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At least 811 records · Page 45Linked to original sources
PubMed1995

Degradation of CD4 induced by human immunodeficiency virus type 1 Vpu protein: a predicted alpha-helix structure in the proximal cytoplasmic region of CD4 contributes to Vpu sensitivity.

The HIV-1-encoded Vpu protein induces a rapid and specific degradation of CD4 molecules in the endoplasmic reticulum (ER). In this study, Vpu-induced degradation of CD4 in the ER was investigated by quantitative immunoprecipitation of CD4 following cotransfection of COS-7 cells with CD4 and Vpu expressors in the presence of brefeldin A, a drug that blocks protein transport from the ER to the Golgi complex. In order to precisely define the sequence(s) or structural element(s) in the CD4 cytoplasmic domain necessary for Vpu-induced degradation, a panel of deletion and substitution mutants in the cytoplasmic domain of CD4 was generated and analyzed. In agreement with previous reports, our deletion analysis indicates that a region encompassing amino acids 411 to 419 (KRLLSEKKT) in the cytoplasmic domain of CD4 was required to confer Vpu sensitivity. However, six specific substitution mutations within this region did not confer CD4 resistance to Vpu, suggesting that neither the amino acid sequence nor the charge of the amino acids in this region was critical to Vpu-induced CD4 degradation. A dileucine motif that is important for internalization of CD4 and Nef-induced CD4 down-regulation was also not required for Vpu-induced CD4 degradation. Interestingly, two substitution mutants (CD4EMKL and CD4MK407,11PP) located in a more proximal cytoplasmic region of CD4 abolished Vpu-induced CD4 degradation. Computer-assisted analysis of the substitution and deletion mutants conferring CD4 resistance to Vpu-induced degradation indicated that these mutations disrupted a putative alpha-helix formed in the proximal cytoplasmic region of CD4. Taken together, these studies strongly suggest that a structural element in the proximal cytoplasmic region of CD4 contributes to Vpu sensitivity.

Amino Acid Sequence↗
PubMed1992

Insect muscle actins differ distinctly from invertebrate and vertebrate cytoplasmic actins.

Invertebrate actins resemble vertebrate cytoplasmic actins, and the distinction between muscle and cytoplasmic actins in invertebrates is not well established as for vertebrate actins. However, Bombyx and Drosophila have actin genes specifically expressed in muscles. To investigate if the distinction between muscle and cytoplasmic actins evidenced by gene expression analysis is related to the sequence of corresponding genes, we compare the sequences of actin genes of these two insect species and of other Metazoa. We find that insect muscle actins form a family of related proteins characterized by about 10 muscle-specific amino acids. Insect muscle actins have clearly diverged from cytoplasmic actins and form a monophyletic group emerging from a cluster of closely related proteins including insect and vertebrate cytoplasmic actins and actins of mollusc, cestode, and nematode. We propose that muscle-specific actin genes have appeared independently at least twice during the evolution of animals: insect muscle actin genes have emerged from an ancestral cytoplasmic actin gene within the arthropod phylum, whereas vertebrate muscle actin genes evolved within the chordate lineage as previously described.

Actins↗
PubMed2002

Molecular diversity of male sterility inducing and male-fertile cytoplasms in the genus Helianthus.

The organisation of mtDNA was investigated for 28 sources of cytoplasmic male sterility (CMS) and a fertile line (normal cytoplasm) of Helianthus annuus by Southern hybridisation. In addition to nine known mitochondrial genes ( atp6, atp9, cob, coxI, coxII, coxIII, 18S, 5S and nd5) three probes for the open reading frames in the rearranged area of PET1, orfH522, orfH708 and orfH873, were used. Genetic similarities of the investigat-ed cytoplasms varied between 0.3 and 1. Cluster analyses using the UPGMA method allowed the distinction of ten mitochondrial (mt) types between the 29 investigated cytoplasms. Most mitochondrial types comprise two or more CMS sources, which could not be further separated, like the PET1-like CMS sources (with the exception of ANO1 and PRR1), or ANN1/ANN2/ANN3, ANN4/ ANN5, ARG3/RIG1, BOL1/EXI1/PEF1/PEP1 and GIG1/ PET2. ANL1, ANL2 and the fertile cytoplasms are also regarded as one mitochondrial type. Unique banding patterns were only observed for ANT1 ( atp6), MAX1 ( atp6, orfH522 and orfH708) and PRR1 ( coxII). However, four of the mitochondrial types showed unique hybridisation signals: ANN4/ANN5 had characteristic bands for atp6 and orfH708, PEF1/PEP1/EXI1/BOL1 for atp6and coxII, and PET2/GIG1 for atp9. The PET1-like cytoplasms all shared the same patterns for orfH522, orfH708and cob (except ANO1). It could be demonstrated that CMS sources, like, e.g., PET2 and PEF1, are different from PET1 in mtDNA organisation and the CMS mechanism. Therefore, these CMS sources represent interesting candidates for the development of new hybrid breeding systems based on new CMS mechanisms.

