The chloroplast import receptor is an integral membrane protein of chloroplast envelope contact sites

A chloroplast import receptor from pea, previously identified by antiidiotypic antibodies was purified and its primary structure deduced from its cDNA sequence. The protein is a 36-kD integral membrane protein (p36) with eight potential transmembrane segments. Fab prepared from monospecific anti-p36 IgG inhibits the import of the ribulose-1,5- bisphosphate carboxylase small subunit precursor (pS) by interfering with pS binding at the chloroplast surface. Anti-p36 IgGs are able to immunoprecipitate a Triton X-100 soluble p36-pS complex, suggesting a direct interaction between p36 and pS. This immunoprecipitation was specific as it was abolished by a pS synthetic transit peptide, consistent with the transit sequence receptor function of p36. Immunoelectron microscopy localized p36 to regions of the outer chloroplast membrane that are in close contact with the inner chloroplast membrane. Comparison of the deduced sequence of pea p36 to that of other known proteins indicates a striking homology to a protein from spinach chloroplasts that was previously suggested to be the triose phosphate-3-phosphoglycerate-phosphate translocator (phosphate translocator) (Flugge, U. I., K. Fischer, A. Gross, W. Sebald, F. Lottspeich, and C. Eckerskorn. 1989. EMBO (Eur. Mol. Biol. Organ.) J. 8:39-46). However, incubation of Triton X-100 solubilized chloroplast envelope material with hydroxylapatite indicated that p36 was quantitatively absorbed, whereas previous reports have shown that phosphate translocator activity does not bind to hydroxylapatite (Flugge, U. I., and H. W. Heldt. 1981. Biochim. Biophys. Acta. 638:296- 304. These data, in addition to the topology and import inhibition data presented in this report support the assignment of p36 as a receptor for chloroplast protein import, and argue against the assignment of the spinach homologue of this protein as the chloroplast phosphate translocator.     

Molecular cloning of the cDNA for a growth factor-inducible gene with strong homology to S-100, a calcium-binding protein

We have identified a cDNA whose sequence is preferentially expressed when quiescent fibroblasts are stimulated to proliferate. The steady-state levels of the mRNA corresponding to this clone, called 2A9, are increased by serum, platelet-derived growth factor, and epidermal growth factor, but not by insulin or platelet-poor plasma. mRNA levels of 2A9 are also increased in human acute myeloid leukemia. The 2A9 cDNA has been molecularly cloned from an Okayama-Berg library, and its complete nucleotide sequence has been determined. It has an open reading frame of 270 nucleotides, which has a 55% homology with the coding sequence of the beta-subunit of the S-100 protein, a calcium-binding protein that belongs (like calmodulin and the vitamin D-dependent intestinal calcium-binding protein) to the family of calcium-modulated proteins and is found in abundance in several human tumors, including melanoma. The S-100 protein and the deduced aminoacid sequence of 2A9 are also partially homologous to the small subunit of a protein complex that serves as a cellular substrate to tyrosine kinase. The partial homology of 2A9 (whose RNA is inducible by growth factors and is overexpressed in human acute myeloid leukemias) to the S-100 protein, other calcium-modulated proteins, and the subunit of a substrate for tyrosine kinase, is particularly interesting in view of the role attributed to calcium and tyrosine kinases in the regulation of cell proliferation.     

Modulation of tyrosine phosphorylation of p36 and other substrates by the S-100 protein

The inhibitory effects of Ca2+-binding proteins on tyrosine phosphorylation of p36 protein isolated from bovine intestinal epithelium by immunoprecipitated p130fps were investigated. S-100 protein dose dependently inhibited the p36 phosphorylation, and calmodulin weakly depressed the phosphorylation, whereas parvalbumin and troponin C had no significant effects. The S-100 preparation purified from bovine brain did not contain phosphatase activity or ATPase activity. The concentration of ATP did not affect the S-100-mediated inhibition of phosphorylation but the substrate protein, p36, reversed the inhibition. S-100 similarly inhibited the tyrosine phosphorylation of p36 by p60src but did not affect the p36 phosphorylation by protein kinase C. S-100 inhibited the tyrosine kinase activity of p130fps using the other substrates tested as well. These results suggest that S-100 interacts with the substrate binding site of retroviral tyrosine-specific protein kinases and may play a regulatory role in the tyrosine phosphorylation.     

