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Macromolecular complexes as depots for releasable regulatory proteins Partho Sarothi Ray, Abul Arif and Paul L. Fox Department of Cell Biology, Lerner Research Institute, Cleveland Clinic, 9500 Euclid Avenue/NC10, Cleveland, OH 44195, USA
Multi-component, macromolecular complexes perform essential cellular functions that require spatial or temporal coordination of activities. Complexes also facilitate coregulation of protein amounts and cellular localization of individual components. We propose a novel function of multi-component complexes as depots for regulatory proteins that, upon release, acquire new auxiliary functions. We further propose that component release is inducible and context-dependent. We describe two cases in which multi-component assemblies – the ribosome and tRNA multi-synthetase complex – function as depots. Both complexes have crucial roles in supporting protein synthesis but they also release regulatory proteins for inflammation-responsive, transcript-specific translational control. Recent evidence indicates that other macromolecular assemblies might be sources for proteins with auxiliary functions, and the depot mechanism might be widespread in nature. Introduction Multi-component, macromolecular complexes are ubiquitous in the three domains of life. A global analysis of Saccharomyces cerevisiae found >500 protein complexes with an average of 4.9 proteins per complex [1]. Complexes can be transient or long-lived. Transient complexes usually transduce signals or transport small molecules from one cell location to another. By contrast, stable macromolecular assemblies facilitate complicated, multi-step cellular processes. Advantages of stable complexes include coordinate control of reaction rates, high reaction efficiency owing to vectorial transfer of substrates and intermediates between components, regulation of cellular compartmentalization, and coordinate regulation of component levels (e.g. by degradation of unbound protein) [2]. Many complexes behave as molecular machines; coordinating sequential reactions while minimizing diffusion of substrates and intermediates. For example, the ribosome, a multi-protein–RNA complex, brings together the mRNA, aminoacylated tRNAs and the elongating peptide chain on the same molecular platform to sequentially perform the peptidyl-transferase reaction [3]. Recent studies indicate that distinctions between transient and stable complexes might be blurred. Macromolecular complexes can be stimulated to release component proteins that acquire non-canonical, or ‘moonlighting’, functions distinct from their primary, canonical Corresponding author: Fox, P.L. (
[email protected]). Available online 23 February 2007. www.sciencedirect.com
activity [4,5]. These results have led us to propose a ‘depot hypothesis’ in which macromolecular assemblies, while maintaining their ordinary activity, acquire the non-canonical capability to release component proteins that perform new functions outside the complex. According to this view, depot complexes are functionally positioned between stable ‘machine-like’ complexes and transient signaling complexes. Here, we define the depot hypothesis, describe the common features of macromolecular depots and their released daughter proteins, and draw attention to several macromolecular complexes that might function as depots. We also formalize criteria that establish depot functions of macromolecular complexes, and speculate on the origins and potential benefits of depot systems. This discussion is particularly timely because recent analyses of cellular proteomes using tools of functional genomics and systems biology have firmly established macromolecular complexes as hubs of protein-interaction networks that control cellular function [1,6]. Moreover, two macromolecular complexes functioning as depots have been discovered recently [4,5]. The ability to function as depots for regulatory proteins adds a new dimension to the functions of macromolecular complexes and indicates additional versatility in their cellular roles. Two depots in eukaryotic translational control Our concept of complexes as depots developed from our own studies of translational control of gene expression [4,5]. Eukaryotic translation is usually regulated at the initiation step, a temporally and spatially coordinated sequence of events that involves several large, multi-component complexes [7]. The regulation can be global and affect most mRNAs, or it can be mRNA-specific. The latter mechanism typically involves interaction of an RNA-binding protein or complex to a structural element in the target transcript. Translation of interferon-g (IFN-g)-induced ceruloplasmin mRNA in human monocytic cells is silenced by a multi-protein, IFN-g-activated inhibitor of translation (GAIT) complex that binds to a structural element (GAIT element) in the 30 -untranslated region (30 UTR) of ceruloplasmin mRNA [8,9]. The four components of the GAIT complex assemble in two steps [4,5] (Figure 1). Glu-ProtRNA synthetase (GluProRS) and NS1-associated protein1 (NSAP1) form an inactive, pre-GAIT complex within 2 h of IFN-g treatment. Approximately 12 h later, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and ribosomal protein L13a join to form the active GAIT complex. The two GAIT-complex components GluProRS and L13a normally