Journal Article↗
PubMed1992

Cytoplasmic accumulation of a normally mitochondrial malonyl-CoA decarboxylase by the use of an alternate transcription start site.

Malonyl-CoA decarboxylase, a normally mitochondrial enzyme, accumulates in the cytoplasm of specialized glands to cause production of multiple methyl-branched fatty acids. Evidence was presented that a single copy of the decarboxylase gene present in the goose genome codes for both the mitochondrial form found in extremely low amounts in the liver and the cytosolic form found in large amounts in uropygial glands. To elucidate how a single gene encodes both forms, the malonyl-CoA decarboxylase gene and the cDNAs for both the mitochondrial (liver) and the cytoplasmic (gland) species were cloned and sequenced. The decarboxylase gene, found in a 21-kb segment of cloned genomic DNA, is composed of five exons of 0.521, 0.118, 0.156, 0.145, and 1.93 kb interrupted by 6.9, 1.5, 0.45, and 9.3-kb introns. Exon 1 revealed two ATGs in frame 150 bp apart. cDNA for the cytoplasmic form and mitochondrial form showed identical nucleotide sequence, except that the latter was longer than the former. The longest cDNA for the cytoplasmic form of the enzyme extended only 44 bp 5' to the second ATG and the position corresponded to the transcription initiation site of the cytoplasmic form revealed by primer extension and RNase protection. The cDNA for the mitochondrial form isolated from the library extended 19 bp further upstream. Primer extension and RNase protection indicated that transcripts for the mitochondrial form initiated upstream from the first ATG. The N-terminal segment of the open reading frame initiated at the first ATG showed an amphipathic signal sequence appropriate for mitochondrial import. A putative full length mRNA for the mitochondrial form of the enzyme when translated in vitro yielded a 55-kDa primary translation product which was processed by removal of about 5 kDa during uptake into goose liver mitochondria. These results strongly suggest that in most tissues transcription initiates 5'- to the first ATG, generating a transcript that would generate a protein with an N-terminal leader for transport into mitochondria. In the uropygial gland the use of an alternate promoter generates transcripts initiated between the two ATGs and the translation product accumulates in the cytoplasm since it lacks a mitochondrial targeting sequence.

Alternative Splicing↗
PubMed1993

Assessment of cytoplasmic effects on the development of mouse embryonic nuclei transferred to enucleated zygotes.

In this study, cytoplasmic effects on the development of nuclear transplant embryos were examined. In addition, the production of offspring from nuclear transplant embryos was attempted. Nuclei from cleavage-stage embryos were transplanted to enucleated zygotes at different cell cycle stages and with different cytoplasmic volumes. A greater developmental rate to the blastocyst stage was observed in reconstituted late stage zygotes that received nuclei from late 2-cell stage embryos than in early stage zygotes (46.3% vs. 16.9%). A further increase in developmental rate to the blastocyst stage (85.5%) and in cell number was obtained in reconstituted late stage zygotes with reduced cytoplasmic volume. However, developmental potential of nuclei from 4- and 8-cell stage embryos was very limited, although they were transferred to enucleated late stage zygotes with reduced cytoplasm. After the transfer of blastocysts derived from nuclear transplant embryos to recipient females, live young were obtained from reconstituted embryos that received nuclei from late 2-cell stage embryos (28.6%). These results confirm that the development of nuclear transplant embryos can be affected by recipient cell cycle stage and cytoplasmic volume. Furthermore, the nuclei from late 2-cell stage embryos in which activation of the embryonic genome had occurred can be reprogrammed to a certain extent when transplanted into enucleated zygotes, especially late stage zygotes with reduced cytoplasmic content.