Different distribution of S-100 protein and glial fibrillary acidic protein (GFAP) immunoreactive cells and their relations with nitrergic neurons in the human fetal small intestine.

The appearance, distribution and some histochemical features of non-neuronal cells (NN cells) associated with the myenteric plexus of human fetal small intestine have been studied by means of S-100 protein and GFAP immunocytochemistry between the 10th and 17th week of gestation. In addition, double labelling immunocytochemistry using an antibody raised against a constitutive isoform of nitric oxide synthase (bNOS) in combination with an S-100 protein antibody was applied to investigate the morphological relations between NN cells and nitrergic neurons in the developing gut wall. Cells with immunoreactivity for both glial-specific proteins are already present in the 10th week of gestation. While cells with S-100 protein immunoreactivity are located within the circular muscle layer as well as in the myenteric, and submucous plexuses, cells with GFAP immunopositivity are mainly restricted to the side of the myenteric plexus adjacent to the longitudinal muscle layer. In contrast to the dense network formed by S-100 protein immunopositive structures the GFAP immunopositive cells appear singly and do not connect into a network. Double-labelling immunocytochemistry reveals nitrergic fibers (NOS-IR) in close relation to the S-100 protein immunoreactive glial network. NOS-IR varicosities are in close association with the surface of those cells both in the circular muscle layer (CM) and in the tertiary plexus. It is concluded that two populations of NN cells with different locations and different immunohistochemical characters appear and develop together with the enteric ganglia in the human fetal intestine. The close morphological relation of NOS-IR fibers with S-100 protein immunopositive cellular network indicate a functional relationship between S-100 protein immunopositive cells and the nitrergic nerves during the early development of human enteric nervous system (ENS).     

The S-100 beta protein in normal human peripheral blood is uniquely present within a discrete suppressor-T-cell compartment

The S-100-positive T lymphocytes, and, particularly, the S-100 beta subunit, are restricted, as demonstrated by quantitative subset analysis and double-labeling (gold-peroxidase) immunoelectron microscopy of T-cell subpopulations, to an unique T8-positive cell subset which interestingly was 9.3-negative and CD11b-positive. Since both the T8-positive, 9.3-negative and the T8-positive, CD11b-positive subpopulations have been demonstrated to show suppressive activities, the S-100-positive T cells seem to be closely restricted to a small T-suppressor-cell compartment. Although functional studies on viable isolated S-100 beta-positive cells are impossible to achieve, due to the lack of this protein on the cell membrane, its presence in a discrete T-suppressor compartment might suggest a possible role for the S-100 beta-positive T cells in the regulation of the immune system.     

S-100 protein stimulates cellular proliferation

Summary S-100 protein (S-100p) is a small, acidic, calcium-binding protein that is present (predominantly) in the cytoplasm of many types of cells including those of neuroectodermal origin, such as glial cells, schwann cells and melanocytes. In human melanoma cells S-100p is abundant relative to the small quantities expressed by normal melanocytes. We investigated the possibility that this protein may be a growth factor. Purified S-100p from bovine brain or human melanoma cells was added exogenously to human melanoma cells and peripheral blood lymphocytes (PBL) and their growth in the presence of different concentrations of S-100p was determined using a [³H]dT-uptake proliferation assay. The growth of melanoma cells was stimulated by S-100p at concentrations of 1.95–31.25 g/ml. Slight inhibition of cell proliferation occurred at high concentrations (125 g/ml). Maximum stimulation of PBL was at 31.25 g/ml. PBL were not inhibited even at high concentrations of S-100p (125 g/ml). PBL stimulation by S-100p did not require the presence of monocytes/macrophages. Though stimulation by S-100p is not restricted to a specific cell type, when released by melanoma cells it may function as an autocrine tumor growth factor. Other cells, such as PBL, coming in contact with S-100p are also stimulated to proliferate.     

