Organization of the multiaminoacyl-tRNA synthetase complex and the cotranslational protein folding

Aminoacyl-tRNA synthetases (ARSs) play an essential role in the protein synthesis by catalyzing an attachment of their cognate amino acids to tRNAs. Unlike their prokaryotic counterparts, ARSs in higher eukaryotes form a multiaminoacyl-tRNA synthetase complex (MARS), consisting of the subset of ARS polypeptides and three auxiliary proteins. The intriguing feature of MARS complex is the presence of only nine out of twenty ARSs, specific for Arg, Asp, Gln, Glu, Ile, Leu, Lys, Met, and Pro, regardless of the organism, cell, or tissue types. Although existence of MARSs complex in higher eukaryotes has been already known for more than four decades, its functional significance remains elusive. We found that seven of the nine corresponding amino acids (Arg, Gln, Glu, Ile, Leu, Lys, and Met) together with Ala form a predictor of the protein α-helicity. Remarkably, all amino acids (besides Ala) in the predictor have the highest possible number of side-chain rotamers. Therefore, compositional bias of a typical α-helix can contribute to the helix''s stability by increasing the entropy of the folded state. It also appears that position-specific α-helical propensity, specifically periodic alternation of charged and hydrophobic residues in the helices, may well be provided by the structural organization of the complex. Considering characteristics of MARS complex from the perspective of the α-helicity, we hypothesize that specific composition and structure of the complex represents a functional mechanism for coordination of translation with the fast and correct folding of amphiphilic α-helices.     

The EMAPII cytokine is released from the mammalian multisynthetase complex after cleavage of its p43/proEMAPII component

Endothelial-monocyte-activating polypeptide II (EMAPII) is an inflammatory cytokine released under apoptotic conditions. Its proEMAPII precursor proved to be identical to the auxiliary p43 component of the aminoacyl-tRNA synthetase complex. We show here that the EMAPII domain of p43 is released readily from the complex after in vitro digestion with caspase 7 and is able to induce migration of human mononuclear phagocytes. The N terminus of in vitro-processed EMAPII coincides exactly with that of the mature cytokine isolated from conditioned medium of fibrosarcoma cells. We also show that p43/proEMAPII has a strong tRNA binding capacity (K(D) = 0.2 microm) as compared with its isolated N or C domains (7.5 microm and 40 microm, respectively). The potent general RNA binding capacity ascribed to p43/proEMAPII is lost upon the release of the EMAPII domain. This suggests that after onset of apoptosis, the first consequence of the cleavage of p43 is to limit the availability of tRNA for aminoacyl-tRNA synthetases associated within the complex. Translation arrest is accompanied by the release of the EMAPII cytokine that plays a role in the engulfment of apoptotic cells by attracting phagocytes. As a consequence, p43 compares well with a molecular fuse that triggers the irreversible cell growth/cell death transition induced under apoptotic conditions.     

p53-independent apoptosis is induced by the p19ARF tumor suppressor

p19(ARF) is a potent tumor suppressor. By inactivating Mdm2, p19(ARF) upregulates p53 activities to induce cell cycle arrest and sensitize cells to apoptosis in the presence of collateral signals. It has also been demonstrated that cell cycle arrest is induced by overexpressed p19(ARF) in p53-deficient mouse embryonic fibroblasts, only in the absence of the Mdm2 gene. Here, we show that apoptosis can be induced without additional apoptosis signals by expression of p19(ARF) using an adenovirus-mediated expression system in p53-intact cell lines as well as p53-deficient cell lines. Also, in primary mouse embryonic fibroblasts (MEFs) lacking p53/ARF, p53-independent apoptosis is induced irrespective of Mdm2 status by expression of p19(ARF). In agreement, p19(ARF)-mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53-independent pathway for p19(ARF)-induced apoptosis that is insensitive to inhibition by Mdm2.     

A component of the multisynthetase complex is a multifunctional aminoacyl-tRNA synthetase.

