Opinion
TRENDS in Biochemical Sciences
Vol.32 No.4
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.
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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
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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
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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
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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
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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
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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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