Recognition of the tumor suppressor protein p53 and other protein targets by the calcium-binding protein S100B

S100B is an EF-hand containing calcium-binding protein of the S100 protein family that exerts its biological effect by binding and affecting various target proteins. A consensus sequence for S100B target proteins was published as (K/R)(L/I)xWxxIL and matches a region in the actin capping protein CapZ (V.V. Ivanenkov, G.A. Jamieson, Jr., E. Gruenstein, R.V. Dimlich, Characterization of S-100b binding epitopes. Identification of a novel target, the actin capping protein, CapZ, J. Biol. Chem. 270 (1995) 14651-14658). Several additional S100B targets are known including p53, a nuclear Dbf2 related (NDR) kinase, the RAGE receptor, neuromodulin, protein kinase C, and others. Examining the binding sites of such targets and new protein sequence searches provided additional potential target proteins for S100B including Hdm2 and Hdm4, which were both found to bind S100B in a calcium-dependent manner. The interaction between S100B and the Hdm2 and/or the Hdm4 proteins may be important physiologically in light of evidence that like Hdm2, S100B also contributes to lowering protein levels of the tumor suppressor protein, p53. For the S100B-p53 interaction, it was found that phosphorylation of specific serine and/or threonine residues reduces the affinity of the S100B-p53 interaction by as much as an order of magnitude, and is important for protecting p53 from S100B-dependent down-regulation, a scenario that is similar to what is found for the Hdm2-p53 complex.     

Primary structure of human, chicken, and Xenopus laevis p11, a cellular ligand of the Src-kinase substrate, annexin II.

The p11 protein is a member of the S-100 family of Ca(2+)-binding proteins and serves within the cell as a ligand of the tyrosine kinase substrate, annexin II. To obtain more structural information on this molecule, we have isolated and characterized p11 cDNA clones from several different species. A comparison of the deduced amino acid (aa) sequences reveals that mammalian and avian p11 are highly similar (at least 90% identical at the aa level), whereas p11 from Xenopus laevis shows a considerable degree of sequence variation (the aa sequence identity drops to approx. 60% when compared to mammalian or chicken p11). Interestingly, the C-terminal 18 aa, which are unique to p11 within the S-100 family, show a relatively high conservation among species. This high evolutionary conservation is in line with a structurally and/or functionally important role of this C terminus, e.g., in annexin II binding.     

Archaeal RNase P has multiple protein subunits homologous to eukaryotic nuclear RNase P proteins

Although archaeal RNase P RNAs are similar in both sequence and structure to those of Bacteria rather than eukaryotes, and heterologous reconstitution between the Bacillus subtilis RNase P protein and some archaeal RNase P RNAs has been demonstrated, no archaeal protein sequences with similarity to any known bacterial RNase P protein subunit have been identified, and the density of Methanothermobacter thermoautotrophicus RNase P in Cs2SO4 (1.42 g/mL) is inconsistent with a single small bacterial-like protein subunit. Four hypothetical open reading frames (MTH11, MTH687, MTH688, and MTH1618) were identified in the genome of M. thermoautotrophicus that have sequence similarity to four of the nine Saccharomyces cerevisiae RNase P protein subunits: Pop4p, Pop5p, Rpp1p, and Rpr2p, respectively. Polyclonal antisera generated to recombinant Mth11p, Mth687p, Mth688p, and Mth1618p each recognized a protein of the predicted molecular weight in western blots of partially purified M. thermoautotrophicus RNase P, and immunoprecipitated RNase P activity from the same partially purified preparation. RNase P in Archaea is therefore composed of an RNA subunit similar to bacterial RNase P RNA and multiple protein subunits similar to those in the eukaryotic nucleus.     

Immunocytochemical study of the distribution of S-100 protein in the parathyroid gland of rats and guinea pigs

The distribution of S-100 protein in the parathyroid cells of normal and hypercalcaemic rats and guinea pigs was investigated. Previous studies had shown that the applied antibodies detect only the beta subunit of S-100 protein. S-100 protein was found in all parathyroid cells of rats aged between 1 and 720 days. In adult guinea pigs, S-100 protein was detectable in only a small proportion of parathyroid cells. The level of S-100 protein in individual cells exhibited considerable variation, particularly in guinea pig. Hypercalcaemia did not affect the distribution of S-100 protein in the parathyroid cells of either rats or guinea pigs. In both species, the presence of small groups of parathyroid cells in the central fragments of thyroid lobes was often noted.     

