National Creative Research Initiatives Center for ARS Network, College of Pharmacy, Seoul National University, Seoul 151-742, Korea
* Author for correspondence (e-mail: sungkim{at}snu.ac.kr)
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Summary |
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Key words: Aminoacyl-tRNA synthetase, Macromolecular protein complex, Multi-functionality, Protein network, Protein synthesis
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Introduction |
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Translation is one of the most complex biological processes, involving diverse protein factors and enzymes as well as messenger and transfer RNAs. As this process is required for the basic operation of cells, many translational factors and enzymes are considered to be housekeeping proteins. Aminoacyl-tRNA synthetases (ARSs) catalyze the ligation of specific amino acids to their cognate tRNAs, which is the initial step in protein synthesis. The aminoacylation reaction proceeds in two stages. First, ARSs activate their substrate amino acids by forming aminoacyl adenylate. Second, the enzyme-bound reaction intermediates are transferred to the 3' acceptor end of the tRNAs docking onto their active sites. Because tRNAs cannot distinguish amino acids conjugated to their ends, the correct recognition of amino acids and tRNAs by these enzymes is a crucial determinant to maintain the fidelity of protein synthesis.
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Moonlighting functions of ARS proteins |
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Mitochondrial YRS of Neurospora crassa and mitochondrial LRS of yeast species are required for the splicing of group I introns (Herbert et al., 1988; Cherniack et al., 1990
; Labouesse, 1990
). By contrast, human YRS can be split by polymorphonuclear (PMN) elastase into two fragments that display distinct cytokine activities (Wakasugi and Schimmel, 1999a
; Wakasugi and Schimmel, 1999b
). One of the fragments (mini-YRS) contains a conserved ELR motif identical to that found in CXC chemokines such as interleukin 8 (IL-8), Gro-
, Gro-ß, Gro-
and NAP-2, which act as angiogenic factors (Herbert et al., 1988
; Clark-Lewis et al., 1991
; Clark-Lewis et al., 1993
; Arenberg et al., 1997
). As expected, mini-YRS induces angiogenesis (Wakasugi et al., 2002a
). Whereas human YRS is converted into two distinct cytokines by proteolytic processing (Wakasugi and Schimmel, 1999a
; Wakasugi and Schimmel, 1999b
), an N-terminally truncated form of WRS, possibly generated through alternative splicing, works as an anti-angiogenic cytokine (Otani et al., 2002
; Wakasugi et al., 2002b
; Kise et al., 2004
).
Human MRS represents another example. It is translocated to the nucleus under proliferative conditions to augment rRNA synthesis in nucleoli, and the presence of MRS in nucleoli depends on the integrity of rRNA and the activity of RNA polymerase I. The addition of MRS stimulates rRNA synthesis, which indicates that it plays a role in rRNA synthesis in nucleoli, although the underlying mechanism is not clearly understood (Ko et al., 2000). Human QRS is recruited to apoptosis signal-regulating kinase 1 (ASK1) to block its kinase activity. The interaction of the two proteins is stimulated by the QRS substrate, glutamine, which can suppress cell death (Ko et al., 2001a
). Note that, in general, the non-canonical activities of ARSs appear to be more prevalent in mammalian systems.
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Mammalian multi-ARS complexes |
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Several lines of evidence have suggested that the translational apparatus in mammalian cells is highly organized. In particular, association of translational components such as tRNA, ARSs and elongation factors with the cytoskeletal framework (Dang et al., 1983; Mirande et al., 1985a
; Sanders et al., 1996
) and colocalization of these components have been described (Barbarese et al., 1995
). ARSs can be classified into two groups based on their structural features (Eriani et al., 1990
; Burbaum and Schimmel, 1991
; Cusack et al., 1991
). Class I ARSs each possess a Rossman fold in their catalytic domains, whereas class II enzymes contain three homologous motifs with degenerate sequence similarity. ARSs can also be grouped on the basis of their ability to form complexes with each other and non-enzymatic factors. Among the complexes formed by ARSs, the mammalian ARS complex is the most intriguing (Robinson et al., 2000
; Kim et al., 2002
; Ko et al., 2002
; Han et al., 2003
). This complex is distinctive compared with other macromolecular protein complexes in that its components are enzymes that carry out similar catalytic reactions simultaneously, and only a subset of ARSs are involved.
