Download ATP-dependent Clp Proteases in Photosynthetic Organisms~ A Cut

Annals of Botany 83 : 593–599, 1999
Article No. anbo.1999.0878, available online at http :\\www.idealibrary.com on
BOTANICAL BRIEFING
ATP-dependent Clp Proteases in Photosynthetic Organisms—
A Cut Above the Rest !
A D R I A N K. C L A R KE*
Department of Plant Physiology, UniŠersity of Umeab , Umeab S-901 87, Sweden
Received : 25 January 1999
Returned for revision : 16 February 1999
Accepted : 2 March 1999
Proteases are critical regulatory factors for many metabolic cellular processes as well as being vital for degrading
proteins damaged during environmental stresses. Many of those responsible for targeted protein degradation require
the hydrolysis of ATP, and one class that has attracted much attention recently are the Clp proteases. They are among
the best characterized proteases to date, and were the first shown to rely on an ATPase regulatory subunit possessing
molecular chaperone activity, which functions both within the proteolytic complex and independently. A range of Clp
proteins has been identified from many different bacteria and eukaryotes, with by far the greatest number and
diversity of forms in oxygenic photobionts such as cyanobacteria and higher plants. Functionally, Clp proteins have
also evolved into one of the more critical proteolytic enzymes within photobionts, and it is now somewhat of a
paradox that we currently know least about Clp protease functions in the photosynthetic organisms, where they have
their most important roles. This discrepancy is now being addressed, with studies on Clp protein in cyanobacteria and,
in an increasing number, in higher plants.
# 1999 Annals of Botany Company
Key words : Chloroplasts, Clp proteins, cyanobacteria, molecular chaperones, proteolysis.
INTRODUCTION
It is only over the last few years that most plant biologists
have become aware of the existence of Clp proteins in
cyanobacteria and plants, and even today many remain
unaware of their importance for protein catabolism in
photobionts. This probably stems from the early characterizations of the Clp protease, done almost exclusively in E.
coli, where genetic evidence suggested it is a minor enzyme
whose loss is readily compensated for by other ATPdependent proteases. The ubiquity of Clp proteins was also
unrecognized for many years and has only recently become
evident as a result of both directed cloning efforts by
individual groups and broad scale genomic sequencing.
From this background, it has emerged that Clp proteins are
indeed present throughout nature, in all eubacteria examined
so far, including cyanobacteria, and in higher eukaryotes
such as plants and mammals. Moreover, such studies have
demonstrated that the importance of Clp proteases is
significantly greater in many organisms than in certain
bacteria such as E. coli.
It is now well recognized that proteases perform vital
functions throughout the cell. As housekeeping enzymes
they are critical for cell homeostasis, removing polypeptides
that denature either spontaneously or as a result of
fluctuating growth environments. Selective protein turnover
is also an important regulatory mechanism, influencing the
activity of short-lived metabolic enzymes and regulatory
proteins, as well as facilitating systemic responses that
involve the removal of pre-existing proteins concomitant
with the synthesis of new ones (Gotesman, 1996). Because
* Fax 46 90 7866676, e-mail Adrian.Clarke!plantphys.umu.se
0305-7364\99\060593j07 $30.00\0
of the potential risk proteases pose to normal protein
components, they need to be tightly controlled within the
cell. As a result, many of the proteases involved in such
targeted proteolysis rely on metabolic energy in the form of
ATP. Three proteolytic systems are now emerging as
important means for protein turnover in bacteria and
eukaryotes. Structurally, all three have similar architecture,
and involve not only proteolytic subunits but also accessory
proteins that facilitate recognition, binding and presentation
of substrates ready for degradation. The ubiquitin\
proteasome system in archeabacteria and within the cytosol
and nucleus of eukaryotes is probably the best known of
these (Hershko and Ciechanover, 1998), with the HslVU (or
ClpQY) protease as its eubacterial counterpart (Rohrwild et
al., 1996), and the ClpP protease widespread in eubacteria
and higher eukaryotes.
THE CLP PROTEASE FROM E. COLI – THE
BASIC MODEL
Most of what we know about Clp protease comes from
studies of the enzyme from E. coli, which has become the
basic model for understanding variants in other organisms.