Structural and functional analyses of a yeast mitochondrial ribosomal protein homologous to ribosomal protein S15 of Escherichia coli.

We have purified a small subunit mitochondrial ribosomal protein, MRPS28p, from the yeast, Saccharomyces cerevisiae. Sequence from the amino terminus of MRPS28p was used to design a degenerate oligonucleotide that was complementary to the MRPS28 gene. The MRPS28 gene was isolated and its sequence determined. The MRPS28 sequence encodes a 28 kDa protein that has a region of homology with ribosomal protein S15 of E. coli. This region spans the entire length of the E. coli protein, but as MRPS28p is larger, includes only the portion of the MRPS28p sequence from amino acids 150 to 238. Based on this homology, we predict that MRPS28p, like E. coli S15, interacts directly with small subunit rRNA and functions as an early protein in ribosome assembly. Cells carrying a disrupted chromosomal copy of MRPS28 are unable to respire and spontaneously lose portions of their mitochondrial genomes at a high frequency. These phenotypes are consistent with an essential role for MRPS28p in the assembly and/or function of the mitochondrial ribosome.     

Annexin II in exocytosis: catecholamine secretion requires the translocation of p36 to the subplasmalemmal region in chromaffin cells

Annexin II is a Ca(2+)-dependent membrane-binding protein present in a wide variety of cells and tissues. Within cells, annexin II is found either as a 36-kD monomer (p36) or as a heterotetrameric complex (p90) coupled with the S-100-related protein, p11. Annexin II has been suggested to be involved in exocytosis as it can restore the secretory responsiveness of permeabilized chromaffin cells. By quantitative confocal immunofluorescence, immunoreplica analysis and immunoprecipitation, we show here the translocation of p36 from the cytosol to a subplasmalemmal Triton X-100 insoluble fraction in chromaffin cells following nicotinic stimulation. A synthetic peptide corresponding to the NH2-terminal domain of p36 which contains the phosphorylation sites was microinjected into individual chromaffin cells and catecholamine secretion was monitored by amperometry. This peptide blocked completely the nicotine-induced recruitment of p36 to the cell periphery and strongly inhibited exocytosis evoked by either nicotine or high K+. The light chain of annexin II, p11, was selectively expressed by adrenergic chromaffin cells, and was only present in the subplasmalemmal Triton X-100 insoluble protein fraction of both resting and stimulated cells. p11 can modify the Ca(2+)- and/or the phospholipid-binding properties of p36. We found that loss Ca2+ was required to stimulate the translocation of p36 and to trigger exocytosis in adrenergic chromaffin cells. Our findings suggest that the translocation of p36 to the subplasmalemmal region is an essential event in regulated exocytosis and support the idea that the presence of p11 in adrenergic cells may confer a higher Ca2+ affinity to the exocytotic pathway in these cells.     

Import and orientation of the MWFE protein in mitochondrial NADH-ubiquinone oxidoreductase

The MWFE subunit of the mitochondrial NADH-ubiquinone oxidoreductase (complex I) is a small, essential membrane protein of 70 amino acids that is made in the cytosol, imported into mitochondria, and assembled without further proteolytic processing. The experiments identify the first approximately 30 amino acids as a minimal mitochondrial targeting sequence, and establish its orientation in the inner membrane and in complex I. This sequence has a highly conserved glutamate at position 4, which is not typical of a mitochondrial targeting signal. However, it is not essential for MWFE function. Within this sequence there is also a 'stop-transfer' signal. The membrane anchor cannot be replaced by that from another subunit within complex I.     