Journal Article↗
PubMed1995

Purification and characterization of cytoplasmic dynein of rabbit liver.

Cytoplasmic dynein is a microtubule-dependent motor protein, which plays a role in intracellular transport. However, there have been few studies regarding the role of cytoplasmic dynein in the liver. Purification of cytoplasmic dynein from rabbit liver took advantage of the affinity of microtubule-dependent motor proteins for microtubules. Purified dynein contained heavy chain (450 kDa), intermediate chain (75 kDa), light chains (45-58 kDa) and dynactin (150 kDa). The subunit composition was consistent with previously reported data on brain cytoplasmic dynein. Microtubules prepared from bovine brain were driven by purified cytoplasmic dynein from rabbit liver, and movements of microtubules were visualized by video-enhanced differential interference contrast microscopy. The mean velocity of the motile microtubules was 1.09 +/- 0.13 microns/s. Our study provides evidence of rapid intracellular transport in hepatocytes controlled by cytoplasmic dynein.

Animals↗
PubMed1994

A limited cytoplasmic region of the prolactin receptor critical for signal transduction.

Prolactin receptors (PRL-R) are members of the cytokine receptor superfamily, which have in common, an absence of any known consensus sequence for signal transduction in their cytoplasmic domains. Four areas of high sequence homology have been identified in the cytoplasmic domains of PRL and growth hormone (GH) receptors, which may be important for signal transduction. The aim of this study was to investigate the role of these cytoplasmic regions in the functional activity of the PRL-R. Several mutant forms of PRL-R were constructed either by truncation or by deletion of the cDNA. Biological activities of these mutant receptors were assayed in CHO cells using a functional assay consisting in the co-transfection of PRL-R cDNA, along with a PRL responsive promoter fused to the coding sequence of the chloramphenicol acetyl transferase (CAT) gene. Progressive truncation of the cytoplasmic domain led to a progressive loss of ability to transactivate the CAT gene. Fully active PRL-R could be obtained when 217 of 358 aa of the cytoplasmic domain were present. Deletion of the first region of homology with the GH-R (residues 245-267) abolished the functional activity of PRL-R, whereas deletion of the second region of homology (residues 322-333) was without effect. These results indicate that a critical cytoplasmic region of 23 residues proximal to the transmembrane domain is essential for PRL signal transduction. There is strong homology within an 8-residue segment of this region with other members of the cytokine receptor superfamily, suggesting it contains a sequence necessary for signal transduction.

Amino Acid Sequence↗
PubMed1982

Estimation of the cytoplasmic catecholamine concentrations in pheochromocytoma cells.

Pheochromocytoma cells contain amine oxidase (flavin-containing), and convert dopamine and norepinephrine to deaminated metabolites. Dihydroxyphenylacetic acid is the major dopamine metabolite produced by the cells, whereas dihydroxyphenylglycol is the predominant metabolite of norepinephrine. Cells incubated under control conditions produce deaminated dopamine metabolites at a rate of about 30 pmol/min per mg protein, and dihydroxyphenylglycol at a rate of approx. 10 pmol/min per mg protein. Activation of tyrosine 3-monooxygenase increases the formation of dihydroxyphenylacetic acid, but does not greatly affect the production of dihydroxyphenylglycol. Inhibition of aromatic-L-amino-acid decarboxylase decreases the production of dihydroxyphenylacetic acid, but does not alter the production of dihydroxyphenylglycol. These results are consistent with the idea that newly synthesized dopamine represents the major source of cytoplasmic dopamine, whereas cytoplasmic norepinephrine is derived largely from catecholamine stores in secretory vesicles. The concentrations of dopamine and of norepinephrine in the cytoplasm of pheochromocytoma cells were estimated by measuring the substrate dependence of amine oxidase activity in extracts of these cells. By this method, the cytoplasmic concentrations of dopamine and of norepinephrine were estimated to be in the range of 0.5 to 1 microM. Incubation of the cells with extracellular norepinephrine or with reserpine results in an increase in the production of dihydroxyphenylglycol, and in inhibition of tyrosine 3-monoxygenase activity. Both of these effects are presumably mediated by a rise in the cytoplasmic norepinephrine concentration. Analysis of the relationship between norepinephrine metabolism and tyrosine 3-monooxygenase activity indicates that the apparent Ki of this enzyme for norepinephrine in intact cells is 10-15-times the basal cytoplasmic concentration of norepinephrine, or approx. 10 microM.