The protease from E. coli was first identified as catalyzing in
Šitro degradation of the substrate casein (KatayamaFujimura, Gottesman and Maurizi, 1987), and hence gained
its designation as Caseinolytic protease (Clp). It consists of
two functionally distinct subunits : a serine-type proteolytic
subunit, ClpP (21 kD), and one of two regulatory ATPase
subunits, ClpA (83 kD) or ClpX (46 kD). Alone, ClpP is
incapable of degrading polypeptides longer than six amino
acids, and requires either ClpA or ClpX to become
# 1999 Annals of Botany Company
594
Clarke—Clp Proteases in Cyanobacteria and Plants
proteolytically active (Woo et al., 1989 ; Wojkowiak,
Georgopoulos and Zylicz, 1993). The proteolytic complex
consists of two central heptameric annuli of ClpP flanked by
one or two hexameric rings of ClpA or ClpX. ClpA has
twice the affinity of ClpX for binding to ClpP, although
mixed complexes of single ClpA and ClpX hexamers
attached to either end of the ClpP core are possible in Šitro
(Grimaud et al., 1998). The ClpAP protease is an endopeptidase that degrades polypeptide substrates without
apparent sequence specificity to short peptides of seven to
ten amino acids (Thompson and Maurizi, 1994 ; Thompson,
Singh and Maurizi, 1994), with the process requiring both
Mg#+ and ATP hydrolysis (Katayama et al., 1988). ATP
binds at two distinct domains of the ClpA protein, one
bound ATP is required for ClpA oligomerization, while
hydrolysis of the other ATP is required for proteolysis
(Singh and Maurizi, 1994). ClpA also recognizes and binds
the protein substrate in some fashion, and then consumes
ATP in steps facilitating the accessibility and binding of the
protein substrate to the ClpP proteolytic active sites (Wang,
Hartling and Flanagan, 1997).
Both ClpA and ClpX are now known to be members of a
new family of molecular chaperones known as Clp\Hsp100.
This family consists of two broad groups, which are
separated further into different types based on specific
sequence signatures (Schrimer et al., 1996). The first group
contains proteins between 85 to 105 kD with two distinct
ATP-binding domains. These are further divided into five
subtypes, designated ClpA to -E. ClpA is found exclusively
in certain Gram-negative bacteria like E. coli, ClpB is
present in all bacteria and eukaryotes, ClpC in most Grampositive bacteria, cyanobacteria and plants, ClpD exclusively in plants, and ClpE in certain Gram-positive bacteria.
The second group has only two representatives to date :
ClpX and ClpY. Both these proteins differ from the first
group in having only a single ATP-binding domain, one
that is more structurally similar to the ATP-2 domain of the
Group 1 Clp proteins (Gottesman et al., 1993). ClpX has
been found in all eubacteria and eukaryotes so far, whereas
ClpY (or HslV) appears to be restricted to certain eubacteria,
functioning as part of the HslVU protease. Excluding ClpY,
varying degrees of evidence for ClpP association and
involvement in Clp proteolysis exist for ClpA, ClpC, ClpE
and ClpX. ClpB is the main exception, however, and is
unlikely to participate directly within a Clp protease complex
(Clarke, 1996). It is currently unclear whether ClpD
possesses the dual protease regulatory\chaperone activity
of most other Hsp100 proteins or if it functions independently of ClpP as does ClpB.
Determining the structure of the Clp protease has been
crucial for our understanding of its action. The architecture
of the holo-ClpAP protease from E. coli resembles that of
the eukaryotic 26S proteasome, while the ClpP peptidase
component resembles that of the 20S proteasome (Kessel et
al., 1995). The two heptomeric ClpP rings are stacked backto-back to form a hollow cylinder (Flanagan et al., 1995 ;
Shin et al., 1996), with the conserved serine-type protease
catalytic sites all facing the inside of the cylinder (Wang et
al., 1997). This central catalytic chamber, 50 A/ in diameter,
is bounded by solid-walls with only two axial openings (Fig.
1). The width of the axial apertures, however, is only 11 A/ ,
which prevents most native polypeptides from entering the
cavity. Activation of ClpP proteolysis is therefore thought
to require unfolding chaperone activity of Hsp100 proteins
such as ClpA for efficient translocation of the protein
substrate into the ClpP active site domain (Beuron et al.,
1998 ; Hoskins et al., 1998). Once bound inside, the unfolded
polypeptide is rapidly degraded due to the number and
distribution of the active sites within the ClpP cylinder
(Wang et al., 1997). Although compelling, several aspects of
this proteolytic model still require experimental verification,
in particular how the Clp\Hsp100 partners interact with
ClpP at its apical surface, and how they recognize, bind and
translocate the protein substrate into the proteolytic
chamber.
ISOMERIC FORMS OF CLP PROTEINS IN
CYANOBACTERIA
As more clp genes are identified in different bacteria, it
becomes evident that the number and functional importance
of Clp proteins is far more diverse than previously thought.