Immunocytochemical study of the distribution of S-100 protein in the parathyroid gland of rats and guinea pigs

Summary The distribution of S-100 protein in the parathyroid cells of normal and hypercalcaemic rats and guinea pigs was investigated. Previous studies had shown that the applied antibodies detect only the subunit of S-100 protein. S-100 protein was found in all parathyroid cells of rats aged between 1 and 720 days. In adult guinea pigs, S-100 protein was detectable in only a small proportion of parathyroid cells. The level of S-100 protein in individual cells exhibited considerable variation, particularly in guinea pigs. Hypercalcaemia did not affect the distribution of S-100 protein in the parathyroid cells of either rats or guinea pigs. In both species, the presence of small groups of parathyroid cells in the central fragments of thyroid lobes was often noted.     

Neuron-specific enolase and S-100 protein immunohistochemistry for defining the structure and topographical relationship of the different enteric nerve plexuses in the small intestine of the pig

Summary The morphological and topographical features of the intramural enteric nervous system in the small intestine of the pig has been studied on whole mounts by means of neuron-specific enolase (NSE) and S-100 protein immu-nohistochemistry. A clear visualization of the myenteric plexus allows the recognition of its characteristic morphology, including the thin tertiary plexus coursing within the smooth muscle layers. In the tela submucosa two ganglionated plexuses, each with its own specific characteristics, can clearly be demonstrated: (1) the plexus submucosus externus (Schabadasch) located near the inner surface of the circular muscle layer at the abluminal side of the submucosal vascular arcades, and (2) the plexus submucosus internus (Meissner) close to the outer surface of the lamina muscularis mucosae at the luminal side of the submucosal vascular arcades. Due to the possibility to trace clearly the perivascular plexuses of these vascular arcades by use of immunohistochemical techniques with antibodies to NSE and S-100 protein, the two submucosal nerve plexuses can be demonstrated with exceptional clarity. This is the first report of an investigation of the intramural nerve plexuses of the small intestine of the pig using the NSE and S-100 immunostaining methods, which is sufficiently detailed to substantiate the characteristic topography and structure of the two submucosal plexuses and their relation to the smooth muscle layers and perivascular plexuses. The level of NSE immunoreactivity for enteric neurons displays great variation, a substantial proportion of the type-II neurons appearing strongly stained. Although little is known of the specific function of these enzymes, proposals are discussed.     

Polyoma small and middle T antigens and SV40 small t antigen form stable complexes with protein phosphatase 2A

We have purified the 36 and 63 kd cellular proteins known to associate with polyomavirus middle and small tumor (T) antigens and SV40 small t antigen. Microsequencing of the 36 kd protein indicated that it was probably identical to the catalytic subunit of protein phosphatase 2A (PP2A). Identity was confirmed by comigration on two-dimensional (2D) gels and by 2D analysis of complete chymotryptic digests. In addition, PP2A-like phosphatase activity was detected in immunoprecipitates of wild-type middle T. Immunoblotting experiments, comigration on 2D gels, and 2D analysis of limit chymotryptic digests demonstrated that the 63 kd protein, present in the middle T complex in approximately equimolar ratio to the 36 kd protein, is a known regulatory subunit of the PP2A holoenzyme. Finally, the 36 kd PP2A catalytic subunit can be immunoprecipitated by anti-pp60c-src antisera only from cells expressing wild-type middle T. These results suggest that complex formation between PP2A and T antigens may be important for T antigen-mediated transformation.     

The molecular cloning of the complementary deoxyribonucleic acid for bovine vitamin D-dependent calcium-binding protein: structure of the full-length protein and evidence for homologies with other calcium-binding proteins of the troponin-C superfamily of proteins

We have cloned the cDNA for bovine intestinal vitamin D-dependent calcium-binding protein and, based on the sequence of the DNA, have deduced the structure of the full-length protein. The sequence of the cDNA clone predicts a protein comprised of 78 amino acids with a mol wt of 8788. The mRNA for the protein in bovine duodenum is about 500-600 bases in length. The protein sequence of bovine intestinal calcium-binding protein is 87% homologous with the sequence of porcine intestinal vitamin D-dependent calcium-binding protein and 81% homologous with the sequence of rat intestinal vitamin D-dependent calcium-binding protein. Hydrophilicity plots of the proteins noted above show that despite differences in amino acid sequence the proteins have similar patterns. In addition, the predicted secondary structure of the proteins is similar. Bovine intestinal calcium-binding protein shows 48.6% homology with the alpha-chain and 38.2% homology with the beta-chain of bovine S-100 protein and a similar high degree of homology with the beta-chain of human S-100 protein. The protein also demonstrates 36-43% homology with parvalbumin alpha and beta from various species and with troponin-C. There is some homology with the 28K vitamin D-dependent calcium-binding proteins. Vitamin D-dependent bovine intestinal calcium-binding protein is closely related to other mammalian intestinal calcium-binding proteins and to the S-100 proteins, parvalbumins, and troponin-C.     