Although there is still some ambiguity about the stoichiometry and total number of components, at least nine different ARSs, including both class I and class II enzymes, have been consistently found in the mammalian complex: EPRS, IRS, LRS, MRS, QRS, RRS, KRS and DRS. Among these, IRS, LRS, MRS, QRS and RRS are monomers, whereas KRS and DRS are dimers. The largest component EPRS harbors two catalytic activities in a single polypeptide. The complex also contains three auxiliary factors of p43, p38 and p18 (Quevillon and Mirande, 1996; Quevillon et al., 1997
; Quevillon et al., 1999
). The complex has been purified to homogeneity from various mammalian tissues, including rat liver, rabbit liver and reticulocytes, sheep liver and spleen (Brevet et al., 1982
; Kellermann et al., 1982
; Cirakoglu and Waller, 1985
; Venema and Traugh, 1991
), as well as from cultured cells such as Chinese hamster ovary (CHO) cells (Mirande et al., 1985b
) and murine erythroleukemia cells (Norcum, 1989
). The complexes purified in these experiments display very similar patterns following gel electrophoresis, comprising 11 polypeptides with molecular weights from 18 kDa to 150 kDa (Kerjan et al., 1992
; Kerjan et al., 1994
). Although all of the enzymatic activities present can be assigned to their corresponding polypeptides, with the exception of PRS (Mirande et al., 1982
; Cirakoglu and Waller, 1985
), the structural organization of this complex has not yet been completely deciphered.
Several approaches have probed the structural organization of this complex. Its components can be partially dissociated under a variety of conditions, such as repeated centrifugation in the presence of high concentrations of phosphate (Dang and Yang, 1979), hydrophobic chromatography (Johnson et al., 1980
) and incubation with chaotropic salts or detergents (Sihag and Deutscher, 1983
; Norcum, 1991
). The gross morphology of the complex has been explored by electron microscopy (Norcum, 1989
; Norcum and Boisset, 2002
; Wolfe et al., 2003
) (Fig. 1), and the nearest neighbors among the component parts have been determined by chemical crosslinking (Norcum and Warrington, 1998
) and genetic approaches (Rho et al., 1996
; Quevillon et al., 1999
).
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The complex-forming and non-complex-forming human ARSs do not display distinct size distributions, structural features, post-translational modifications, expression profiles or chromosomal locations (Table 2 and data not shown). Moreover, comparison of functional motifs present in ARSs provides few clues as to what is responsible for complex formation. Interestingly, domains homologous to glutathione S-transferase (GST) are present in the N-terminal extensions of MRS and EPRS (Quevillon and Mirande, 1996; Quevillon et al., 1999
) among the complex-forming enzymes, as well as the C-terminal regions of p18 and p38 among the non-enzymatic cofactors (Galani et al., 2001
) (Fig. 2). Although these domains are also observed in other ARSs, such as mammalian VRS (Fig. 2) and the putative ERSs of Schizosaccharomyces pombe and Arabidopsis thaliana, these enzymes are also likely to be involved in different types of complex. Mammalian VRS is associated with elongation factor subunits that also contain the GST homology domain (Bec et al., 1989
; Bec et al., 1994
; Brandsma et al., 1995
), and a CRS isoform can potentially associate with elongation factor subunits (Kim, J. E. et al., 2000
). In yeast, ERS forms a primitive complex with MRS and Arc1p, and the importance of the GST-homology domain for complex formation has been shown experimentally (Deinert et al., 2001
; Galani et al., 2001
). Thus, the presence of the GST domain might be a crucial determinant for complex formation.