Adrenal Gland Neoplasms↗
PubMed2005

The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae.

Gram-negative bacteria use type II secretion systems for the transport of virulence factors and hydrolytic enzymes through the outer membrane. These sophisticated multi-protein complexes reach from the pore in the outer membrane via the pseudopilins in the periplasm and a multi-protein inner-membrane sub-complex, to an ATPase in the cytoplasm. The human pathogen Vibrio cholerae uses such a secretion machinery, called the Eps-system, for the export of its major virulence factor cholera toxin into the intestinal tract of the human host. Here, we describe the 2.4 A structure of the hetero-tetrameric complex of the N-terminal domain of the ATPase EpsE and the cytoplasmic domain of the inner membrane protein EpsL, which constitute the major cytoplasmic components of the Eps-system. A stable fragment of EpsE in complex with the cytoplasmic domain of EpsL was identified via limited proteolysis and facilitated the crystallization of the complex. This first structure of a complex between two different proteins of the type II secretion system reveals that the N-terminal domain of EpsE and the cytoplasmic domain of EpsL form a hetero-tetramer, in which EpsL is the central dimer and EpsE binds on the periphery. The dimer of EpsL in this complex is very similar to the dimer seen in the crystal structure of the native cytoplasmic domain of EpsL, suggesting a possible physiological relevance despite a relatively small 675 A2 buried solvent accessible surface. The N-terminal domain of EpsE, which forms a compact domain with an alpha+beta-fold, places its helix alpha2 in a mostly hydrophobic cleft between domains II and III of EpsL burying 1700 A2 solvent accessible surface. This extensive interface involves several residues whose hydrophobic or charged nature is well conserved and is therefore likely to be of general importance in type II secretion systems.

Amino Acid Sequence↗
PubMed2002

The integrin beta1 subunit cytoplasmic tail forms oligomers: a potential role in beta1 integrin clustering.

Integrins are alpha/beta heterodimeric cell surface receptors devoid of enzymatic activity. Signal transduction therefore requires the association of cytosolic and cytoskeletal proteins with the integrin subunit intracellular regions. This association is initiated upon ligand binding to the integrin receptor and includes clustering of the integrins and recruitment of focal adhesion-associated proteins. Whether integrin clustering is solely dependent on ligand binding to the integrin extracellular parts or involves also interactions between the intracellular tails of integrins is so far unknown. To investigate intracellular events in integrin clustering, we have used peptides corresponding to the integrin beta1 cytoplasmic region. Loading of cells with the peptides results in a decreased cell adhesion and in an inhibition of cell spreading in agreement with the previously reported dominant negative effect of the beta1 integrin cytoplasmic tail on integrin clustering. Direct protein-protein interaction studies by surface plasmon resonance demonstrate that integrin beta1 cytoplasmic peptides self-associate in contrast to integrin beta3 cytoplasmic tails. Size exclusion chromatography and SDS-PAGE analysis of the peptides further show that the integrin beta1 cytoplasmic parts form oligomers and that they assume alpha helical conformation to the extent of about 13% and that this fraction is increased upon aggregation. Thus self-association of the integrin beta1 subunit cytoplasmic regions may be central to beta1 integrin clustering.

Cell Adhesion↗
PubMed1995

Comparison of the motile and enzymatic properties of two microtubule minus-end-directed motors, ncd and cytoplasmic dynein.