Complex of pregnancy-associated plasma protein-A and the proform of eosinophil major basic protein. Disulfide structure and carbohydrate attachment

Pregnancy-associated plasma protein-A (PAPP-A) is a metzincin superfamily metalloproteinase responsible for cleavage of insulin-like growth factor-binding protein-4, thus causing release of bound insulin-like growth factor. PAPP-A is secreted as a dimer of 400 kDa but circulates in pregnancy as a disulfide-bound 500-kDa 2:2 complex with the proform of eosinophil major basic protein (pro-MBP), recently shown to function as a proteinase inhibitor of PAPP-A. Except for PAPP-A2, PAPP-A does not share global similarity with other proteins. Three lin-notch (LNR or LIN-12) modules and five complement control protein modules (also known as SCR modules) have been identified in PAPP-A by sequence similarity with other proteins, but no data are available that allow unambiguous prediction of disulfide bonds of these modules. To establish the connectivities of cysteine residues of the PAPP-A.pro-MBP complex, biochemical analyses of peptides derived from purified protein were performed. The PAPP-A subunit contains a total of 82 cysteine residues, of which 81 have been accounted for. The pro-MBP subunit contains 12 cysteine residues, of which 10 have been accounted for. Within the 2:2 complex, PAPP-A is dimerized by a single disulfide bond; pro-MBP is dimerized by two disulfides, and each PAPP-A subunit is connected to a pro-MBP subunit by two disulfide bonds. All other disulfides are intrachain bridges. We also show that of 13 potential sites for N-linked carbohydrate substitution of the PAPP-A subunit, 11 are occupied. The large number of disulfide bonds of the PAPP-A.pro-MBP complex imposes many restraints on polypeptide folding, and knowledge of the disulfide pattern of PAPP-A will facilitate structural studies based on recombinant expression of individual, putative PAPP-A domains. Furthermore, it will allow rational experimental design of functional studies aimed at understanding the formation of the PAPP-A.pro-MBP complex, as well as the inhibitory mechanism of pro-MBP.     

Two subunits of the canine signal peptidase complex are homologous to yeast SEC11 protein

Canine microsomal signal peptidase activity was previously isolated as a complex of five subunits (25, 22/23, 21, 18, and 12 kDa). Two of the signal peptidase complex (SPC) subunits (23/23 and 21 kDa) have been cloned and sequenced. One of these, the 21-kDa subunit, was observed to be a mammalian homolog of SEC11 protein (Sec11p) (Greenburg, G., Shelness, G. S., and Blobel, G. (1989) J. Biol. Chem. 264, 15762-15765) a gene product essential for signal peptide processing and cell growth in yeast (B?hni, P.C., Deshqies, R.J., and Schekman, R.W. (1988) J. Cell Biol. 106, 1035-1042). cDNA clones for the 18-kDa SPC subunit have now been characterized and found to encode a second SEC11p homolog. Both the 18- and 21-kDa canine SPC subunits are integral membrane proteins by virtue of their resistance to alkaline extraction. Upon detergent solubilization, both proteins are found in a complex with the 22/23 kDa SPC subunit, the only SPC subunit containing N-linked oligosaccharide. No steady-state pool of canine Sec11p-like monomers is detected in microsomal membranes. Alkaline extraction of microsomes prior to solubilization or solubilization at alkaline pH causes partial dissociation of the SPC. The Sec11p-like subunits displaced from the complex under these conditions demonstrate no signal peptide processing activity by themselves. The existence of homologous subunits is common to a number of known protein complexes and provides further evidence that the association between SPC proteins observed in vitro may be physiologically relevant to the mechanism of signal peptide processing and perhaps protein translocation.