ATP-Dependent Protease Complexes
ATP-dependent protease complexes are large, oligomeric
complexes composed of chaperone and proteolytic subunits (Clp and proteasome
families). In some cases the chaperone and proteolytic parts are located in the
same polypeptide chain (Lon and FtsH families). These proteases are built on
the principle of self-compartmentalization – the proteolytic active sites are
buried inside a narrow channel in the oligomeric structure; only substrates
unfolded and translocated inside by the chaperone part are accessible to
degradation, thus the surrounding native proteins are protected from breakdown
(Schmidt et al., 1999). This structural organization, in which the protease
active sites are hidden into the interior chamber, allows only processive
degradation of unfolded polypeptide chains - gradual degradation to small
peptides (5-10 amino acids) and to amino acids, coupled to ATP-dependent,
chaperone-assisted unfolding and translocation of the unfolded polypeptide.
Energy-dependent
proteases, in cooperation with the chaperone system, act as protein quality
control systems in maintaining the functional state of cell proteins in
essential compartments such as cytosol, endoplasmic reticulum, mitochondria and
chloroplasts (Leidhold and Voos 2007). As peptide bond hydrolysis is an
exergonic reaction, ATP hydrolysis is most probably linked to specific
recognition, regulation and control over proteolysis. Besides ATP
coupling, these proteases are also able to degrade poorly structured or
oxidatively modified proteins in an ATP-independent manner (Kurepa et al.,
2009).
The Proteasome
The
main proteolytic degradation system of eukaryotes, operating in the cytoplasm
and nucleus, is the ubiquitin/26S proteasome pathway. The proteasome plays
a crucial role in the turnover of regulatory proteins, cellular house-keeping
and stress tolerance (Kurepa and Smalle, 2008). The structure and activities of
proteasomes are highly conserved among eukaryotes, suggesting essential
functions in protein homeostasis. This system is an extremely large and complex
route for protein degradation, accounting for nearly 6% of the Arabidopsis
thaliana transcriptome (Vierstra, 2009).
Proteins targeted for degradation are covalently tagged with
the highly conserved 76 residue peptide ubiquitin (Ub) by a cascade of three
types of enzymes ‒ E1 (ubiquitin-activating enzyme), E2
(ubiquitin-conjugating enzyme) and E3 (ubiquitin ligase) ‒
that catalyse the conjugation of Ub monomer to a lysine residue of the target
protein. E3 controls the specificity of Ub conjugation. In the Arabidopsis
genome, 2 E1s, at least 37 E2s and over 1,400 different E3s are expressed
(Vierstra, 2009). Ub conjugation is a reversible reaction - detachment of Ub is
catalysed by Ub-specific proteases that form a group of multiple enzymes,
controlling ubiquitination and recycling Ub for reuse. While
mono-ubiquitination of proteins facilitates interaction with ubiquitin binding
domains in specific target proteins and has a regulatory function,
polyubiquitinated proteins (at least four Ubs linked to target proteins by the
residue lysine 48 of Ub) are recognized by specific receptors within the 26S
proteasome or by adaptor proteins associated with the proteasome and are
degraded in an ATP-dependent manner (Miura and Hasegava, 2010).
The 26S proteasome controls proteolysis of key components of
numerous signalling pathways, regulated proteolysis of functional proteins and
removal of misfolded and damaged proteins (Smalle and Vierstra, 2004; Vierstra,
2009). Besides ubiquitin, there is a set of other ubiquitin-like proteins involved
in posttranslational modification of proteins such as RUB1/Nedd8, SUMO, HUB,
ISG15, and ATG (Catala et al., 2007). SUMO (small ubiquitin-related modifiers)
tagging of proteins proceeds in a pathway similar to that for ubiquitination,
with sequential action of analogous E1, E2 and E3 enzymes. SUMO-specific
proteases cleave off SUMO for re-use (Novatchkova et al., 2004). SUMOylation
regulates protein degradation and localization, protein–protein interactions,
transcriptional activity and counteracts the Ub-mediated degradation by the
proteasome (Novatchkova et al., 2004).
