Download A GTPase gate for protein import into chloroplasts

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A GTPase gate for protein import into
chloroplasts
© 2002 Nature Publishing Group http://structbio.nature.com
Felix Kessler and Danny J. Schnell
Protein import into chloroplasts is regulated by the binding and hydrolysis of GTP at two homologous GTPases,
Toc34 and Toc159. The crystal structure of the Toc34 GTP-binding domain suggests that GTP-regulated dimerization
of the Toc GTPase domains controls the targeting and translocation of preproteins at the chloroplast envelope.
The intracellular trafficking of newly synthesized polypeptides is governed by a set
of organelle-specific soluble and/or membrane-bound receptor systems that recognize targeting signals within passenger
proteins and direct them to protein
translocation machines (translocons) at
the boundary membrane of the appropriate organelle. Although the major signal
receptors and translocon components for
most organelles have been identified, the
communication mechanisms assuring the
fidelity of targeting within these systems
remain largely mysterious.
Chloroplast biogenesis requires the
import of hundreds of nuclear-encoded
preproteins across the double membrane
envelope from the cytosol. These preproteins are targeted to the organelle by
cleavable N-terminal transit sequences. A
multimeric translocon at the outer membrane (Toc complex) functions in the
recognition of the transit sequence. The Toc
complex associates with the translocon at
the inner membrane (Tic complex) to provide a direct conduit for preproteins from
the cytosol to the internal stromal compartment1–3 (Fig. 1). Two homologous GTPases,
Toc34 and Toc159, mediate the recognition
of transit sequences at the chloroplast surface4,5 and regulate the presentation of preproteins to the translocation channel6,7. On
page 95 of this issue of Nature Structural
Biology, Chwan-Deng Hsiao and coworkers8 offer a tantalizing glimpse into the role
of GTP in regulating this novel class of
GTPases by providing the crystal structure
of the GTP binding domain of Toc34. The
structure indicates that GTP binding and
hydrolysis at Toc34 are regulated by novel
homodimeric interactions. These data suggest how GTP-regulated dimerization may
regulate access of preproteins to the protein
translocation machinery of the chloroplast
envelope.
with Toc75, the major component of the
translocation channel12–14. The G-domains
of Toc159 and Toc34 are homologous and
define a unique subgroup of the GTPase
superfamily. Toc159 exists in both membrane-bound and cytosolic forms15 and is
the major receptor for transit sequences of
newly synthesized preproteins4. Toc34 is an
integral membrane protein whose GTPase
activity is required for translocation of
bound preproteins across the outer envelope membrane7. On the basis of these
observations, Toc159 is proposed to target
preproteins to the outer envelope membrane and Toc34 is proposed to act as a
GTP-regulated gate to the translocation
channel. Therefore, the structure of the Gdomain of Toc34 provides an entry point to
defining the role of GTP in regulating the
activities of both Toc GTPases.
Novel Toc34 G-domain structure
Sun et al.8 determined the structure of the
GDP-bound form of the 28 kDa Toc34
cytosolic G-domain. The construct lacks
only the 52-amino acid C-terminal membrane anchor. The first intriguing feature of
the structure is the unique arrangement of
motifs that define the GTP/GDP binding
pocket. The overall fold resembles that of
the canonical GTP-binding protein
H-ras-p21 (Ras)16. However, unlike Ras, six
insertions considerably enlarge the structure of the Toc34 G-domain. With one
exception, all of these insertions are of relevance for dimerization and GTPase activity.
Five motifs (G1–G5) within Ras are
involved in GTP binding and hydrolysis.
With the exception of G1 (P loop), the corresponding motifs either do not exist in
Toc34 or diverge significantly from
Ras–GDP, suggesting that Toc34 may have
evolved novel mechanisms of GTP binding and hydrolysis. For example, novel G4
and G5 motifs within Toc34 coordinate
GTP via the guanine ring. Although Toc34
Toc complex
has an apparent G4 motif (216NKxD219)
The core of the Toc complex consists of similar to that of Ras (117NKxD120), it does
Toc159 and Toc34 (refs 9–11) in association not appear to be involved in GDP binding.
nature structural biology • volume 9 number 2 • february 2002
Fig. 1 A model for the import of proteins into
chloroplasts. Nuclear-encoded chloroplast preproteins are synthesized on free cytosolic ribosomes and bind to the outer envelope
membrane via an interaction of their intrinsic
transit peptides with the Toc complex (green).