Cytoplasmic dynein and ncd, a kinesin-related protein from Drosophila, are motor proteins that move toward the minus ends of microtubules, while kinesin moves to the microtubule plus end. In previous work, we examined the nucleotide dependence of motility and enzymatic activity by kinesin [Shimizu, T., Furusawa, K., Ohashi, S., Toyoshima, Y. Y., Okuno, M., Malik, F., & Vale, R. D., (1991) J. Cell Biol. 112, 1189-1197]. In this study, we examined these activities of the cytoplasmic dynein from bovine brain and ncd in order to explore what enzymatic features might be shared by these two minus-end-directed motors. Both ncd and cytoplasmic dynein demonstrated an activation of ATPase activity upon the addition of microtubules (30-fold and 6-fold, respectively). A significant difference between ncd and cytoplasmic dynein was their relative sensitivity to vanadate and to aluminum fluoride. In contrast to cytoplasmic dynein, ncd polypeptide was not cleaved by UV-vanadate treatment, and its ATPase and motility were unaffected by vanadate (up to 0.1 mM). When the nucleotide requirement for movement as examined using a battery of 20 nucleotides and nucleotide analogues, cytoplasmic dynein was found to exhibit a specificity very similar to that of axonemal dyneins from Tetrahymena. Surprisingly, however, the nucleotide specificities of in vitro motility produced by ncd or its construct, GST/MC1 (a fusion protein of glutathione S-transferase and 210-700 of the predicted ncd amino acid sequence), were quite distinct from that of kinesin. Thus, the nucleotide specificity profiles of members of the kinesin motor superfamily do not appear to be identical.

Aluminum Compounds↗
PubMed1980

Structure, translation, and metabolism of the cytoplasmic copia ribonucleic acid of Drosophila melanogaster.

We have characterized the copia RNA in the cytoplasm of cultured Drosophila cells. Copia RNA was detected and purified by hybridization to DNA of the plasmid cDm 1142, which contains the copia sequence. A large fraction (2.2%) of the total cytoplasmic poly(A)+ RNA was found to be copia RNA. Cytoplasmic copia RNA displays all the characteristics expected for a messenger RNA. It possesses a poly(A) tract identical in length with that of total poly(A)+ cytoplasmic RNA. It is associated with polysomes and can be released from this association by treatment with EDTA. When purified copia RNA is added to an mRNA-dependent rabbit reticulocyte lysate, three polypeptides of 51000, 33000, and 21000 daltons are seen. We have not determined if these are different polypeptides or if the two smaller polypeptides are fragments of the 51000-dalton polypeptide. The half-life of copia cytoplasmic RNA was determined in pulse--chase experiments to be 9.5 h; this is 1.6 times longer than the half-life of the intermediate decay class of total poly(A)+ cytoplasmic RNA. These properties provide strong evidence that copia RNA functions in vivo as a messenger RNA.

Animals↗
PubMed1997

The last two cytoplasmic loops in the lactose permease of Escherichia coli comprise a discontinuous epitope for a monoclonal antibody.

Monoclonal antibody (mAb) 4B11 binds to a conformational epitope in the lactose permease that is exposed on the cytoplasmic face of the membrane with a KD of 2.8 x 10(-7) M. By studying binding of 4B11 to permease mutants containing six contiguous His residues in each cytoplasmic loop, inserted factor Xa protease sites, or a C-terminal deletion, the cytoplasmic loops between helices VIII and IX (loop VIII/IX) and between helices X and XI (loop X/XI) are shown to comprise the epitope. Subsequently, Cys-scanning mutagenesis in conjunction with thiol modification was carried out in order to identify specific residues involved in 4B11 recognition. Glu342 and Arg344 in loop X/XI are primary determinants for 4B11 binding, while Ile283 in loop VIII/IX and Phe334 and Lys335 in loop X/XI are secondary determinants. Consistently, binding of avidin to biotinylated single-Cys replacements in loop VIII/IX or loop X/XI blocks 4B11 binding, but avidin binding to biotinylated Cys residues in other cytoplasmic loops or insertion of cytochrome b562 into cytoplasmic loop VI/VII has no significant effect. The studies demonstrate that the last two cytoplasmic loops in lactose permease comprise a discontinuous epitope for monoclonal antibody 4B11 and thereby provide independent evidence for the conclusion that helices VIII-XI are in close proximity.

Antibodies, Monoclonal↗
PubMed2006

Expression of cytoplasmic and nuclear Survivin in primary and secondary human glioblastoma.