Although most is known about E. coli Clp proteins, their
genetic disruption causes few phenotypic changes, either
during normal growth or under stresses such as heat shock
(Kroh and Simon, 1990 ; Maurizi et al., 1990 a). In many
other bacteria such as Bacillus subtilis, however, Clp
proteases are intricately involved in a variety of processes,
ranging from developmental changes to stress tolerance
(reviewed in Porankiewicz, Wang and Clarke, 1999). As in
these bacteria, Clp proteins in cyanobacteria are also vital
for growth and stress acclimation. As in most bacteria,
cyanobacteria possess two different Clp\Hsp100 proteins
likely to participate in Clp proteolytic complexes. In addition
to ClpX, cyanobacteria have a homologue to the E. coli
ClpA, known as ClpC. Little is yet known about the exact
functions of either protein, or their involvement in different
Clp proteolytic complexes in cyanobacteria. The cyanobacterial ClpC, however, is approx. 90 % similar (i.e. 80 %
identical and 10 % functionally conserved) in amino acid
sequence to ClpC in plant chloroplasts, and both proteins
are constitutively expressed and their levels remain essentially unaffected by different stress conditions (Shanklin,
DeWitt and Flanagan, 1995 ; Clarke and Eriksson, 1996).
Genetic interference with ClpC synthesis is also lethal for
both kinds of photobionts (Shanklin et al., 1995 ; Clarke
and Eriksson, 1996), demonstrating that its chaperone
activity, whether independent or as part of a Clp protease,
is essential.
An added complexity within cyanobacteria is the existence
of isomeric forms of ClpP. In the unicellular strains,
Synechocystis sp. PCC 6803 and Synechococcus sp. PCC
7942, three distinct ClpP isomers have been found (Kaneko
et al., 1996 ; Clarke, Schelin and Porankiewicz, 1998 ; Clarke
and Schelin, unpubl. res.). Originally a fourth clpP gene was
assigned within the Synechocystis genome (Kaneko et al.,
1996), although the sequence similarity to other clpP genes
was relatively low. Analysis of the predicted polypeptide,
however, shows it lacks all three conserved residues that
constitute the serine-type catalytic active site, and is thus
Clarke—Clp Proteases in Cyanobacteria and Plants
595
F 1. Structure of E. coli ClpP complex. Shown are space-filling representations of the ClpP tetradecamer as seen from end-on, depicting the
narrow opening to the proteolytic chamber (A), the side, showing the arrangement of the two heptameric ClpP rings (B), and a cutaway view,
exposing the large, cylindrical proteolytic chamber (C). Although each subunit is identical, for clarity each is shown in a different colour. Pictures
come from Wang, Hartling and Flanagan (1999) and are reproduced with permission.
unlikely to function within a Clp protease complex. This
protein may still function as a protease with characteristics
different from ClpP and is, as such, distinguished as ClpR
(Porankiewicz et al., 1999). Interestingly, genes coding for
two distinct polypeptides homologous to ClpR have been
identified from Arabidopsis thaliana (Clarke, unpubl. res.),
suggesting these proteins may constitute a new protease
family unique to photosynthetic organisms.
Of the three cyanobacterial genes, two are monocistronic
(clpP1 and clpP3) while the other (clpP2) is apparently
arranged in an operon with clpX, similar to the E. coli
homologue (Kaneko et al., 1996 ; Clarke et al., 1998). All
studies on cyanobacteria Clp proteins have so far come
from Synechococcus sp. PCC 7942, of which to date most is
known about ClpP1. ClpP1 is synthesized constitutively in
Synechococcus at a relatively low level but, surprisingly, it is
not heat-shock inducible, as are the known ClpP proteins in
all other eubacteria, nor is it involved in acquired thermotolerance (Clarke et al., 1998). Instead, ClpP1 appears to
play an important role during steady-state growth and for
long-term acclimation to other types of environmental
changes, rather than being involved in resistance to severe,
transient stresses.
In cyanobacteria and plants, light is a critical environmental variable. ClpP1 content increases dramatically
during both short exposures to extreme high light resulting
in photoinhibition, and during acclimation to moderate but
non-inhibitory light. Inactivation of clpP1, however, has no
effect on the susceptibility of Synechococcus to photoinhibition but does produce pleiotrophic changes during
steady-state growth. These include slower growth rates,
particularly at higher light intensities, as well as changes in
pigment composition and the formation of many
filamentous cells similar to those formed during stationary
phase in wild type cells (Clarke et al., 1998). Reduced
growth rates and filamentous morphology were also
observed in the corrsponding ∆clpP strain from B. subtilis
(Msadek et al., 1998 ; Gerth et al., 1998) suggesting protein
turnover mediated by this class of ClpP protein is involved
in cell division in some bacteria.
In addition to high light, ClpP1 is also strongly induced
during acclimation of Synechococcus to temperature drops
(37 to 25 mC) or to moderate UV-B supplementation of the
growth light (Porankiewicz, Schelin and Clarke, 1998). In
these instances, inactivation of ClpP1 synthesis produced a
much more severe phenotype. While wild type
Synechococcus readily acclimates to both treatments, the
∆clpP1 strain could not, being incapable of either resuming
growth or recovering photosynthetically after the initial
stress period (Porankiewicz et al., 1998). This again
demonstrates the importance of ClpP proteolysis in stress
acclimation in cyanobacteria, to a greater extent than in any
other eubacteria studied.