0968-0004/$ – see front matter ß 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2007.02.003
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Figure 1. Ribosome and tRNA multi-synthetase complexes are depots for translational control proteins. IFN-g induces phosphorylation and release of GluProRS (green) from the tRNA multi-synthetase complex. Phosphorylated (red sphere) GluProRS (P-GluProRS) joins NSAP1 (pink) to form an inactive, pre-GAIT complex. Subsequently, ribosomal protein L13a (dark blue) is phosphorylated (P-L13a) and exits the large ribosomal subunit (light blue). P-L13a joins GAPDH (brown) and the pre-GAIT complex to form the active GAIT complex, which binds to the GAIT element in the 30 UTR of the target mRNA and inhibits its translation by targeting the translation initiation complex (orange) and blocking ribosome recruitment [47]. The tRNA multi-synthetase complex and ribosome might be joined by mutual interactions with eukaryotic elongation factor-1 (eEF1; orange) [48].
reside in other stable, multi-component complexes but, on IFN-g stimulation, become associated with the GAIT complex and participate in translation inhibition outside their parent complexes. GluProRS, the only bifunctional tRNA synthetase, catalyzes acylation of both glutamic acid and proline to cognate tRNAs. GluProRS resides in the 1.5-mDa tRNA multi-synthetase complex (MSC) that contains seven other tRNA synthetases and three non-synthetase proteins [10]. GluProRS in the GAIT complex can originate from several sources: the MSC, a pre-existing free pool or newly synthesized protein. The appearance of GluProRS in the preGAIT complex coincides temporally and quantitatively with its disappearance from the MSC, indicating the MSC as the source [5]. The unbound pool of GluProRS is an unlikely source because it contains much less GluProRS than that found in the pre-GAIT complex. Lastly, newly synthesized GluProRS is excluded because GluProRS is found in the pre-GAIT complex even in the absence of protein synthesis. The mechanism of release from the MSC is unknown; however, IFN-g induces serine phosphorylation of GluProRS just before its release, and release is blocked by Ser/Thr kinase inhibitors [5]. Electron-microscopy studies place GluProRS at the exterior of the MSC, which is consistent with susceptibility to inducible release [11]. The appearance of L13a in the GAIT complex coincides with its disappearance from the ribosome, indicating the eukaryotic large ribosomal subunit as source [4,5]. The mechanism of L13a release from the ribosome is unknown, but IFN-g-induced phosphorylation of L13a coincides with, and is required for, its release. X-ray crystallography of the archaeal large ribosomal subunit of Haloarcula marismortui shows that L13, the archaeal homolog of eukaryotic www.sciencedirect.com
L13a, resides entirely on the ribosome RNA surface, having essentially no contact with adjacent or underlying proteins [12] (Figure 2a). L13 lacks the long, rRNA-penetrating extensions that are characteristic of many other archaeal large ribosomal subunit proteins (Figure 2d–f), and is distant from the interior rRNA domains responsible for ribosome catalysis. Similar to many other ribosomal proteins, no role for L13 in ribosome function has been reported. Surface localization of the protein could facilitate its release while minimizing disruption of the remaining parent complex. If these observations can be extended to the eukaryotic homolog, then the structure and location of L13a are consistent with unhindered escape from the ribosome without disruption of global protein synthesis. In summary, elucidation of the GAIT pathway has revealed that two macromolecular assemblies – the MSC and ribosome – are induced to release specific component proteins to form a new regulatory complex. Remarkably, the entire cellular complement of L13a and approximately half of the GluProRS escape from their parent complexes, yet total protein synthesis continues unperturbed. These findings form the experimental underpinnings of the ‘depot hypothesis’, which holds that macromolecular assemblies, in addition to functioning as machines that coordinate complex tasks, also function as depots for releasable regulatory proteins. In the cases described here, the function of released daughter proteins (i.e. transcript-selective translational control) is intimately related to the function of the parent complexes (i.e. protein synthesis). Therefore, parent macromolecular complexes and released daughter proteins are active in the same locale, indicating that a principal function of depot complexes might be to localize regulatory proteins in appropriate intracellular compartments, where they are released upon appropriate signals.
