19 did not function in these assays. These results show that the 21-aa sequence in protein 4.1 is critical to mechanical integrity of the red cell membrane. These results also allow the role of protein 4.1 in membrane mechanics to be interpreted primarily in terms of its spectrin-actin binding function. Alternatively expressed sequences within the 10-kDa domain of nonerythroid protein 4.1 are suggested to have different, yet to be defined functions.

Actins↗
PubMed1993

An isoform-specific mutation in the protein 4.1 gene results in hereditary elliptocytosis and complete deficiency of protein 4.1 in erythrocytes but not in nonerythroid cells.

Multiple protein 4.1 isoforms are expressed in a variety of tissues through complex alternative pre-mRNA splicing events, one function of which is to regulate use of two alternative translation initiation signals. Late erythroid cells express mainly the downstream initiation site for synthesis of prototypical 80-kD isoforms; nonerythroid cells in addition use an upstream site to encode higher molecular mass isoform(s). In this study, we examined the effects of a 5' gene rearrangement in a family with hereditary elliptocytosis and complete deficiency of erythrocyte 4.1 protein on 4.1 isoform expression in erythroid vs. nonerythroid cells. Patient 4.1 mRNAs from reticulocytes, fibroblasts, and B lymphocytes were amplified by reverse transcriptase/polymerase chain reaction techniques and shown to exhibit a 318-nucleotide deletion that encompasses the downstream AUG, but leaves intact the upstream AUG. Immunoblot analysis revealed a total deficiency of 4.1 in patient red cells and a selective deficiency of 80-kD isoform(s) but not high molecular weight 4.1 in patient nonerythroid cells. Thus, the 4.1 gene mutation in this family produces an isoform-specific deficiency that is manifested clinically in tissue-specific fashion, such that red cells are affected but other cell types are unaffected because of tissue-specific differences in RNA splicing and translation initiation.

B-Lymphocytes↗
PubMed1992

Human erythroid 5-aminolevulinate synthase. Gene structure and species-specific differences in alternative RNA splicing.

Erythroid 5-aminolevulinate synthase (ALAS) is expressed exclusively in differentiating erythroid cells as the principal isoform of the enzyme to catalyze the first step of the heme biosynthetic pathway. The human gene encoding this isozyme was isolated from a cosmid library, and its structure was characterized with restriction mapping followed by sequencing of fragments. The gene is 22 kilobases long and has 11 exons. Exon 2 encodes the N-terminal signal sequence required for mitochondrial import, exons 3 and 4 encode a variable portion of the N-terminal end, and exons 5-11 the highly conserved C-terminal portion of the mature protein, respectively. Enzymatic amplification of human reticulocyte RNA using PCR techniques revealed two erythroid ALAS mRNA transcripts predicted to encode both the prototypical 64-kDa isoform as well as a novel smaller isoform with a deletion of 37 amino acids near the N terminus. The two mRNA isoforms are generated by alternative splicing of exon 4 and are expressed in fetal erythroid cells as well as at all stages of erythroid development tested, so that there is no evidence of differentiation-specific regulation of exon 4 splicing. However, striking species-specific differences were observed in that alternative splicing of exon 4 was found in man but not dog or mouse; also, the previously described alternative splicing within exon 3 in mouse was not observed in man. This transcript heterogeneity suggests the existence of erythroid ALAS protein isoforms with potentially distinct functional or regulatory roles. The occurrence of species-specific splicing in the least conserved region of the enzyme may reflect another mechanism of gene evolution in eukaryotes.

5-Aminolevulinate Synthetase↗
PubMed1991

Hereditary elliptocytosis due to both qualitative and quantitative defects in membrane skeletal protein 4.1.

Protein 4.1 is an important structural component of the membrane skeleton that helps determine erythrocyte morphology and membrane mechanical properties. In a previous study we identified a case of human hereditary elliptocytosis (HE) in which decreased membrane mechanical stability was due to deletion of 80 amino acids encompassing the entire 10-Kd spectrin-actin binding domain. A portion of this domain (21 amino acids) is encoded by an alternatively spliced exon that is expressed in late but not early erythroid cells. We now report a case of canine HE in which the abnormal phenotype is caused by failure to express this alternative peptide in the mature red blood cell (RBC) membrane skeleton, in conjunction with quantitative deficiency of protein 4.1. Western blotting of RBC membranes from a dog with HE showed a truncated protein 4.1 that did not react with antibodies directed against the alternative peptide. In addition, sequencing of cloned reticulocyte protein 4.1 cDNA showed a precise deletion of 63 nucleotides comprising this exon. Normal dog reticulocytes did express this exon. Expression of this 21 amino acid peptide during erythroid maturation is therefore essential for proper assembly of a mechanically competent membrane skeleton, because RBCs lacking this peptide have unstable membranes.