Toc159 (159) is the primary transit peptide
receptor. Binding may be facilitated by interactions of the transit peptide with outer membrane lipids and cytoplasmic factors. GTP
hydrolysis at the G-domains (G) of Toc159
and/or Toc34 (34) results in translocation of the
preprotein across the envelope. ATP hydrolysis
is also required for the translocation step and
has been attributed to molecular chaperones
(Com70 (C70), Hsp70 IAP (H70), Hsp93 (93), and
Cpn60 (60)) that maintain the unfolded import
competence of the preprotein during import.
Upon preprotein insertion across the outer
membrane, the Toc complex associates with the
Tic complex (blue) consisting of Tic20 (20), Tic22
(22), Tic40 (40), Tic55 (55) and Tic110 (110). The
Toc–Tic supercomplex facilitates direct translocation of preproteins from the cytosol to the
chloroplast stroma. The transit peptide is
removed by the stromal processing peptidase
(SPP), and newly imported proteins fold and
assemble or undergo subsequent sorting to the
proper suborganellar compartment.
Rather, Thr 162 and His 163 of Toc34
form contacts with the ribose and guanine
ring of GDP in a manner analogous to the
canonical G4 motif. These two residues
may therefore form the functional G4
motif of Toc34. Asp 119 of the Ras G5
motif, which forms two hydrogen bonds
to the guanine ring, is absent from Toc34.
Instead, Glu 210 and Asn 211 make strong
and weak interactions with the ring, thereby defining a unique G5 motif.
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© 2002 Nature Publishing Group http://structbio.nature.com
Fig. 2 Detailed view of one of two identical GTP binding sites in the Toc34 homodimer. The GTP
binding pocket of one subunit (blue ribbon) lies at the dimer interface in the GDP-bound form of
the molecule. Pro 169, Asp 170, Tyr 132 and Arg 133 of the second subunit (green ribbon) make
direct contacts with the bound GDP (red). This arrangement of residues is characteristic of the
arginine fingers of GTPase activating proteins, such as RhoGAP, RapGap and YptGAP, when bound
to their corresponding G-proteins. Disruption of dimer formation by mutation of Arg 128, a
residue that does not participate in GDP binding, reduces Toc34 GTPase activity. The figure was
generated by J.F. Swain using Insight II (Molecular Simulations, Inc.).
Additionally, Toc34 does not have a
classic G2 motif as defined by the invariant Thr 35 of Ras; rather, alternative
residues of Toc34 (Ser 72 or Ser 68) are
predicted to interact with the γ-phosphate
of GTP. Finally, the G3 motif that contributes a catalytic glutamine (Gln 61) to
the active site of Ras contains a leucine
(Leu 97) in the corresponding position in
Toc34, suggesting a novel mechanism of
hydrolysis. These observations not only
define Toc34 as a model for the unique
Toc GTPase family, but also will allow in
depth functional analysis of nucleotide
binding and hydrolysis in the import reaction through effective mutagenesis.
A GTPase switch?
The most exciting implications of the
structure relate to the activity of Toc34
within the Toc complex. A particularly
striking feature is that the G-domain forms
a dimer with residues of one monomer
contributing to the formation of the GDP
binding pocket of the other monomer.
Remarkably, the dimer interface resembles
the interface between small GTPases and
their GTPase-activating proteins (GAPs)17
(Fig. 2). Such GAPs contribute a catalytic
‘arginine finger’ to the active site of small
GTPases upon binding. Arg 133 of Toc34 is
deeply inserted into the GDP binding
region of the other monomer, suggesting
that it may function as an arginine finger to
stimulate hydrolysis (Fig. 2). The authors
further show that mutation of a second
arginine, Arg 128, which forms critical
hydrogen bonds at the dimer interface, disrupts dimer formation in solution and
greatly reduces GTPase activity relative to
wild type. These findings suggest a novel
reciprocal activation of the Toc34 GTPase
subunits in the homodimer.