It is an apparent paradox that isomeric forms of ClpP
exist in certain organisms, given that ClpP lacks substratebinding domains and alone is proteolytically inactive. One
explanation is differential associations of the ClpP isomers
with the various Clp\Hsp100 proteins. This proposal arises
from theoretical studies whereby the amino acid differences
between each cyanobacterial ClpP protein isomer and ClpP
from E. coli are highlighted on the structural model for the
E. coli protein (Porankiewicz et al., 1999). This comparison
clearly shows that for each cyanobacterial ClpP, most
amino acid variations are on the outer surfaces of the
heptameric ring structure, away from the central proteolytic
cavity, suggesting each cyanobacterial homologue possesses
similar proteolytic activity. When the variations distinguishing the three cyanobacterial ClpP proteins are compared, significant differences appear on the apical surface,
the region thought to interact with the Hsp100 partner in
the holocomplex. This suggests each ClpP isomer may have
different affinities for the different Clp\Hsp100 proteins.
Although this proposal is appealing, there are only two
likely Clp\Hsp100 partners in cyanobacteria (i.e. ClpC and
ClpX) to interact with the three ClpP isomers. Despite the
two ClpB isomers in cyanobacteria, there is no evidence to
date from any organism to suggest ClpB can associate with
ClpP to form an active protease. One solution to this
stoichiometric conundrum may be the apparent promiscuous affinity of ClpP for other chaperones that have protein
596
Clarke—Clp Proteases in Cyanobacteria and Plants
F. 2. Diagrammatic representation of the types, sizes, genetic origin and localization of Clp proteins in the higher plant, Arabidopsis thaliana.
Intracellular distribution of Clp proteins is based on experimental observations except for those indicated by question marks where currently only
predicted sequence evidence supports localization assignments.
Clarke—Clp Proteases in Cyanobacteria and Plants
unfolding activity, as was shown in Šitro by the activation of
E. coli ClpP proteolysis by GroEL (Kandor et al., 1994).
Another possible explanation for the multiple ClpP isomers
is that they may differ in their specificities for short peptides,
since one unresolved question about ClpP is whether its
peptidase activity has any functional significance in ŠiŠo,
perhaps in the degradation of peptide fragments. Despite
the attraction of such suggestions, it cannot yet be excluded
that the sequence variations between the ClpP isomers have
simply accumulated during evolution and thus have no
functional significance, especially since most are situated
outside of the proteolytic cavity. The main difference
between these proteins may be in their genetic regulation,
with each being differentially expressed during growth and
in response to various stresses.
DIVERSITY AND MULTITUDE OF CLP
PROTEINS IN HIGHER PLANTS
ATP-dependent proteolysis within chloroplasts was demonstrated over 15 years ago (Hammond and Preiss, 1983),
but specific proteases have only been identified within the
last 5 years. Three main compartmentalized proteolytic
systems are known from chloroplasts : soluble Clp proteases
in the stroma (Shanklin et al., 1995), FtsH protease within
thylakoid membranes (Lindahl et al., 1996 ; Ostersetzer and
Adam, 1997), and DegP protease inside the thylakoid lumen
(Itzhaki et al., 1998). Other chloroplastic proteolytic
activities have been reported (Sokolenko et al., 1997), but
specific proteins or corresponding genes for such proteases
have not yet been identified.
To date four distinct Clp proteins have been found inside
plant chloroplasts, two Hsp100 types (ClpC and ClpD)
which are both nuclear-encoded, and two ClpP proteins
(Fig. 2). The best characterized of these so far is ClpC
together with one of the two ClpP proteins. As in
cyanobacteria, ClpC appears to be the main Clp\Hsp100
protein in plant chloroplasts, and the homologues share
similar characteristics (Shanklin et al., 1995 ; Clarke and
Eriksson, 1996). Although two clpC genes were identified in
tomato (Gottesman et al., 1990), both code for near
identical proteins and thus there is probably only one
functional form of ClpC protein.
In contrast to the nuclear-encoded ClpC, the first ClpP
protein identified in plants was one encoded by the plastid
genome (pClpP) (Gray, Hird and Dyer, 1990 ; Maurizi et al.,
1990 b). In most plant species, the pclpP gene is cotranscribed along with two ribosomal protein genes (5hrps12 and rpl20), and is later post-transcriptionally processed to produce the mature monocistronic transcript
(Koller et al., 1987 ; Clarke, Gustafsson and Lidholm,
1994) ; the main exception to this pattern is pclpP from
Arabidopsis thaliana, which is monocistronic and split into
two exons. Like ClpC, the pClpP protein is localized in the
stroma and is synthesized constitutively in all plant tissues
(Shanklin et al., 1995 ; Ostersetzer and Adam, 1996 ;
Ostersetzer et al., 1996). Its function is also apparently
essential for chloroplasts since its disruption prevents
phototrophic growth in green algae (Huang et al., 1994).