The 26S proteasome is a 32-subunit, multicatalytic protease
that degrades polyubiquitinated protein targets (Smalle and Vierstra 2004;
Vierstra, 2009). It is composed of two subcomplexes - the barrel-shaped,
proteolytically active core 20S proteasome (about 700 kDa in size) and the 19S
regulatory particle (700 kDa) that recognizes, unfolds and channels Ub-targeted
proteins into the 20S proteasome for degradation (Kurepa and Smalle, 2008). In
the absence of ATP the 26S proteasome dissociates into 20S and 19S subcomplexes
(Vierstra, 1996). The proteolytic core belongs to the threonine-class of
proteases and possesses trypsin-like, chymotrypsin-like and peptidyl
glutamyl-peptide-hydrolase activities. The core particle is built of four
rings: two inner rings of seven beta-subunits each, three of them
proteolytically active (the proteolytic chamber), and two outer rings of seven
alpha-subunits (the entrance to the proteolytic chamber) which interact with
the regulatory particle. The regulatory particle contains two subcomplexes, the
lid and the base. The base contains six different AAA ATPases that form a
ring-like structure and uses ATP hydrolysis to unfold target proteins, together
with non-ATPase subunits that function as docking sites for proteins. The lid,
an 8-subunit, non-ATPase assembly linked to the core particle by the base, is
required for ubiquitin-conjugate degradation and is closely related to the
signalosome (Schmidt et al., 1999). The structural composition of the
proteasome is heterogeneous and some subunits are subjected to phosphorylation,
acetylation and other post-translational modifications. The main functions of
Ub-dependent proteolysis are degradation of misfolded or damaged proteins and
of inherently unstable proteins that carry specific degradation signals,
quality control by removal of proteins with translational errors, and
inactivation of regulatory proteins. Phosphorylation and dephosphorylation in
the signal transduction cascades often alter protein stability and affect the
affinity of E3 for the respective target proteins. Further, the proteasome
exerts control on metabolic fluxes by degrading key enzymes whose stability may
depend on changes in metabolite concentrations (Kurepa and Smalle, 2008).
Ub-independent proteolysis is involved in degradation of oxidized proteins -
thus 20S may play an important role in tolerance to the secondary oxidative
stress accompanying many primary abiotic stresses. In addition, plant proteasomes
have RNAse activity, which is important in protection against pathogenic
viruses.
Rotease Complexes in Chloroplasts and Mitochondria
Chloroplasts and mitochondria contain their own
proteolytic systems, which are less complex than the ubiquitin/26S
proteasome pathway. They are homologous to the bacterial proteases and
have evolved in plants from single genes into multigene families (Adam, 2007;
Janska et al., 2010). In Arabidopsis chloroplasts, at least 11 different
types of protease families, encoded by more than 50 genes, have been found,
amounting to about 2.3% of the chloroplast proteins (Sakamoto, 2006). The Clp
complex is an abundant proteolytic system in chloroplast stroma. Its
proteolytic subunits are encoded in Arabidopsis by 6 different genes,
one of the products being targeted to mitochondria and 5 to chloroplasts. One
of these genes is in the plastome and the others in the nuclear genome.
Additional genes code for 4 non-catalytic ClpP-related ClpR subunits. The
chaperone part of Clp in Arabidopsis is coded by three genes for
chloroplasts (ClpC1, ClpC2, ClpD) and three for mitochondria (ClpX). There are
an additional two copies of ClpS (unique for land plants) and one ClpT copy
(Adam, 2007).
The structure of the Clp protease complex resembles that of
the 26S proteasome. It consists of two functional elements: one, a
barrel-shaped, hetero-oligomeric proteolytic core complex of 325-350 kDa,
composed of two stacked heptameric rings of ClpP, ClpR and ClpS subunits with
narrow (about 10 Ǻ) axial openings, forming a chamber which hides inside
serine-type proteolytic active sites (catalytic triad Ser-His-Asp), and the
other, two hexameric rings at the openings of the proteolytic core, composed of
chaperones of the AAA+ superfamily of ATPases (Clp/Hsp100). The mitochondrial
Clp core complex is a 320 kDa homotetradecamer of ClpP2 associated with ClpX
chaperones (Peltier et al., 2004). ClpC and ClpX chaperones appear to be
constitutively expressed, while ClpD is responsive to drought stress and
senescence (Sakamoto, 2006).