Clinically, human glioblastoma (GBM) may develop de novo or from a low-grade glioma (secondary GBM), and molecular alterations in the two pathways may differ. This study examined the status of Survivin expression and apoptosis in 30 primary and 26 secondary GBMs. Our results show that cytoplasmic Survivin positivity was significantly (P<0.001) more frequent in primary GBMs (83%) than that in secondary GBMs (46%). In addition, an inverse correlation of cytoplasmc Survivin positivity with GBM apoptotic index, and a positive association between cytoplasmic Survivin and size of the tumours were observed. These results suggest that cytoplasmic Survivin, via its antiapoptotic function, may be involved in the tumorigenesis of many primary GBMs, but only in a small fraction of secondary GBMs. Furthermore, the overall progression times from low-grade precursor lesions to secondary GBMs were significantly shorter (P<0.05) in cytoplasmic Survivin-positive cases (mean, 15.6 months) than those in Survivin-negative cases (mean, 23.8 moths), and the positive expression level of Survivin in cytoplasm was upregulated in most secondary GBMs when compared to matched pre-existing low-graded lesions. These results suggest that the increased accumulation of Survivin in the cytoplasm of more malignant glioma cells may prove to be a selective advantage, thus accelerating progression to a more aggressive phenotype.

Adolescent↗
PubMed1999

cis-Determinants in the cytoplasmic domain of CEACAM1 responsible for its tumor inhibitory function.

CEACAM1, also known as C-CAM, BGP and CD66a, is a member of the carcinoembryonic antigen (CEA) family which is itself part of the immunoglobulin supergene family. CEACAM1 is involved in intercellular adhesion, signal transduction and tumor cell growth regulation. CEACAM1 is down-regulated in colon and prostate carcinomas, as well as in endometrial, bladder and hepatic tumors, and 30% of breast cancers. We have shown in a mouse colon tumor model that CEACAM1 with a long cytoplasmic domain inhibited the development of tumors whereas a splice variant lacking the cytoplasmic domain did not. In this study, we define the subregions of the long cytoplasmic domain participating in the tumor inhibition phenotype of CEACAM1. We show that a single point mutation of Tyr488, conforming to an Immunoreceptor Tyrosine Inhibition Motif (ITIM), was sufficient to reverse the in vivo tumor cell growth inhibition. Substitution or deletion of residues in the C-terminal region of the CEACAM1 cytoplasmic domain also led to reversal of tumor cell growth inhibition. This result is in agreement with our previous studies demonstrating the C-terminal region of the cytoplasmic domain influences the levels of CEACAM1 Tyr phosphorylation and its association with the protein Tyr phosphatases SHP-1 and SHP-2. Furthermore, removal of the N-terminal domain of CEACAM1, essential for intercellular adhesion, did not impair the tumor inhibitory effect. These results suggest that Tyr phosphorylation or dephosphorylation of the CEACAM1 cytoplasmic domain represents a crucial step in the control of epithelial cell proliferation.

3T3 Cells↗
PubMed1995

Physicochemical characterization of the cytoplasmic domain of the epidermal growth factor receptor and evidence for conformational changes associated with its activation by ammonium sulphate.

The physiochemical properties of the purified cytoplasmic domain of the epidermal growth factor (EGF) receptor, its self-phosphorylation and peptide phosphorylation activities, and its activation by ammonium sulphate have been studied. Highly efficient purification procedures for the isolation of the recombinant cytoplasmic domain (Met644-Ala1186) of the EGF receptor, expressed in the baculovirus/insect cell system, are described. Physicochemical characterization of the protein included investigation of its isoelectric and hydrodynamic properties, stability, oligomeric status, and secondary structure using far-u.v. circular dichroism. The recombinant protein was not recognized by anti-phosphotyrosine antibodies, unless first self-phosphorylated in vitro. Tryptic phosphopeptide maps of self-phosphorylated recombinant cytoplasmic domain and the EGF-stimulated A431-membrane receptor were very similar, suggesting that the recombinant had similar self-phosphorylation capacity and specificity. The preparations were characterized by high specific activity towards peptide tyrosine phosphorylation. Although the cytoplasmic domain was isolated as a homogeneously monomeric protein, storage at 4 degrees C led to slow, spontaneous aggregation with reduction in specific activity. Both high activity and monomeric state were maintained by storage below 0 degree C. The dependence of the initial rate of self-phosphorylation on protein concentration was consistent with cross-phosphorylation but not with the known oligomerization-induced activation of holoreceptor. The peptide phosphorylation activity was stimulated by Mn2+, Mg2+ and (NH4)2SO4 at high concentrations. The substrate specificity of (NH4)2SO4 activation was studied using synthetic peptides. Self-phosphorylation was inhibited by (NH4)2SO4 in the range 0-0.25 M but activated at 1.0-1.5 M, possibly as a result of ionic and hydrophobic protein interactions respectively. Phosphopeptide maps of cytoplasmic domain phosphorylated in the presence of high (NH4)2SO4 showed that the protein was more extensively phosphorylated than in the absence of salt, or than the native receptor. Far-u.v. circular-dichroism spectra of the cytoplasmic domain changed dramatically at 1 M (NH4)2SO4, raising the possibility that (NH4)2SO4 activates the kinase catalytic domain by inducing conformational changes.