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Figure 2. Two classes of large ribosomal subunit proteins. High-resolution structural data for the eukaryotic ribosome is lacking, but a high-resolution structure for the archaeal Haloarcula marismortui ribosome has been published. The ribosomal proteins shown here have eukaryotic homologs (indicated in parentheses). The first class of ribosomal proteins, (a) L13, (b) L5 and (c) L14 (yellow spheres), lack protrusions into the rRNA core (gray tubes) and have only minimal contact with other ribosomal proteins (blue spheres) and are candidates for release from the archaeal large ribosomal subunit. The second class of ribosomal proteins, (d) L2, (e) L3 and (f) L15, have long protrusions into the rRNA core and important interactions with other proteins, and are poor release candidates.
Characteristics of depot systems A depot system consists of a ‘parent’ complex and a released ‘daughter’ protein. The ribosome and the MSC, and their daughter proteins L13a and GluProRS, respectively, can be used as prototypes for formalization of criteria to establish a depot function for a cellular complex (Box 1). Depot parent and daughter relationships could exhibit certain characteristics. For example, the daughter protein is likely to reside at the surface of the parent complex, with minimal penetrations into the core, to facilitate release and reduce perturbation of the daughterless parent complex. Archaeal large ribosomal subunit proteins L13, L5 and L14 (and potentially their eukaryotic homologs L13a, L11 and L23, respectively) satisfy this criterion and are candidates for release (Figure 2a–c). By contrast, archaeal proteins L2, L3 and L15 (and eukaryotic homologs L8, L3 and L27a, respectively) have long protrusions into the RNA core and are poor candidates for release (Figure 2d–f). However, the complex-penetrating domains of such proteins might be removed by the activation of specific proteases. Likewise, a scaffolding protein that binds to multiple components of a macromolecular complex, for example, p38 of the MSC, is www.sciencedirect.com
an unlikely candidate because its release would disrupt the integrity of the parent complex [13]. Multiple release mechanisms are possible. For L13a and GluProRS, protein phosphorylation is crucial [4,5]. In the simplest mechanism, daughter-protein phosphorylation could decrease its affinity for the parent complex, possibly by a conformational change. Alternatively, daughter-protein phosphorylation might increase its affinity for a non-depotbinding partner, and facilitate release from the parent complex. This mechanism could pertain to GluProRS, which, upon phosphorylation, binds to NSAP1 and is released from the MSC [5]. Because post-translational modifications such as phosphorylation are usually regulatable, it might be a principal mechanism by which stimulus- or context-dependent signals are transduced to induce daughter-protein release. Alternative triggers for daughter-protein release are also possible. These include proteolytic cleavage of domains that anchor the daughter protein to the parent complex or conformational change of the daughter protein due to interaction with non-depot proteins or nucleic acids. Daughter-protein release from a parent complex provides a unique, stimulus-dependent mechanism of
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Box 1. Criteria and classification of depot systems Criteria for establishing depot function for a macromolecular complex The protein donor is a multi-component, macromolecular complex that is stable under normal conditions; for example, the ribosome, proteasome and multi-enzyme complexes. This criterion excludes transient assemblies, for example, signaling complexes. The daughter protein(s) is released from the parent complex. To rigorously establish a release mechanism, free daughter protein appearance should correspond temporally and quantitatively to its disappearance from the parent complex. De novo synthesis can be ruled out with protein-synthesis inhibitors. A free, non-bound pool can be eliminated by showing that it is smaller than the daughter pool. The parent complex remains structurally intact after daughterprotein release. Structural integrity can be assessed by activity or by size fractionation of the complex and biochemical identification of the remaining components. Daughter-protein release is context- and/or stimulus-dependent. The released protein is functional, and functions independently of the parent complex. The daughter protein, by itself or in association
with other proteins or DNA or RNA, has a cellular function that is distinct from that of the parent complex. Three classes of depot systems Depot systems can be subdivided into three classes based on the functional relationship of the parent complex and daughter protein (Figure I). Type 1: the daughter protein is inactive in the parent complex but acquires function upon release. For example, the ribosomal protein L13a does not have a known function in the mature ribosome but acquires translation-repression activity upon release [4]. Type 2: the daughter protein performs the same function in the parent complex and after release. For example, the ATPases of the 19S proteasome-regulatory subunit bind to and remodel transcription-initiation machinery at specific promoters [44]. Type 3: the daughter protein performs different functions in and out of the parent complex. For example, the GluProRS catalyzes tRNA aminoacylation in the parent MSC but mediates translational silencing upon release [5]. Both functions might be mediated by different domains or by a single domain that undergoes a conformational switch.