Amino Acid Sequence↗
PubMed1991

Tissue- and development-specific alternative RNA splicing regulates expression of multiple isoforms of erythroid membrane protein 4.1.

Protein 4.1, a multifunctional structural protein originally described as an 80-kDa component of the erythroid membrane skeleton, exhibits tissue- and development-specific heterogeneity in molecular weight, subcellular localization, and primary amino acid sequence. Earlier reports suggested that some of this impressive heterogeneity is generated by alternative RNA splicing (Conboy, J. G., Chan, J., Mohandas, N., and Kan, Y. W. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 9062-9065; Tang, T. K., Leto, T., Marchesi, V. T., and Benz, E. J. (1990) J. Cell Biol. 110, 617-624). We have now completed a systematic analysis of 4.1 mRNA isoforms expressed in erythroid cells, and have generated an "alternative splicing map" which summarizes diagrammatically a multitude of polypeptide isoforms potentially generated by combinatorial splicing of nine alternative exons. Complex 5' splicing events yield mRNA isoforms that may initiate translation at different sites and thus generate elongated or truncated NH2 termini; elongated approximately 135-kDa and prototypical approximately 80-kDa species were detected in both erythrocytes and T-lymphocytes, but in very different ratios. Among the functional domains of 4.1 responsible for interaction with other membrane skeletal elements, four variants of the 10-kDa spectrin-actin-binding region and four variants of the putative 30-kDa glycophorin-binding region are predicted. Developmentally controlled alternative RNA splicing in the spectrin-actin-binding region may help regulate remodeling of membrane architecture and mechanical properties that occur during erythropoiesis.

Amino Acid Sequence↗
PubMed1988

Multiple protein 4.1 isoforms produced by alternative splicing in human erythroid cells.

Protein 4.1 is a multifunctional structural protein located in the erythrocyte membrane skeleton and in many nonerythroid cells. Molecular characterization of cloned protein 4.1 sequences from human reticulocytes has revealed the existence of multiple transcripts of the protein 4.1 gene that may encode a family of closely related protein isoforms. Several independently isolated cDNAs were sequenced and demonstrated to encode four different protein 4.1 species having identical primary sequences, except for the presence or absence of discrete peptides in the 8-kDa spectrin/actin binding domain (21 amino acids) and near the carboxyl terminus (43 and 34 amino acids). The same four protein 4.1 isoforms were detected when reticulocyte protein 4.1 mRNA sequences were reverse transcribed into cDNA and enzymatically amplified in vitro by using protein 4.1-specific oligonucleotide primers and the polymerase chain reaction. The finding of multiple protein 4.1 isoforms raises the possibility that the many binding functions ascribed to protein 4.1 may reside in distinct structural isoforms. Since only a single protein 4.1 gene appears to be expressed in erythrocytes, it is likely that these isoforms are produced by alternative mRNA splicing from a common protein 4.1 pre-mRNA. Multiple RNA splicing pathways are thus operative in the protein 4.1 gene even within a single cell lineage, human erythroid cells.

Amino Acid Sequence↗
PubMed1987

Isolation and characterization of cDNA clones for human erythrocyte beta-spectrin.

Spectrin is an important structural component of the membrane skeleton that underlies and supports the erythrocyte plasma membrane. It is composed of nonidentical alpha (Mr 240,000) and beta (Mr 220,000) subunits, each of which contains multiple homologous 106-amino acid segments. We report here the isolation and characterization of a human erythroid-specific beta-spectrin cDNA clone that encodes parts of the beta-9 through beta-12 repeat segments. This cDNA was used as a hybridization probe to assign the beta-spectrin gene to human chromosome 14 and to begin molecular analysis of the gene and its mRNA transcripts. RNA transfer blot analysis showed that the reticulocyte beta-spectrin mRNA is 7.8 kilobases in length. Southern blot analysis of genomic DNA revealed the presence of restriction fragment length polymorphisms (RFLPs) within the beta-spectrin gene locus. The isolation of human spectrin cDNA probes and the identification of closely linked RFLPs will facilitate analysis of mutant spectrin genes causing congenital hemolytic anemias associated with quantitative and qualitative spectrin abnormalities.