In higher eukaryotes, nine aminoacyl-tRNA synthetases are associated within a multienzyme complex which is composed of 11 polypeptides with molecular masses ranging from 18 to 150 kDa. We have cloned and sequenced a cDNA from Drosophila encoding the largest polypeptide of this complex. We demonstrate here that the corresponding protein is a multifunctional aminoacyl-tRNA synthetase. It is composed of three major domains, two of them specifying distinct synthetase activities. The amino and carboxy-terminal domains were expressed separately in Escherichia coli, and were found to catalyse the aminoacylation of glutamic acid and proline tRNA species, respectively. The central domain is made of six 46 amino acid repeats. In prokaryotes, these two aminoacyl-tRNA synthetases are encoded by distinct genes. The emergence of a multifunctional synthetase by a gene fusion event seems to be a specific, but general attribute of all higher eukaryotic cells. This type of structural organization, in relation to the occurrence of multisynthetase complexes, could be a mechanism to integrate several catalytic domains within the same particle. The involvement of the internal repeats in mediating complex assembly is discussed.     

Characterization of p40/GPR69A as a peripheral membrane protein related to the lantibiotic synthetase component C

The 40 kDa erythrocyte membrane protein p40/GPR69A, previously assigned to the G-protein-coupled receptor superfamily, was now identified by peptide-antibodies and characterized as a loosely associated peripheral membrane protein. This result is in striking contrast to the proposed seven-transmembrane protein structure and function and therefore we wish to correct our previous proposal. p40 is located at the cytoplasmic side of the membrane and is neither associated with the cytoskeleton nor lipid rafts. Refined sequence analysis revealed that p40 is related to the LanC family of bacterial membrane-associated proteins which are involved in the biosynthesis of antimicrobial peptides. Therefore, we rename p40 to LanC-like protein 1 (LANCL1) and suggest that it may play a similar role as a peptide-modifying enzyme component in eukaryotic cells.     

p19ARF determines the balance between normal cell proliferation rate and apoptosis during mammary gland development

This study demonstrated, for the first time, the following events related to p19(ARF) involvement in mammary gland development: 1) Progesterone appears to regulate p19(ARF) in normal mammary gland during pregnancy. 2) p19(ARF) expression levels increased sixfold during pregnancy, and the protein level plateaus during lactation. 3) During involution, p19(ARF) protein level remained at high levels at 2 and 8 days of involution and then, declined sharply at day 15. Absence of p19(ARF) in mammary epithelial cells leads to two major changes, 1) a delay in the early phase of involution concomitant with downregulation of p21(Cip1) and decrease in apoptosis, and 2) p19(ARF) null cells are immortal in vivo measured by serial transplantion, which is partly attributed to complete absence of p21(Cip1) compared with WT cells. Although, p19(ARF) is dispensable in mammary alveologenesis, as evidenced by normal differentiation in the mammary gland of pregnant p19(ARF) null mice, the upregulation of p19(ARF) by progesterone in the WT cells and the weakness of p21(Cip1) in mammary epithelial cells lacking p19(ARF) strongly suggest that the functional role(s) of p19(ARF) in mammary gland development is critical to sustain normal cell proliferation rate during pregnancy and normal apoptosis in involution possibly through the p53-dependent pathway.     

p120, a p120-related protein (p100), and the cadherin/catenin complex

Cadherins and catenins play an important role in cell-cell adhesion. Two of the catenins, beta and gamma, are members of a group of proteins that contains a repeating amino acid motif originally described for the Drosophila segment polarity gene armadillo. Another member of this group is a 120-kD protein termed p120, originally identified as a substrate of the tyrosine kinase pp60src. In this paper, we show that endothelial and epithelial cells express p120 and p100, a 100-kD, p120- related protein. Peptide sequencing of p100 establishes it as highly related to p120. p120 and p100 both appear associated with the cadherin/catenin complex, but independent p120/catenin and p100/catenin complexes can be isolated. This association is shown by coimmunoprecipitation of cadherins and catenins with an anti-p120/p100 antibody, and of p120/p100 with cadherin or catenin antibodies. Immunocytochemical analysis with a p120-specific antibody reveals junctional colocalization of p120 and beta-catenin in epithelial cells. Catenins and p120/p100 also colocalize in endothelial and epithelial cells in culture and in tissue sections. The cellular content of p120/p100 and beta-catenin is similar in MDCK cells, but only approximately 20% of the p120/p100 pool associates with the cadherin/catenin complex. Our data provide further evidence for interactions among the different arm proteins and suggest that p120/p100 may participate in regulating the function of cadherins and, thereby, other processes influenced by cell-cell adhesion.     