Figure I. Criteria and classification of depot systems. Daughter proteins (blue) and parent complexes (yellow) are shown for each depot system type, which are classified by functional relationship.
protein activation. The depot system has the advantage of speed because it does not require transcription or protein synthesis for activation. In addition, the system is potentially reversible and, therefore, energetically conservative. Moreover, the parent complex can convey the daughter protein to the appropriate intracellular region, thereby establishing an energetically favorable network in which the parent complex reduces diffusion of the daughter protein away from its site of activity, replacing energydependent pathways of transport. Alternatively, the parent complex could sequester the daughter protein from its site of action, particularly in cases in which the protein can cause cell injury if ectopically present. The parent complex could mask or alter the active site of the daughter protein. Lastly, the parent complex could regulate turnover of the daughter protein by protecting it from inactivating post-translational modification or from degradation by proteases. Other macromolecular complexes as depot candidates Many proteins exist in complex-bound and -free forms. Therefore, on the basis of the criteria outlined here, multiple macromolecular complexes might exhibit depot www.sciencedirect.com
functions. However, experimental evidence for protein release from the parent complex is lacking. Not all multi-protein complexes will necessarily exhibit depot functions; however, our criteria can form a useful framework for experiments to confirm the role of specific macromolecular complexes as depots. tRNA multi-synthetase complex The MSC is a multi-protein complex that contains nine aminoacyl-tRNA synthetases and three non-synthetase proteins [10]. Several of these synthetases have non-canonical functions unrelated to aminoacylation [14], including GluProRS. Lys-tRNA synthetase (LysRS) is an integral MSC component that is secreted in response to tumor necrosis factor-a and triggers a pro-inflammatory response in target macrophages; however, release from the MSC has not been shown [15]. LysRS also activates the transcription factors MITF (microphthalmia-associated transcription factor) and USF2 (upstream stimulatory factor-2) in the nucleus of activated mast cells [16]. Two other MSC synthetases are candidate daughter proteins. Met-tRNA synthetase translocates to the nucleolus in response to growth factors and enhances rRNA synthesis [17], whereas
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Gln-tRNA synthetase interacts with the apoptosis signal-regulating kinase-1 in a glutamine-dependent manner and inhibits its activity, thereby having an anti-apoptotic role [18]. Two other tRNA synthetases, Tyr-tRNA synthetase and Trp-tRNA synthetase, generate fragments that have cytokine activity [19]. Their residence in the MSC has not been shown, but possibly less disruptive purification procedures would show an association with the MSC. In addition, the three non-synthetase MSC proteins have extra-MSC functions. p43 is a potent cytokine that is secreted from endothelial and immune cells. The C-terminal domain of p43 [known as endothelial-monocyte-activating polypeptide II (EMAP II)] is released from the MSC following caspase cleavage under apoptotic conditions [20]. EMAP II induces mononuclear phagocyte migration and inhibits endothelial cell proliferation [20,21]. p38 and p18 also function independently of the MSC; however, their source seems to be de novo synthesis rather than the MSC because their release, especially release of the scaffolding protein p38, might compromise the integrity of the depot [13]. Ribosome The ribosome is a macromolecular complex composed of multiple rRNAs and proteins. Most of the ribosome mass consists of rRNA, and the absence of proteins near the peptidyl-transferase catalytic center indicates that the ribosome is a ribozyme [22]. More than half of the archaeal 50S proteins are candidates for release because, like L13a, they lack tethering extensions into the rRNA core [12]. Several ribosomal proteins have extra-ribosomal functions, often involving interactions with nucleic acids or with other proteins [23]. For example, eukaryotic small ribosomal subunit protein S3a, also known as v-fos transformation effector (FTE), interacts with the CHOP/GADD153 (CCAAT/enhancer-binding protein homologous protein or growth arrest- and DNA-damage-inducible protein) transcription factor [24]. The eukaryotic large ribosomal subunit proteins L5, L11 and L23 stabilize p53 by inhibiting the proteolytic activity of HDM2 (human homolog of murine double minute 2) [25–27]. Also, DNA damage induces L26 binding to