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Multi-functionality of non-enzymatic components |
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p43 plays a complex role in angiogenesis. Although it induces migration of endothelial cells at low concentration, it suppresses angiogenesis by blocking the proliferation and triggering apoptosis of endothelial cells at high concentrations (Park, S. G. et al., 2002). p43 contains a caspase-cleavage site, which is targeted upon apoptosis; this releases the C-terminal domain of p43 (previously known as EMAPII) from the complex. The process was thought to trigger the secretion of the cytokine component from p43, causing the disintegration of the multi-ARS complex to block protein synthesis. However, p43 processing does not appear to affect the function of the complex, and it turns out that the uncleaved form of the p43 is the active cytokine (Ko et al., 2001b
). The role of proteolytic cleavage of p43 at apoptosis is thus unclear at this point.
p38 also has an unexpected additional role. It can bind to FUSE-binding protein (FBP), a transcriptional activator of the gene encoding Myc, which promotes its ubiquitylation and proteasome-dependent degradation (Kim et al., 2003). When the expression of endogenous p38 is abolished by insertion of a gene-trap vector in the p38-encoding gene, Myc is overexpressed owing to the lack of p38-mediated suppression, which causes hyperproliferation of lung cells. The consequent malfunction of the lung causes p38/ mice to die at birth, although they survive development through the prenatal stage.
It is not known yet whether the smallest cofactor, p18, is also multi-functional. It shares limited sequence similarity with elongation factor subunits (Quevillon and Mirande, 1996), thus it could have a role connecting the aminoacylation and translational machineries. Considering the functional diversity of the two other factors, it would not be surprising to find other crucial activities of this factor.
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The evolution of ARS complexes |
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Trbp111, a homolog of Arc1p, is present in the extreme thermophile Aquifex aeolicus (Morales et al., 1999) and binds to single tRNA molecules as a dimer (Swairjo et al., 2000
). However, it is not yet known whether this protein forms any specific complex with ARSs. Recently, Lipman et al. copurified a novel protein, Mj1338, with PRS from the archaeon M. jannaschii. Mj1338 also has a general affinity for tRNA and the potential to interact with KRS and DRS, in addition to PRS (Lipman et al., 2000
; Lipman et al., 2003
) (Fig. 3C). Interestingly, it is predicted to be a metabolic protein related to the members of the H2-forming N5, N10-methylene tetrahydromethanopterin (5,10-CH2-H4MP) dehydrogenase family, which catalyze an intermediate step in the C1 unit metabolism of the methanogen.
p43 shares structural similarity with Trbp111 and other tRNA-binding proteins present in lower organisms (Renault et al., 2001), and is capable of binding to tRNA (Shalak et al., 2001
) to help the tRNA dock onto the bound ARS (Park et al., 1999
) (Fig. 4A). Clear homologs of p38 and p18 have not been found in lower eukaryotes or bacteria. However, the C-terminal domains of p18 and p38 share significant sequence similarity with the N-terminus of Arc1p (Galani et al., 2001
), which might thus combine features of the three non-enzymatic members of the mammalian complex.
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Interestingly, many ARSs also have cis-acting domains that appear to facilitate the recruitment of tRNAs to their catalytic sites (Cahuzac et al., 2000; Frugier et al., 2000
; Kaminska et al., 2001
; Francin et al., 2002
; Francin and Mirande, 2003
). For example, the C-terminus of E. coli MRS (Morales et al., 1999
), and the N-terminus of the E. coli FRS ß-subunit (Simos et al., 1996
) (Fig. 2) share structural similarity with the nonenzymatic factors in the mammalian ARS complex, and thus they probably function similarly to their trans-acting counterparts (Valenzuela and Schulman, 1986
; Kim et al., 1993
; Mosyak et al., 1995
; Goldgur et al., 1997
). It is thus likely that the transacting tRNA-binding proteins derive from the ARSs (Fig. 2, Fig. 4A) but have acquired more functional flexibility during evolution. However, we cannot exclude the alternative possibility that they were inserted into the ARS structure to augment the catalytic efficiency of the enzymes. Some ARSs also contain cis-acting motifs that are structurally unrelated to the common domains found in Arc1p, p43 and Trbp111 (Cahuzac et al., 2000
; Frugier et al., 2000
; Kaminska et al., 2001
; Francin et al., 2002
; Francin and Mirande, 2003
) (Figs 2, 4). They usually form amphiphatic helices in which one side of the helix displays an array of basic residues for interaction with tRNAs (Fig. 4B).