Plant organelles also have FtsH and Lon ATP-dependent
proteases in which the proteolytic active site and chaperone domain are parts
of the same polypeptide chain. The FtsH gene family in Arabidopsis
contains 12 members, 4 of whose products are targeted to mitochondria and 9 to
chloroplasts. FtsH proteases have a 400-450 kDa hexameric ring-like structure
formed by identical or closely related subunits. These provide an internal
channel harbouring the proteolytic sites, access to which is controlled by the
chaperone part. Each 74 kDa FtsH monomer consists of two trans-membrane helices
at the N-terminus which anchor the protein to membranes. The stroma-facing
AAA+ATPase domain and the proteolytic domain possess a conserved zinc-binding
motif (His-Glu-X-X-His) at the C-terminus, typical of metalloproteases. FtsH
complexes are heteromeric and its subunits are partially functionally redundant
(Sakamoto, 2006).
Arabidopsis possesses four genes for Lon protease
which are targeted to mitochondria, chloroplasts and peroxisomes (Adam, 2007;
Janska et al., 2010). Lon is a hexameric ATP-dependent serine protease with a
Ser-Lys dyad in the active site. Chaperone and catalytic domains are located in
the same polypeptide.
The interesting hexameric DegP serine protease, with 16
paralogs, is coded for in the Arabidopsis genome. It is not involved in
the ATP-dependent proteolytic pathway but switches between chaperoning and
proteolytic activity under the influence of high temperature (Adam, 2007). The
DegP monomers of 48 kDa are composed of a proteolytic domain at the N-terminus
with the catalytic triad Ser-His-Asp, and two tandem PDZ domains at the
C-terminus which regulate protein-protein interactions and recognition of the
substrate proteins. In the chaperone form the active site of the protease is
blocked by an auto-inhibitory segment. The temperature-induced conformational
change pulls apart this segment, making the active centre accessible to
substrates (Adam, 2007).
The main functions ascribed to the processive ATP-dependent
proteases in organelles are i) to degrade excess subunits of proteins, ii) to
eliminate photo-oxidatively damaged proteins, and iii) to exert protein quality
control (Sakamoto, 2006). The involvement of FtsH and DegP in the repair cycle
of PSII in chloroplasts under high light, by degradation of D1 protein, is well
documented (Kato and Sakamoto, 2009). Mitochondrial Lon and FtsH proteases are
involved in the biogenesis and maintenance of the oxidative phosphorylation
system (Janska et al., 2010).
Relation of Expression of ATP-Dependent Protease Complexes to Function
The proteasome is essential for proper cellular function,
removing proteins containing translation errors, improper processing and
irreparable damage. It participates in protein quality control for both
cytosolic and endoplasmic located proteins via the ER-associated
degradation pathway, involving the assistance of ER chaperones and retrograde
transport back to the cytoplasm (Kurepa and Smalle, 2008). Up to 30% of the
translation products are rapidly removed by the proteasome under non-stress
conditions (Smalle and Vierstra, 2004). Various pathway mutants are lethal or
hypersensitive to stresses that damage or denature cell proteins (Smalle and
Vierstra, 2004). In addition, the Ub-proteasome pathway controls hormonal
signalling, including stress signal transduction, as well as cell metabolism,
by removing short-lived regulatory proteins and enzymes that direct
rate-limiting steps of metabolite pathways (Vierstra, 2009). These
indispensable housekeeping functions and the great complexity of the proteasome
favour its primarily constitutive expression and a role in acclimation to
stress rather than of rapid stress induction.
Plant cells contain 26S and 20S proteasomes which mediate
Ub-dependent and Ub-independent proteolysis, respectively. It is established
that increased 26S biogenesis supports increased growth and increased tolerance
to misfolded protein stresses (heat-shock stress for example). In contrast, increase
in the relative abundance of 20S leads to decreased growth and better
performance in oxidative stress conditions, since 20S degrades oxidatively
modified proteins in an ATP-independent manner (Kurepa et al., 2009).
Chloroplast Clp protease is principally a constitutively
expressed enzyme that degrades numerous stromal proteins. Among its putative
substrates are enzymes involved in metabolic pathways such as photosynthetic
carbon fixation, nitrogen metabolism and chlorophyll/haem biosynthesis. Other
putative substrates of Clp have been described that are involved in
housekeeping roles such as RNA maturation, protein synthesis and maturation,
and recycling processes (Stanne et al., 2009). Constitutive expression of ClpP
proteins, without significant changes, has been reported under various stress
conditions. It appears that plastid and mitochondrial proteolysis is regulated,
not through regulation of ClpP gene expression, but rather through interaction
of the core complex with ClpS1,2 and other chaperone-like molecules, as well as
through substrate recognition mechanisms (Peltier et al., 2004). FtsH
transcript levels in chloroplasts are highly responsive to strong light stress
while temperature stresses have no effect on FtsH transcript abundance (Adam,
2007). Expression of mitochondrial ATP-dependent proteases is rather constant
during their development, with some changes found in flowers and seeds (Janska
et al., 2010). Expression of the Deg protease, which is not dependent on ATP,
is increased under salt, light and temperature stresses (Sakamoto, 2006).