the 50 UTR of p53 mRNA and enhances its translation [28]. Although release has only been shown for L13a, the ribosomal proteins described here are candidates for induced release as they are surface-located and lack penetrating tails. Proteasome The 26S proteasomal complex selectively degrades poly-ubiquitylated proteins [29]. It consists of a 20S proteolytic core and two 19S regulatory lid subunits [30] each containing 18 proteins, including six AAA-family member ATPases [31]. The ATPases unfold and transport proteins into the proteolytic core for degradation [32]. Independently of the 26S proteasome or the 19S subunit, proteasomal ATPases interact with the transcription initiation factor TATA-binding protein [33]. After galactose induction, the 19S ATPases Rpt1–6, together with two proteins that form the ‘base’ of the 19S, Rpn 1 and 2, bind to the GAL1–10 promoter in a Gal4-dependent manner and www.sciencedirect.com
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function as authentic transcriptional co-regulators [34]. These ATPases, known as APIS (AAA proteins independent of 20S) proteins, could be released as a group from the parent complex and facilitate energy-dependent remodeling of the transcription machinery during initiation, thereby enabling promoter escape and elongation [35,36]. Release from the parent has not been shown but the short, 10-min interval between transcription induction and promoter association of three of the ATPases Rpt1, Rpt4 and Rpt5 argues against de novo synthesis but for recruitment of pre-existing proteasomal ATPases [34]. The proteasome might, therefore, be a depot for a group of ATPases that have extra-proteasomal roles in transcription. Signalosome The COP9 signalosome complex is a nuclear, multi-protein complex that is involved in development [37]. The signalosome component Jab1-CSN5 is a co-activator of c-Jun [38], and is involved in nuclear export and subsequent degradation of the cyclin-dependent kinase inhibitor p27Kip1 [39]. Jab1 is present in two complexes in mammalian cells: a 450-kDa nuclear COP9 signalosome and a 100-kDa cytoplasmic complex. Importantly, the cytoplasmic complex disappears when nuclear export is blocked by leptomycin B, suggesting that the nuclear signalosome is the source of cytoplasmic Jab1. Other signalosome components are also present in the Jab1-containing cytoplasmic complex [39]. Therefore, the signalosome might be a depot for proteins that shuttle to the cytoplasm and facilitate export and degradation of p27. Components of other multi-protein complexes also function independently of their parent complexes. Several spliceosomal proteins such as Clf1p and Prp8 function independently of the spliceosome [40,41]. Similarly, subunits of the exosome, a macromolecular complex that has important roles in RNA processing and turnover, associate with protein complexes distinct from the exosome [42,43]. Further experiments are necessary to determine whether these proteins exist independently in these distinct complexes or are released from the parent complex. Origin and evolution of depots Several pathways for the evolution of depot systems can be envisioned (Figure 3): The accretion model In the ‘accretion’ scenario, a free protein with a pre-existing function is incorporated into a macromolecular complex during evolution. Gradual acquisition of proteins over evolutionary time is a phenomenon that is common to many macromolecular complexes. For example, the bacterial ribosome has 54 proteins, whereas the eukaryotic ribosome has accumulated an additional 26 (totaling 80) proteins [44]. Release of component proteins could have begun as an equilibrium-driven process, and later evolved into a stimulus-inducible mechanism. Molecular ‘symbiosis’ between the macromolecular complex and the incorporated protein could have conferred a selective advantage to the expanded complex by increasing functional efficiency, and to the daughter protein by enhancing cell compartmentalization, stability and speed
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Figure 3. Evolution of depot systems. Three scenarios for the evolution of depot systems are shown: ‘accretion’, ‘coalescence’ and ‘gain-of-release’. In the accretion scenario, a free protein with a pre-existing function is incorporated into a macromolecular complex. In coalescence, multiple dual-function proteins unite to form a complex that functions as a depot for their storage and inducible release. In gain-of-release, the component protein(s) of a complex acquires novel domains or undergoes modifications in existing domains, resulting in independent function and inducible release. Daughter proteins (blue) and parent complexes (yellow) are shown for each pathway, and release is indicated by the broken arrow.