Amino Acid Sequence↗
PubMed1981

Pre-ornithine transcarbamylase. Properties of the cytoplasmic precursor of a mitochondrial matrix enzyme.

Biogenesis of the mitochondrial matrix enzyme, ornithine transcarbamylase, has been shown to begin with synthesis on cytoplasmic ribosomes of a precursor, designated pre-ornithine transcarbamylase, which is approximately 4000 daltons larger than its corresponding mitochondrial subunit, followed by post-translational uptake and proteolytic processing of the precursor to its mature counterpart by mitochondria. We now report initial studies on the structure and properties of preornithine transcarbamylase. When this precursor is labeled at the NH2 terminus with N-formyl[35S]methionine and processed by mitochondria, no label is recovered with the mature subunit. This demonstrates that the amino acid extension which is characteristic of the precursor and which is removed during mitochondrial processing is NH2-terminal. This NH2-terminal extension is found intact in two peptides produced by limited proteolysis of the labeled precursor. Moreover, this amino acid extension modifies the behavior of the precursor during immunoprecipitation in the presence of ionic detergents and plays a critical role in facilitating uptake of the precursor by mitochondria.

Animals↗
PubMed1981

Posttranslational uptake and processing of in vitro synthesized ornithine transcarbamoylase precursor by isolated rat liver mitochondria.

The mitochondrial matrix enzyme ornithine transcarbamoylase (OTCase; ornithine carbamoyltransferase; carbamoylphosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3) is encoded by a nuclear gene on the X chromosome, synthesized on cytoplasmic ribosomes, and translocated across both mitochondrial membranes. Using specific immunoprecipitation, we presented evidence previously that the primary in vitro translation product of OTCase in rat liver is a polypeptide about 4000 daltons larger than the "mature" OTCase augment subunit purified from homologous mitochondria. In this report we augment the immunological identification of this cell-free translation product (pOTCase) with structural information and show, by electrophoresis of proteolysis products, that pOTCase is structurally similar to mitochondrial OTCase. Moreover, we now demonstrate that, when pOTCase is incubated posttranslationally with isolated rat liver mitochondria, it is converted to the size of mature OTCase and is sequestered within the mitochondria in such a way that it becomes resistant to externally added proteases. Such posttranslational processing is catalyzed specifically by the mitochondrial fraction of rat liver cells and is dependent both on the duration of incubation with mitochondria and on the amount of mitochondrial protein added. We conclude that pOTCase is indeed the bona fide precursor of mitochondrial OTCase and that use of this simplified cell-free system will facilitate analysis of OTCase biogenesis at both the cellular and the molecular level.

Animals↗
PubMed1979

In vitro synthesis of a putative precursor of mitochondrial ornithine transcarbamoylase.

Ornithine transcarbamoylase (OTCase; ornithine carbamoyltransferase; carbamoyl phosphate:L-ornithine carbamoyltransferase, EC 2.1.3.3), a major mitochondrial matrix enzyme in ureotelic animals, is synthesized on cytoplasmic ribosomes and translocated across both mitochondrial membranes to the matrix. In an attempt to identify the primary translation product (or an early intermediate) that is the substrate for this transport process, we translated rat liver polysomal RNA in vitro by using the rabbit reticulocyte lysate system. Immunoprecipitation of the [35S]methionine-labeled translation mixture was performed by using monospecific OTCase antiserum and the immunoadsorbent Staphylococcus aureus. Approximately 0.3% of total trichloroacetic acid-insoluble 35S-labeled material was specifically precipitated. Analysis of the precipitate by fluorography of a dried sodium dodecyl sulfate/polyacrylamide gel showed a single major translation product whose mobility corresponded to a polypeptide of 43,000 daltons, a value approximately 4000 daltons greater than that noted for the "mature" OTCase subunit isolated from rat liver. This translation product was not precipitated by preimmune rabbit serum, and excess unlabeled mature OTCase competed with it for interaction with OTCase antiserum. These results suggested that rat liver OTCase, like a number of other cytoplasmically synthesized organellar proteins, is initially made as a larger precursor that contains an amino acid sequence necessary to confer on OTCase its transport properties. The potential application of these findings to the study of inherited complete OTCase deficiency in humans is discussed.

Animals↗