Web-ware bioinformatical analysis and structure modelling of N-terminus of human multisynthetase complex auxiliary component protein p43

Human multisynthetase complex auxiliary component, protein p43 is an endothelial monocyte-activating polypeptide II precursor. In this study, comprehensive sequence analysis of N-terminus has been performed to identify structural domains, motifs, sites of post-translation modification and other functionally important parameters. The spatial structure model of full-chain protein p43 is obtained.     

Cytochrome c is released in a single step during apoptosis

Release of cytochrome c from mitochondria is a central event in apoptotic signaling. In this study, we utilized a cytochrome c fusion that binds fluorescent biarsenical ligands (cytochrome c-4CYS (cyt. c-4CYS)) as well as cytochrome c-green fluorescent protein (cyt. c-GFP) to measure its release from mitochondria in different cell types during apoptosis. In single cells, the kinetics of cyt. c-4CYS release was indistinguishable from that of cyt. c-GFP in apoptotic cells expressing both molecules. Lowering the temperature by 7 degrees C did not affect this corelease, but further separated cytochrome c release from the subsequent decrease in mitochondrial membrane potential (DeltaPsi(m)). Cyt. c-GFP rescued respiration in cells lacking endogenous cytochrome c, and the duration of cytochrome c release was approximately 5 min in a variety of cell types induced to die by various forms of cellular stress. In addition, we could observe no evidence of caspase-dependent amplification of cytochrome c release or changes in DeltaPsi(m) preceding the release of cyt. c-GFP. We conclude that there is a general mechanism responsible for cytochrome c release that proceeds in a single step that is independent of changes in DeltaPsi(m).     

CARF is a novel protein that cooperates with mouse p19ARF (human p14ARF) in activating p53

The INK4a locus on chromosome 9p21 encodes two structurally distinct tumor suppressor proteins, p16(INK4a) and the alternative reading frame protein, ARF (p19(ARF) in mouse and p14(ARF) in human). Each of these proteins has a role in senescence of primary cells and activates pathways for cell cycle control and tumor suppression. The current prevailing model proposes that p19(ARF) activates p53 function by antagonizing its degradation by MDM2. It was, however, recently shown that stabilization of p53 by p14(ARF) occurs independent of the relocalization of MDM2 to the nucleolus. We have identified a novel collaborator of ARF, CARF. It co-localizes and interacts with ARF in the nucleolus. We demonstrate that CARF is co-regulated with ARF, cooperates with it in activating p53, and thus acts as a novel component of the ARF-p53-p21 pathway.     

Multiple domains of the mouse p19ARF tumor suppressor are involved in p53-independent apoptosis

The ARF (p19ARF for the mouse ARF consisting of 169 amino acids and p14ARF for the human ARF consisting of 132 amino acids) genes upregulate p53 activities to induce cell cycle arrest and sensitize cells to apoptosis by inhibiting Mdm2 activity. p53-independent apoptosis also is induced by ectopic expression of p19ARF. We constructed various deletion mutants of p19ARF with a cre/loxP-regulated adenoviral vector to determine the regions of p19ARF which are responsible for p53-independent apoptosis. Ectopic expression of the C-terminal region (named C40) of p19ARF whose primary sequence is unique to the rodent ARF induced prominent apoptosis in p53-deficient mouse embryo fibroblasts. Relatively low-grade but significant apoptosis also was induced in p53-deficient mouse embryo fibroblasts by ectopic expression of p19ARF1-129, a p19ARF deletion mutant deficient in the C40 region. In contrast, ectopic expression of the wild-type p14ARF did not induce significant apoptosis in human cells. Taken together, we concluded that p53-independent apoptosis was mediated through multiple regions of the mouse ARF including C40, and the ability of the ARF gene to mediate p53-independent apoptosis has been not well conserved during mammalian evolution.     

p54nrb is a component of the snRNP-free U1A (SF-A) complex that promotes pre-mRNA cleavage during polyadenylation