of activation. The depot function of the ribosome might have evolved by the accretion mechanism. The heterogeneity of ribosomal protein structures indicates they did not arise to have a single role, or even related roles, but have been appended gradually during evolution. Proteins with nucleic-acid-binding activities are particularly good candidates for recruitment; therefore, the preponderance of nucleic-acid-binding motifs [23]. In agreement with this concept, several proteins that reside in ribosomes regulate processes that involve nucleic acids, for example, translational regulation by L13a [4] and L26 [28]. The coalescence model In the ‘coalescence’ model, multiple dual-function proteins could coalesce to form a complex that functions as a depot for their storage and inducible release. The tRNA MSC might be an example of this evolutionary mechanism because several synthetases have dual functions. Possibly, the selective advantage that drove these proteins to form the MSC was tRNA channeling, whereby aminoacylated tRNAs are directly transferred from tRNA synthetases to the elongation factors to the ribosomes, without diffusion into the cellular fluid [45]. The complex could also synchronize turnover of the components because MSC disruption by depletion of the core protein p38 causes rapid degradation of component proteins [13]. Moreover, the complex can function as a depot for these proteins, permitting development of inducible release mechanisms, and maintaining a dynamic equilibrium between the novel regulatory activities and protein synthesis. The gain-of-release model It is possible that tRNA synthetases in the MSC might have acquired novel domains or undergone modifications in existing domains, resulting in independent functions and inducible release of certain components. This suggests an alternative pathway for the evolution of depot www.sciencedirect.com
function, namely, ‘gain-of-release’, that is, acquisition of auxiliary function by a protein that is already present in the parent complex. In the MSC example, localization of the complex in a specific subcellular compartment, or its involvement in specific processes, could have driven the acquisition of independent, but related, functions of component proteins, for example, mRNA-binding and translation repression by GluProRS [5]. Concluding remarks The depot model establishes a new paradigm of macromolecular complex function. The depot system represents a unique stratagem adopted by cells to use ubiquitous molecular machines as reservoirs for regulatory proteins, to be released when conditions demand. Future studies of macromolecular complexes are likely to identify new depot systems and provide insights into the circumstances that induce release of daughter proteins and their release mechanisms. It is noteworthy that multiple MSC components are involved in the inflammatory response, for example, GluProRS regulates the levels of inflammatory proteins, and LysRS and p43 function as cytokines [5,15,46]. By contrast, candidate depot proteins that originate in the proteasome are primarily involved in transcription [34]. Therefore, each depot system might be programmed to participate in a specific physiological or pathological process. The failure of a depot system to release a protein, or unregulated or ectopic release, might have pathological consequences. Identifying these relationships might provide novel insights into the role of macromolecular complexes in health and disease. Acknowledgements We thank Ira Wool (University of Chicago) and Aparna K. Sapra (Max Planck Institute of Molecular Cell Biology and Genetics) for helpful discussions. This work was supported by funds from the National Institutes of Health (to P.L.F.), and by a Postdoctoral
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Fellowship from the American Heart Association, Ohio Valley Affiliate (to A.A.).
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