There is now evidence for a structural association between
597
pClpP and ClpC (Halperin and Adam, 1996 ; Desimone et
al., 1997 ; Sokolenko et al., 1998), suggesting they form an
active Clp protease in the chloroplast stroma. The potential
for such a protease has already been demonstrated in Šitro,
with recombinant plant ClpC being able to activate E. coli
ClpP proteolytic activity, and conversely E. coli ClpA
activating plant pClpP proteolysis (Shanklin et al., 1995).
Less is known about the remaining chloroplast Clp
proteins, although their existence implies there is more
complexity in the types of Clp proteases than previously
thought. The ClpD protein is synthesized constitutively but
to a much lesser degree than ClpC (Zheng and Clarke,
unpubl. res.). It was first identified in Arabidopsis as a
desiccation-inducible protein, Erd1 (Kiyosue, YamaguchiShinozaki and Shinozaki, 1993), and has since been shown
to also be induced by high salt concentrations, dark-induced
etiolation, and at the onset of senescence (Nakashima et al.,
1997).
Apart from the plastid-encoded protein, a second ClpP
protein occurs in plant chloroplasts. This ClpP protein
(nClpP1) is nuclear-encoded but post-translationally
imported into the organelle, where it is again localized in the
stroma (Sokolenko et al., 1998 ; Zheng and Clarke, unpubl.
res.). This ClpP isomer was first identifed from a gene clone
from a tomato wound-inducible cDNA library (Schaller
and Ryan, 1995), although recent data suggests nClpP1 is
primarily a constitutively synthesized protein like pClpP
(Zheng and Clarke, unpubl. res.). The nClpP1 protein also
appears to form oligomeric structures with ClpC, as well as
mixed complexes with pClpP (Sokolenko et al., 1998),
suggesting it may participate in Clp proteolytic activity
within plant chloroplasts.
The importance of individual Clp proteins for chloroplast
function has been demonstrated through genetic inactivations (Huang et al., 1994 ; Shanklin et al., 1995), and
structural associations between different chloroplastic Clp
subunits have been shown. Nevertheless, functional evidence
for an active stromal Clp protease remains scant. The
specific role of such a Clp protease remains speculative,
whether it functions mainly in housekeeping to remove and
recycle damaged or otherwise aberrant polypeptides, or
whether it instead targets specific stromal enzymes and
regulatory proteins. If the latter, then one potential stromal
target is Rubisco, whose subunit levels are regulated
proteolytically to maintain correct stoichiometry (Schmidt
and Mishkind, 1983). Incorrectly processed precursors
imported from the cytosol are also degraded in the stroma
by enzymes with the functional characteristics of Clp
proteases (Halperin and Adam, 1996). Recognition of such
mistargeted polypeptides may be further facilitated by the
apparent association of a small proportion of ClpC with the
chloroplast inner-membrane import machinery (Nielsen et
al., 1997), possibly enabling it to efficiently screen precursor
proteins following membrane translocation. Supracomplexes within thylakoid membranes may also be subject
to Clp proteolysis under certain conditions, particularly the
cytochrome b \f complexes during nitrogen starvation in
'
algal chloroplasts (Vallon, pers. comm.).
Apart from the chloroplast proteins, there are at least
four other ClpP isomers in higher plants, all of which are
598
Clarke—Clp Proteases in Cyanobacteria and Plants
nuclear-encoded and are known to date from Arabidopsis
(Fig. 2). Little is yet known about any of these additional
ClpP isomers, although circumstantial evidence suggests
one or more may form part of a Clp protease within
mitochondria. A similar possibility has already been
proposed in humans, where the single known ClpP protein
is localized within mitochondria (Corydon et al., 1998).
Sequence alignments of all known ClpP proteins show that
one of the isomers from Arabidopsis (nClpP2) is closely
related to the human mitochondrial protein, and also
includes a potential transit peptide (Porankiewicz et al.,
1999). Furthermore, in humans, the only known Hsp100\
Clp partner for the lone ClpP protein is ClpX, for which a
cDNA clone was recently identified. A similar clpX cDNA
was recently cloned from Arabidopsis, and although the
protein product was originally thought to be chloroplastic,
studies now suggest it may instead be localized inside
mitochondria (Adam, pers. comm.). A ClpXP protease,
therefore, is likely to be present in human and plant
mitochondria, meaning that Clp proteases are not exclusive
to plastids in plants.