The U1 snRNP-A (U1A) protein has been known for many years as a component of the U1 snRNP. We have previously described a form of U1A present in human cells in significant amounts that is not associated with the U1 snRNP or U1 RNA but instead is part of a novel complex of non-snRNP proteins that we have termed snRNP-free U1A, or SF-A. Antibodies that specifically recognize this complex inhibit in vitro splicing and polyadenylation of pre-mRNA, suggesting that this complex may play an important functional role in these mRNA-processing activities. This finding was underscored by the determination that one of the components of this complex is the polypyrimidine-tract-binding protein-associated splicing factor, PSF. In order to further our studies on this complex and to determine the rest of the components of the SF-A complex, we prepared several stable HeLa cell lines that overexpress a tandem-affinity-purification-tagged version of U1A (TAP-tagged U1A). Nuclear extract was prepared from one of these cell lines, line 107, and affinity purification was performed along with RNase treatment. We have used mass spectrometry analysis to identify the candidate factors that associate with U1A. We have now identified and characterized PSF, p54(nrb), and p68 as novel components of the SF-A complex. We have explored the function of this complex in RNA processing, specifically cleavage and polyadenylation, by performing immunodepletions followed by reconstitution experiments, and have found that p54(nrb) is critical.     

Adenylate kinase of Escherichia coli, a component of the phage T4 dNTP synthetase complex

Adenylate kinase, which catalyzes the reversible ATP-dependent phosphorylation of AMP to ADP and dAMP to dADP, can also catalyze the conversion of nucleoside diphosphates to the corresponding triphosphates. Lu and Inouye (Lu, Q., and Inouye, M. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5720-5725) showed that an Escherichia coli ndk mutant, lacking nucleoside diphosphate kinase, can use adenylate kinase as an alternative source of nucleoside triphosphates. Bacteriophage T4 can reproduce in an Escherichia coli ndk mutant, implying that adenylate kinase can meet a demand for deoxyribonucleoside triphosphates that increases by up to 10-fold as a result of T4 infection. In terms of kinetic linkage and specific protein-protein associations, NDP kinase is an integral component of T4 dNTP synthetase, a multienzyme complex containing phage-coded enzymes, which facilitates the synthesis of dNTPs and their flow into DNA. Here we asked whether, by similar criteria, adenylate kinase of the host cell is also a specific component of the complex. Experiments involving protein affinity chromatography, immunoprecipitation, optical biosensor measurements, and glutathione S-transferase pulldowns demonstrated direct interactions between adenylate kinase and several phage-coded enzymes, as well as E. coli nucleoside diphosphate kinase. These results identify adenylate kinase as a specific component of the complex. The rate of DNA synthesis after infection of an ndk mutant was found to be about 40% of the rate seen in wild-type infection, implying that complementation of the missing NDP kinase function by adenylate kinase is fairly efficient, but that adenylate kinase becomes rate-limiting for DNA synthesis when it is the sole source of dNTPs.     

Complementation of yeast Arc1p by the p43 component of the human multisynthetase complex does not require its association with yeast MetRS and GluRS

Yeast Arc1p, human p43 and plant methionyl-tRNA synthetase (MetRS) possess an EMAPII-like domain capable of non-specific interactions with tRNA. Arc1p interacts with MetRS (MES1) and GluRS and operates as a tRNA-interacting factor (tIF) in trans of these two synthetases. In plant MetRS, the EMAPII-like domain is fused to the catalytic core of the synthetase and acts as a cis-acting tIF for aminoacylation. We observed that the catalytic core of plant MetRS expressed from a centromeric plasmid cannot complement a yeast arc1(-) mes1(-) strain. Overexpression of the mutant enzyme from a high-copy number plasmid restored cell growth, suggesting that deletion of its C-terminal tIF domain was responsible for the poor aminoacylation efficiency of that enzyme in vivo. Accordingly, expression of full-size plant MetRS from a centromeric plasmid, but also of fusion proteins between its catalytic core and the EMAPII-like domains of yeast Arc1p or of human p43 restored cell viability. These data showed that homologous tIF domains from different origins are interchangeable and may act indifferently in trans or in cis of the catalytic domain of a synthetase. Unexpectedly, co-expression of Arc1p with the catalytic core of plant MetRS restored cell viability as well, even though Arc1p did not associate with plant MetRS. Because Arc1p also interacts with yeast GluRS, restoration of cell growth could be due at least in part to its role of cofactor for that enzyme. However, co-expression of human p43, a tIF that did not associate with plant MetRS or with yeast GluRS and MetRS, also restored cell viability of a yeast strain that expressed the catalytic core of plant MetRS. These results show that p43 and Arc1p are able to facilitate tRNA aminoacylation in vivo even if they do not interact physically with the synthetases. We propose that p43/Arc1p may be involved in sequestering tRNAs in the cytoplasm of eukaryotic cells, thereby increasing their availability for protein synthesis.