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Assembly and disassembly of ARSs and cofactors |
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The function of the multi-ARS complex |
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Components of the EF-1 complex could be attracted to the charged tRNAs by their direct affinity for ARSs as well as tRNAs (Sivaram and Deutscher, 1990; Negrutskii and Deutscher, 1991
; Negrutskii and Deutscher, 1992
; Stapulionis and Deutscher, 1995
). ARSs such as DRS, FRS, LRS, HRS, EPRS, ARS, KRS and WRS have been observed to interact with subunits of EF-1H (Reed and Yang, 1994
; Negrutskii et al., 1996
; Lee et al., 2002
). The multi-ARS complex could thus generate a channel for the delivery of tRNAs (Calado et al., 2002
; Simos et al., 2002
; Hopper and Phizicky, 2003
).
Another complex, in which VRS is associated with the four subunits of EF-1H, is also present (Bec et al., 1989; Bec et al., 1994
; Brandsma et al., 1995
; Negrutskii et al., 1999
; Galani et al., 2001
). In this complex, the N-terminal extension of VRS is bound to the EF-1H subunits (ß,
and
) that are responsible for guanine nucleotide exchange. Careful kinetic analyses of the VRS EF-1H complex demonstrated that the catalytic activity of VRS is enhanced about twofold by the addition of the
subunit (Negrutskii et al., 1999
).
A systematic trafficking network involving mammalian ARSs and the translational machinery might thus exist (Negrutskii and Deutscher, 1991). Indeed, the primitive complex consisting of Arc1p, MRS and ERS found in yeast provides supporting evidence for this model (Simos et al., 1996
; Simos et al., 1998
). However, clustering of different ARSs within a complex might not necessarily positively affect the flow of tRNAs. The macromolecular assembly might sterically hinder efficient movement of large tRNA substrates. Thus, it will be interesting to see how ARSs are spatially arranged within the complex so that different tRNAs can move into and out of their cognate catalytic cores without colliding.
Alternatively, complex formation might contribute to the subcellular localization of ARSs. The ARS complex has been found in the nucleus (Nathanson and Deutscher, 2000), and the catalytic activities of the ARSs are thought to help the proofreading of newly synthesized nuclear tRNAs (Lund and Dahlberg, 1998
). Another function of the complex might be to control the cellular turnover of the components. The chaperone Hsp90 facilitates assembly of multi-ARS complexes (Kang et al., 2000
). Blocking its activity suppresses the incorporation of nascent ARSs, which are subsequently degraded. This indicates that their association is required to protect the components from degradation. The finding that dissociation of the components by depletion of the p38 protein results in their rapid degradation further supports this idea (Kim et al., 2002
). Thus, their association to form a complex might control the cellular turnover of ARSs.
Last, complex formation might be used to control non-canonical activities of the components. As already mentioned, several ARSs have additional functions (Table 1). Similarly, two of the non-enzymatic factors, p43 and p38, also play unique moonlighting roles. Thus, cells must somehow control the dynamic equilibrium between ARSs and cofactors used for protein synthesis and those used for novel regulatory activities. The multi-ARS complex may thus function as a molecular reservoir for distribution of these enzymes and cofactors. Further work is clearly needed if we are to understand the functions of this complex. Note that the roles suggested above need not be mutually exclusive, and additional functions are of course possible.