FUTURE PERSPECTIVES
Genetic evidence to date suggests ATP-dependent Clp
proteases are vital for protein catabolism in both cyanobacteria and higher plants, under both optimal and stress
conditions. Despite recent progress in this area, many
questions still remain unanswered, especially the specific
biochemical functions and compositions of different Clp
proteases. Future work must characterize the regulatory
Clp\Hsp100 subunits, which confer protein substrate
recognition, binding and translocation into the ClpP
proteolytic cavity. Similarly, the identity and nature of
those protein substrates remain unclear in both cyanobacteria and plant organelles. Another puzzling feature is
the number of Clp proteins in photobionts ; what are the
functions of these isomers ? In the case of plant Clp\Hsp100
proteins, an obvious role would be to confer differences in
substrate specificities and to accommodate sub-organellar
compartmentalization of different Clp proteases. For ClpP
isomers, the possible functions are less evident, although
one possibility is differential specificities for Clp\Hsp100
proteins. Regulatory questions also need investigation, to
determine whether the different Clp isomers are differentially
expressed or assembled within cyanobacteria and plants,
and if so, exactly which signals are responsible ; whether
developmental or environmental. Details of the specific
cellular locations and targets for these multiple Clp proteases
in photosynthetic organisms will greatly advance our
knowledge about this new and undoubtedly important
family of proteases.
A C K N O W L E D G E M E N TS
The author would like to thank Dr Jimin Wang for the
structural models of the E. coli ClpP complex shown in Fig.
1, Drs Zach Adam and Olivier Vallon for access to
unpublished data, and Dr Douglas Campbell for critical
reading of the manuscript. This work is supported by grants
from the Swedish Natural Science Research Council, the
Swedish Agricultural and Forestry Resource Council and
the Centre for Forest Biotechnology and Chemistry.
LITERATURE CITED
Beuron F, Maurizi MR, Belnap DM, Kocsis E, Booy FP, Kessel M,
Steven AC. 1998. At sixes and sevens : Characterization of the
symmetry mismatch of the ClpAP chaperon-assisted protease.
Journal of Structural Biology 123 : 248–259.
Clarke AK. 1996. Variations on a theme : Combined molecular
chaperone and proteolysis functions in Clp\HSP100 proteins.
Journal of Biosciences 21 : 161–177.
Clarke AK, Eriksson M-J. 1996. The cyanobacterium Synechococcus
sp. PCC 7942 possesses a close homologue to the chloroplast ClpC
protein of higher plants. Plant Molecular Biology 31 : 721–730.
Clarke AK, Gustafsson P, Lidholm JC. 1994. Identification and
expression of the chloroplast clpP gene from the conifer Pinus
contorta. Plant Molecular BioloŠy 26 : 851–862.
Clarke AK, Schelin J, Porankiewicz J. 1998. Inactivation of the clpP1
gene for the proteolytic subunit of the ATP-dependent Clp
protease in the cyanobacterium Synechococcus limits growth and
light acclimation. Plant Molecular Biology 37 : 791–801.
Corydon TJ, Bross P, Holst HU, Neve S, Kristiansen K, Gregersen N,
Bolund L. 1998. A human homologue of Escherichia coli ClpP
caseinolytic protease : recombinant expression, intracellular processing and subcellular localization. Biochemical Journal 331 :
309–316.
Desimone M, Weiß-Wichert W, Wagner E, Altenfeld U, Johanningmeier
U. 1997. Immunochemical studies on the Clp-protease in chloroplasts : evidence for the formation of a ClpC\P complex. Botanica
Acta 110 : 234–239.
Flanagan JM, Wall JS, Capel MS, Schneider DK, Shanklin J. 1995.
Scanning transmission electron microscopy and small-angle
scattering provide evidence that native Echerichia coli ClpP is a
tetradecamer with an axial pore. Biochemistry 34 : 10910–10917.
Gerth U, Kru$ ger E, Derre! I, Msadek T, Hecker M. 1998. Stress
induction of the Bacillus subtilis clpP gene encoding a homologue
of the proteolytic component of the Clp protease and the
involvement of ClpP and ClpX in stress tolerance. Molecular
Microbiology 28 : 787–802.
Gottesman S. 1996. Proteases : and their targets in Escherichia coli.
Annual ReŠiews in Genetics 30 : 465–506.
Gottesman S, Clark WP, de Crecy-Lagard V, Maurizi MR. 1993. ClpX,
an alternative subunit for the ATP-dependent Clp protease of
Escherichia coli. Journal of Biological Chemistry 268 : 22618–22626.
Gottesman S, Squires C, Pichersky E, Carrington M, Hobbs M, Mattick
JS, Darlymple B, Kuramitsu H, Shiroza T, Foster T, Clark WP,
Ross B, Squires CL, Maurizi MR. 1990. Conservation of the
regulatory subunit for the Clp ATP-dependent protease in
prokaryotes and eukaryotes. Proceedings of the National Academy
of Sciences USA 87 : 3513–3517.
Gray JC, Hird SM, Dyer TA. 1990. Nucleotide sequence of a wheat
chloroplast gene encoding the proteolytic subunit of an ATPdependent protease. Plant Molecular Biology 15 : 947–950.
Grimaud R, Kessel M, Beuron F, Stevens AC. 1998. Enzymatic and
structural similarities between the Escherichia coli ATP-dependent
proteases, ClpXP and ClpAP. Journal of Biological Chemistry 273 :
12476–12481.