Ammonium Sulfate↗
PubMed1995

Mutagenic structure/function analysis of the cytoplasmic cysteines of the insulin receptor.

Native human insulin receptor (hIR) has been reported to contain only one free thiol group proposed to lie near the ATP-binding. domain of its beta-subunit [Finn, Ridge and Hofmann (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 419-423]. The present study investigated the role of the six cytoplasmic cysteines of the beta-subunit of the hIR using a mutagenic approach in which insulin receptors, mutated at each cytoplasmic cysteine (to alanine) in turn, were transfected into Chinese hamster ovary (CHO) cells. Cell lines expressing hIR mutation at high level were obtained which, by both flow-cytometric analysis towards an hIR-specific monoclonal antibody (83-7) and insulin-binding analysis, were similar to the well-characterized CHOT cell line which overexpresses native hIR. The ED50 and Kd values of the mutant receptors were the same as those of the wild-type hIR. Each of the mutant receptors signalled insulin action to stimulate receptor autophosphorylation and kinase activity as well as glucose utilization to levels appropriate for the receptor level expressed. In contrast, insulin-stimulated thymidine uptake and glucose-transport responses of two of the six mutant cell lines, those expressing Cys981Ala and Cys1245Ala, were impaired compared with that of the native hIR-expressing cell line, CHOT. The beta-subunits of each of the hIR cytoplasmic cysteine mutant cell lines could be alkylated specifically with N-[3H]ethylmaleimide. The kinase activity of each receptor was inhibited by N-ethylmaleimide and stimulated by iodoacetamide, indicating that none of the cytoplasmic cysteines alone contributes the single free thiol group to the hIR structure. We conclude that the cytoplasmic cysteines of the hIR have a predominantly passive role in hIR activity although Cys-981 and Cys-1245 do affect mitogenic and glucose-transport responses of the receptor. Our findings indicate that the stoicheiometry of a single free thiol group/mol of insulin-binding activity noted in previous studies is either spread fractionally over a number of the cytoplasmic cysteines or is one of the four cysteines in the ectodomain of the hIR beta-subunit. Alternatively, the mutagenesis performed in the present study may enable differential exposure of a second titratable cysteine in wild-type and mutant receptors.

Amino Acid Sequence↗
PubMed2004

Tomographic reconstruction of treponemal cytoplasmic filaments reveals novel bridging and anchoring components.

An understanding of the involvement of bacterial cytoplasmic filaments in cell division requires the elucidation of the structural organization of those filamentous structures. Treponemal cytoplasmic filaments are composed of one protein, CfpA, and have been demonstrated to be involved in cell division. In this study, we used electron tomography to show that the filaments are part of a complex with a novel molecular organization that includes at least two distinct features decorating the filaments. One set of components appears to anchor the filaments to the cytoplasmic membrane. The other set of components appears to bridge the cytoplasmic filaments on the cytoplasmic side, and to be involved in the interfilament spacing within the cell. The filaments occupy between 3 and 18% of the inner surface of the cytoplasmic membrane. These results reveal a novel filamentous molecular organization of independent filaments linked by bridges and continuously anchored to the membrane.

Bacterial Proteins↗