Previous biochemical analyses indicate
that GTP binding alters the interaction of
Toc34 with preproteins5,18. In addition,
GTP hydrolysis at Toc34 is required to initiate preprotein translocation through the
Toc complex7. These two observations in
conjunction with the structural data sug82
gest a possible model in which GTP-regulated dimerization of Toc34 functions as
part of the molecular switch controlling
the commitment of bound preproteins to
translocation. In the GDP-bound dimer
of Toc34, Glu 73 of the G2 motif occupies
the predicted position of the γ-phosphate
of a bound GTP. Furthermore, there is no
apparent entrance/exit site for nucleotide
in the dimer. Thus, a significant conformational shift or perhaps dimer dissociation may be required for GDP/GTP
exchange. As a consequence, the conformational state of Toc34 may be directly
regulated by bound nucleotide. It is
tempting to speculate that this shift coupled with subsequent dimer-activated
GTP hydrolysis could constitute at least
part of the GTP-dependent switch
required for the initiation of membrane
translocation.
One possible mechanism for coupling
the GTP binding and hydrolytic events at
Toc34 to other components of the Toc
complex is also suggested by the crystal
structure. The single cysteine at position
215 in Toc34 is located in the longest loop
region in the structure. Cys 215 can be
oxidatively crosslinked directly to other
Toc components19, in particular Toc75,
indicating close contact to the channel
component. The newly assigned G5 is
located on the longest loop as well, suggesting that this loop may transmit events
at the GTP binding site to other translocon subunits. Conversely, the contacts at
the Cys 215 loop provide a mechanism for
other Toc components to participate in
nucleotide exchange, possibly by acting as
GTP/GDP exchange factors (GEFs).
The Toc34 G-domain structure suggests
a second possible interaction with implications for preprotein targeting to the Toc
complex. The Toc159 receptor contains a
G-domain with significant sequence similarity to that of Toc34 (ref. 9). The most
conserved regions between Toc34 and
Toc159 lie in their GTP-binding motifs
and the regions responsible for dimer formation. The putative arginine finger is
conserved in all known Toc159
sequences8. This raises the possibility that
Toc159 also forms homodimers with reciprocal GTPase activating properties.
Another, more intriguing possibility is
heterodimer formation between Toc34
and Toc159. This proposal is supported
by the observation that the G-domain of
the Arabidopsis ortholog of Toc34 competes for the insertion of the soluble form
of Toc159 in the chloroplast outer membrane15. The new structural data therefore
suggest the possibility that Toc34 may
serve as a GTP-regulated docking site for
the cytosolic form of the Toc159 receptor
as it targets to the Toc complex in the
outer membrane. In fact, Sun et al.8 report
that the GDP-bound form of the Toc34
G-domain exists in solution at a
dimer:monomer ratio of 1:4. Although
this observation could be an artifact of
measuring association external to the two
dimensional lipid bilayer of the outer
membrane, it provides additional indirect
evidence that homodimerization is
dynamic and opens the door for
Toc159–Toc34 heterodimer formation.
The scenario of a preprotein targeting
cycle involving cognate GTPases bears an
uncanny resemblance to the targeting of
the ribosome–nascent chain complexes
(RNC) to the endoplasmic reticulum (ER)
membrane by the signal recognition particle (SRP) and SRP receptor (SR)20. SRP
associates with RNCs in the cytoplasm
and the SRP–RNC is targeted to the ER
protein translocon by docking at SR.
Upon binding, SRP and the SR α-subunit
act as reciprocal GTPase activators in a
GTP-dependent reaction that ensures unidirectional transfer of the RNC to the
translocation channel of the translocon21.
The reciprocal GAP activities are proposed to involve conserved arginine finger
domains similar to those observed in the
Toc34 dimer. Although it remains to be
demonstrated that the Toc34–Toc159
interaction represents a step in preprotein
targeting to chloroplasts, the potential
conservation of GTP-dependent mechanisms in these two targeting reactions is
very compelling. As such, the structure of
Toc34 provides a bounty of new testable
hypotheses to account for the GTP-dependent protein targeting mechanisms at the
outer chloroplast membrane and other
intracellular protein targeting pathways.