Halperin T, Adam Z. 1996. Degradation of mistargeted OEE33 in the
chloroplast stroma. Plant Molecular Biology 30 : 925–933.
Hammond JBW, Preiss J. 1983. ATP-dependent proteolytic activity
from spinach leaves. Plant Physiology 73 : 902–905.
Hershko A, Ciechanover A. 1998. The ubiquitin system. Annual ReŠiew
of Biochemistry 67 : 425–479.
Hoskins JR, Pak M, Maurizi MR, Wickner S. 1998. The role of the
ClpA chaperone in proteolysis by ClpAP. Proceedings of the
National Academy of Sciences USA 95 : 12135–12140.
Huang C, Wang S, Chen L, Lemieux L, Otis C, Turmel M, Liu X-Q.
1994. The Chlamydomonas chloroplast clpP gene contains translated large insertion sequences and is essential for cell growth.
Molecular and General Genetics 244 : 151–159.
Clarke—Clp Proteases in Cyanobacteria and Plants
Itzhaki H, Naveh L, Lindahl M, Cook M, Adam Z. 1998. Identification
and characterization of DegP, a serine protease associated with the
luminal side of the thylakoid membrane. Journal of Biological
Chemistry 273 : 7094–7098.
Kandror O, Busconi L, Sherman M, Goldberg AL. 1994. Rapid
degradation of an abnormal protein in Escherichia coli involves
the chaperones GroEL and GroES. Journal of Biological Chemistry
269 : 23575–23582.
Kaneko T, Sato S, Kotani H, Tanaka A, Asamizu E, Nakamura Y,
Miyajima N, Hirosawa M, Sugiura M, Sasamoto S, Kimura T,
Hosouchi T, Matsuno A, Muraki A, Nakazaki N, Naruo K,
Okumura S, Shimpo S, Takeuchi C, Wada T, Watanabe A, Yamada
M, Yasuda M, Tabata S. 1996. Sequence analysis of the genome of
the unicellular cyanobacterium Synechocystis sp. strain PCC 6803.
DNA Research 3 : 109–136.
Katayama-Fujimura Y, Gottesman S, Maurizi MR. 1987. A multiplecomponent, ATP-dependent protease from Escherichia coli.
Journal of Biological Chemistry 262 : 4477–4485.
Katayama Y, Gottesman S, Pumphrey J, Rudikoff S, Clark WP. 1988.
Two-component, ATP-dependent Clp protease of Escherichia coli.
Journal of Biological Chemistry 263 : 15226–15236.
Kessel M, Maurizi MR, Kim B, Kocsis E, Trus B, Singh SK, Steven AC.
1995. Homology in structural organization between E. coli ClpAP
protease and the eukaryotic 26S proteasome. Journal of Molecular
Biology 250 : 587–594.
Kiyosue T, Yamaguchi-Shinozaki K, Shinozaki K. 1993. Characterization of cDNA for a dehydration-inducible gene that encodes
a ClpA, B-like protein in Arabidopsis thaliana L. Biochemical and
Biophysical Research Communications 196 : 1214–1220.
Koller B, Fromm H, Galun E, Edelman M. 1987. Evidence for in ŠiŠo
trans splicing of pre-mRNAs in tobacco chloroplasts. Cell 48 :
111–119.
Kroh HE, Simon LD. 1990. The ClpP component of Clp protease is the
σ$#-dependent heat shock protein F21.5. Journal of Bacteriology
172 : 6026–6034.
Lindahl M, Tabak S, Cseke L, Pichersky E, Andersson B, Adam Z.
1996. Identification, characterization, and molecular cloning of a
homologue of the bacterial FtsH protease in chloroplasts of higher
plants. Journal of Biological Chemistry 271 : 29329–29334.
Maurizi MR, Clark WP, Katayama Y, Rudikoff S, Pumphrey J. 1990 a.
Sequence and structure of ClpP, the proteolytic component of the
ATP-dependent Clp protease of Escherichia coli. Journal of
Biological Chemistry 265 : 12536–12545.
Maurizi MR, Clark WP, Kim S-H, Gottesman S. 1990 b. ClpP represents
a unique family of serine proteases. Journal of Biological Chemistry
265 : 12546–12552.
Msadek T, Dartois V, Kunst F, Herbaud ML, Denizot F, Rapoport G.
1998. ClpP of Bacillus subtilis is required for competence
development, motility, degradative enzyme synthesis, growth at
high temperature and sporulation. Molecular Microbiology 27 :
899–914.
Nakashima K, Kiyosue T, Yamaguchi-Shinozaki K, Shinozaki K. 1997.
A nuclear gene, erd1, encoding a chloroplast-targeted Clp protease
regulatory subunit homolog is not only induced by water stress but
also developmentally up-regulated during senescence in
Arabidopsis thaliana. Plant Journal 12 : 851–861.