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The dynamic balance of complex components |
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Protein-protein interactions can be regulated by post-translational modifications such as phosphorylation. There are many phosphorylation sites for different kinases in ARSs. Damuni et al. have shown that the catalytic activities of the complex-forming ARSs can be modulated by phosphorylation in vivo and in vitro (Damuni et al., 1982). Five enzymes (DRS, QRS, ERS, MRS and KRS) are phosphorylated in reticulocytes. QRS and DRS are selectively phosphorylated in response to 8-bromo-cAMP (Pendergast et al., 1987
), and DRS, ERS, MRS and KRS are phosphorylated in vitro by casein kinase I (Pendergast and Traugh, 1985
). Phosphorylation by casein kinase I reduces the aminoacylation activity and alters the binding of the ARS complex to tRNA-Sepharose. Protein kinase C selectively phosphorylates QRS in rabbit reticulates stimulated by tumor-promoting phorbol esters (Venema and Traugh, 1991
). Phosphorylation by protein kinase C in vivo also inhibits aminoacylation activity. However, no solid evidence is yet available that the assembly or disassembly of the multi-ARS complex is regulated by phosphorylation (Pendergast et al., 1987
).
Since p38 is indispensable for the maintenance of the multi-ARS complex, it is unlikely that p38 embedded in the multi-ARS complex is dispatched to other sites, since this would destabilize the complex. To prevent a shortage of p38 in the multi-ARS complex when it needs to be delivered to other target sites, the level of p38 should be dynamically regulated. Indeed, the level of p38 is significantly increased by transforming growth factor (TGF)-ß, which generates additional p38 that is not bound to the multi-ARS complex (Kim et al., 2003). This fraction of p38 localizes to the nucleus, as determined by cell fractionation and immunofluorescence staining to control Myc expression (Kim et al., 2003
). Thus, at least the `static model' seems to apply to p38. However, because p38 is the main switch for assembly of the complex, it can also be used to control complex formation.
As mentioned above, one of the complex-forming ARSs, QRS, can bind to ASK1, and this interaction is enhanced by increases in the level of glutamine without changing the total cellular level of QRS (Ko et al., 2001a). In this case, glutamine might control the dynamic equilibrium between QRS in the ARS complex and at its alternative target site. Perhaps QRS shuttles between the multi-ARS complex and ASK1, depending on the conditions or glutamine concentration. These observations favor the partial association/dissociation model. Thus, different models appear to apply, depending on the component.
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Conclusions and perspectives |
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Why did these multi-functional factors become part of the ARS community? It could simply be an evolutionary coincidence: they happened to associate with the ARSs, through their GST-homology domains (Deinert et al., 2001; Galani et al., 2001
), and subsequently other enzymes joined the primitive complex. In fact, p38 and p18 interact with each other, and p18 specifically interacts with MRS (Quevillon et al., 1999
). In addition, the deletion of the GST-homology domain in the C-terminal region of p38 results in the dissociation of EPRS and MRS from the complex (Kim et al., 2002
).
Alternatively, these cofactors might have become linked to ARSs for functional reasons. ARSs can be good sensors of cellular conditions because they use amino acids as their reaction substrates. Interestingly, many ARSs can undergo conformational changes following binding to amino acids (Kornelyuk et al., 1995; Onesti et al., 2000
). In the case of QRS, its anti-apoptotic interaction with ASK1 is stimulated by the increase in glutamine levels (Ko et al., 2001a
). In addition, the concentration of charged/uncharged tRNAs is a crucial determinant for protein synthesis (Rojiani et al., 1990
; Kimball, 2001
). Therefore, these factors might monitor the condition of the cell by being physically linked to ARSs, in addition to enhancing the stability and catalytic capability of ARSs or controlling their cellular trafficking.
The evolutionary paradox in gene evolution is that higher eukaryotic cells harbor much more DNA than necessary. Conversely, the number of encoded proteins in these organisms appears to be much smaller than we used to predict. How can this limited number of proteins meet the demand for functional diversity that highly differentiated multicellular organisms require? Mammalian systems appear to take advantage of subcellular compartments, in which the same protein can be placed in a different physical environment or combined with a different repertoire of proteins. Perhaps higher organisms have evolved to maximize the safety and flexibility of genetic information by having extra DNA, yet economize by using one protein for many different purposes. In this regard, the mammalian ARSs and their associated factors provide a fascinating example of such multi-functionality.
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Acknowledgments |
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Footnotes |
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References |
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