Felix Kessler is in the Institute of Plant
Sciences, Plant Physiology and Biochem-
nature structural biology • volume 9 number 2 • february 2002
© 2002 Nature Publishing Group http://structbio.nature.com
news and views
istry Group, ETH Zürich Universitätstrasse
2, 8092 Zürich, Switzerland. Danny J.
Schnell is in the Department of
Biochemistry and Molecular Biology,
University of Massachusetts, Amherst,
Massachusetts 01003, USA. Correspondence should be addressed to F.K. email:
[email protected] or D.J.S.
email: [email protected].
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Bacterial Esperanto
Stephen C. Winans
The structure of a bacterial pheromone bound to its receptor sheds light on cell–cell signaling in bacteria.
Many groups of bacteria, which until
recently were thought to live rather reclusive lives, are now known to communicate
with each other by means of diffusible
chemical signals. Bacteria are thought to
use chemical pheromones to take a census
of their population size and to express particular target genes only at high cell densities, a phenomenon sometimes referred to
as quorum sensing1. These bacterial
pheromones are required for diverse
behaviors, including bioluminescence, the
production of pathogenesis factors, antibiotics and other secondary metabolites, the
horizontal transfer of DNA, and the formation of biofilms2. Although most known
examples of bacterial signaling occur within a single species, Bonnie Bassler and colleagues3 at Princeton University several
years ago discovered a chemical signal,
called autoinducer-2 (AI-2), that is released
by many groups of eubacteria, raising the
possibility of a universal chemical lexicon
employed by multitudes of diverse bacteria. Although the gene encoding the AI-2
synthase (luxS) was subsequently identified and found to be widely conserved4, the
chemical structure of this signal has until
now remained elusive. In a recent issue of
Nature, Hughson (also at Princeton),
Bassler and colleagues5 solved the structure
of AI-2, remarkably through X-ray crystallography of its protein receptor.
Fig. 1 Production and detection of a universal bacterial pheromone. In most bacteria, utilization of
S-adenosylmethionine (SAM) as a donor of methyl groups results in production of S-adenosylhomocysteine (SAH), which is metabolized in two steps by Pfs and LuxS to create homocysteine,
adenine, and a molecule that spontaneously rearranges to form Pro-AI-2. Pro-AI-2 is released from
the bacteria and accumulates in the cell supernatant. Vibrio harveyi and probably many other
bacterial genera can detect pro-AI-2 as a pheromone. In the case of V. harveyi, pro-AI-2 forms a
complex with borate, and this complex binds to LuxP, an extracytoplasmic protein that resembles
the ribose binding protein of enteric bacteria. LuxP–AI-2 complexes transduce a signal to LuxQ, a
transmembrane kinase, which phosphorylates LuxO (indirectly via LuxU, not shown). LuxO-P indirectly causes induction of a biolumenscence operon, resulting in light production. LuxO can also be
phosphorylated by a second kinase, LuxN, in response to a separate pheromone (not shown).
groups of Gram-positive bacteria use
either oligopeptides or γ-butyrolactones
as signals2. At least one cyanobacterium
also signals via oligopeptides6. In contrast,
most signaling in proteobacteria is
accomplished using N-acylhomoserine
lactones7. However, earlier studies of AI-2,
first discovered in the bioluminescent
A rich chemical lexicon for bacterial marine bacterium Vibrio harveyi, suggestsignaling
ed that it was unlikely to resemble any of
Chemical signaling has evolved many these molecules. Bassler and colleagues8
times among the prokaryotes. Many previously showed that AI-2 is formed
nature structural biology • volume 9 number 2 • february 2002
during the catabolism of S-adenosylmethionine (SAM), a universal donor of
methyl groups. Demethylation of SAM
yields a compound, S-adenosylhomocysteine (SAH) that is further processed to
yield adenine, homocysteine, and a compound (derived from the ribosyl moiety of
SAM) that spontaneously rearranges to
yield AI-2 (Fig. 1). AI-2 should therefore
structurally resemble ribose.
AI-2 is detected by V. harveyi through
binding to an extracytoplasmic receptor
83