Nielsen E, Akita M, Davila-Aponte J, Keegstra K. 1997. Stable
association of chloroplastic precursors with protein translocation
complexes that contain proteins from both envelope membranes
and a stromal Hsp100 molecular chaperone. EMBO Journal 16 :
935–946.
Ostersetzer O, Adam Z. 1996. Effects of light and temperature on
expression of ClpC, the regulatory subunit of chloroplastic Clp
protease, in pea seedlings. Plant Molecular Biology 31 : 673–676.
Ostersetzer O, Adam Z. 1997. Light-stimulated degradation of an
unassembled Rieske FeS protein by a thylakoid-bound protease :
The possible role of the FtsH protease. The Plant Cell 9 : 957–965.
599
Ostersetzer O, Tabak S, Yarden O, Shapira R, Adam Z. 1996.
Immunological detection of proteins similar to bacterial proteases
in higher plant chloroplasts. European Journal of Biochemistry
236 : 932–936.
Porankiewicz J, Schelin J, Clarke AK. 1998. The ATP-dependent Clp
protease is essential for acclimation to UV-B and low temperature
in the cyanobacterium Synechococcus. Molecular Microbiology 29 :
275–284.
Porankiewicz J, Wang J, Clarke AK. 1999. New insights into the ATPdependent ClpP protease : E. coli and beyond. Molecular Microbiology (in press).
Rohrwild M, Coux O, Huang H-C, Moerschell RP, Yoo SJ, Seol JH,
Chung CH, Goldberg AL. 1996. HslV-HslU : A novel ATPdependent protease complex in Escherichia coli related to the
eukaryotic proteasome. Proceedings of the National Academy of
Sciences USA 93 : 5808–5813.
Schaller A, Ryan CA. 1995. Cloning of a tomato cDNA encoding the
proteolytic subunit of a Clp-like energy dependent protease. Plant
Physiology 108 : 1341–1342.
Schmidt GW, Mishkind ML. 1983. Rapid degradation of unassembled
ribulose 1,5-bisphosphate carboxylase small subunit in chloroplasts. Proceedings of the National Academy of Sciences USA 80 :
2632–2636.
Schrimer EC, Glover JR, Singer MA, Lindquist S. 1996. HSP100\Clp
proteins : a common mechanism explains diverse functions. TIBS
21 : 289–295.
Shanklin J, DeWitt ND, Flanagan JM. 1995. The stroma of higher
plant plastids contain ClpP and ClpC, functional homologs of
Escherichia coli ClpP and ClpC : an archetypal two-component
ATP-dependent protease. Plant Cell 7 : 1713–1722.
Shin DH, Lee CS, Chung CH, Suh SW. 1996. Molecular symmetry of
the ClpP component of the ATP-dependent Clp protease, an
Escherichia coli homolog of 20 S proteasome. Journal of Molecular
Biology 262 : 71–76.
Singh SK, Maurizi MR. 1994. Mutational analysis demonstrates
different functional roles for the two ATP-binding sites in ClpAP
protease from Escherichia coli. Journal of Biological Chemistry
269 : 29537–29545.
Sokolenko A, Altschmied L, Herrmann RG. 1997. Sodium dodecyl
sulfate-stable proteases in chloroplasts. Plant Physiology 115 :
827–832.
Sokolenko A, Lerbs-Mache S, Altschmied L, Herrmann RG. 1998. Clp
protease complexes and their diversity in chloroplasts. Planta 207 :
286–295.
Thompson MW, Maurizi MR. 1994. Activity and specificity of
Escherichia coli ClpAP protease in cleaving model peptide
substrates. Journal of Biological Chemistry 269 : 18201–18208.
Thompson MW, Singh SK, Maurizi MR. 1994. Processive degradation
of proteins by the ATP-dependent Clp protease from Escherichia
coli : requirement for the multiple array of active sites in ClpP but
not ATP hydrolysis. Journal of Biological Chemistry 269 :
18209–18215.
Wang J, Hartling JA, Flanagan JM. 1997. The structure of ClpP at
2n3 A/ resolution suggests a model for ATP-dependent proteolysis.
Cell 91 : 447–456.
Wang J, Hartling JA, Flanagan JM. 1999. The ab inito structure
determination of E. coli ClpP : A self-compartmentalizing ATPdependent protease. Journal of Structural Biology (in press).
Wojtkowiak D, Georgopoulos C, Zylicz M. 1993. Isolation and
characterisation of ClpX, a new ATP-dependent specificity
component of the Clp protease of Escherichia coli. Journal of
Biological Chemistry 268 : 22609–22617.
Woo KM, Chung WJ, Ha DB, Goldberg AL, Chung CH. 1989. Protease
Ti from Escherichia coli requires ATP hydrolysis for protein
breakdown but not for hydrolysis of small peptides. Journal of
Biological Chemistry 264 : 2088–2091.