Involvement of ATP-Dependent Protease Complexes in the Response to Drought
Water
deficit resulted in decreased total ATP-dependent proteolytic activity in wheat
genotypes (Wisniewski and Zagdanska, 2001), while acclimation to drought
prevented this decline. ATP-dependent protein degradation under drought was
greater in the leaves of acclimated than of non-acclimated stressed plants
(Zagdanska and Wisniewski, 1998; Wisniewski and Zagdanska, 2001). This activity
is associated mainly with the proteasome in the cytosol and nucleus and the
ATP-dependent proteases in chloroplasts and mitochondria.
ATP
consumption for energy-dependent proteolysis amounts to about half that
required for protein synthesis, and increases substantially under water deficit
conditions (Zagdanska, 1995).
Regulation of proteasome activity includes adjustment of
both total cellular proteolytic potential and target specificity. Changes in
total proteasome activity can be caused by altered proteasome abundance or by
posttranslational modification of subunits without affecting proteasome
content. Changes in target specificity can be brought about by subunit
modification and by the incorporation of subunit variants (Kurepa and Smalle,
2008). Plomion et al. (2006) reported induction by drought of members of the
protein degradation machinery (i.e. 20S proteasome, polyubiquitin) in poplar
trees, at the transcript level and the proteome level (26S protease regulatory
subunit). Up-regulation of the 20S proteasome subunit was found in a proteomic
study of drought-treated alfalfa plants (Aranjuelo et al., 2011). Wan et al.
(2011) described up-regulation of a ubiquitin-conjugating enzyme gene AhUBC2 in
dehydrated peanut plants; constitutive expression of this gene in Arabidopsis
resulted in improved water-stress tolerance. The AtAIRP1 gene, encoding an E3
Ub ligase, was rapidly and significantly induced by ABA and by abiotic stresses
including drought, low temperature, and high salinity. AtAIRP1-overexpressing
transgenic plants were highly resistant to severe water stress (Ryu et al.,
2010). It appears that the sumoylation system is highly responsive to
environmental cues and plays a regulatory role in plant stress responses. Low
and high temperatures, drought, salt, and oxidative stresses induce SUMO
conjugation to protein substrates (Reed et al., 2010; Miura and Hassegava,
2010). Arabidopsis plants exposed to drought stress accumulate increased
levels of sumoylated proteins by an ABA-independent pathway which is, in part,
dependent on the transient increase of E3 SUMO ligase levels in response to
drought (Catala et al., 2007).
Transcript analysis of ClpP/ClpR genes indicates
constitutive expression in roots and leaves of A. thaliana with rather
minor changes under stress and in senescence (Zheng et al., 2002;
Sinvany-Villalobo et al., 2004). Short-term moderate and severe stresses
(desiccation, high salt, cold, heat, oxidation, wounding and high light) all
failed to elicit significant or rapid increases in any chloroplast Clp protein.
However, increases in mRNA and protein content for ClpD and several ClpP
isomers did occur during long-term high light and cold acclimation of Arabidopsis
plants. These results reveal the great complexity of Clp proteins within the
stroma of plant chloroplasts (Nakashima et al., 1997; Zheng et al., 2002).
Immunoblotting analysis showed an enhancement of Clp proteases in drought
treated wheat plants (Demirevska et al., 2008a).
Both FtsH genes in maize were constitutively expressed, the
expression level of ZmFtsH2B transcripts being higher than that of ZmFtsH2A.
Under polyethylene glycol, cold, high salt and ABA treatments, ZmFtsH2B
transcription in leaves was markedly up-regulated, while ZmFtsH2A was
constitutively expressed in both leaves and roots. However, drought tolerance
of transgenic tobaccos overexpressing ZmFtsH2A and ZmFtsH2B was not greater
than that of wild-type controls (Yue et al., 2010). Decreased abundance of FtsH
was established in a susceptible Kentucky bluegrass cultivar under drought
stress (Xu and Huang, 2010). Li et al. (2010) established that Arabidopsis
Lon protease (atlon4) mutant is more sensitive to drought stress than wild-type
plants.
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