We demonstrate the operation of the Sec and Tat pathways in cyanelles and show for the first time in vitro protein import across cyanobacteria-like thylakoid membranes and protease protec
Trang 1The cyanelle of Cyanophora paradoxa
Juergen M Steiner1, Juergen Bergho¨fer2, Fumie Yusa1, Johannes A Pompe1, Ralf B Klo¨sgen2 and Wolfgang Lo¨ffelhardt1
1 Max F Perutz Laboratories, University Departments at the Vienna Biocenter, Department of Biochemistry and Molecular Cell Biology and Ludwig Boltzmann Research Unit for Biochemistry
2 Martin-Luther-Universitaet Halle-Wittenberg, Institute for Plant Physiology, Vienna, Austria
The photosynthetic apparatus of cyanobacteria and
chloroplasts are organized in an analogous way, with
some differences in detail [1], and comprise stromal
proteins that temporarily attach to the thylakoid
surface, a number of integral thylakoid membrane
proteins, and some soluble or loosely
membrane-asso-ciated proteins of the thylakoid lumen In addition,
there are certain proteins that possess a thylakoid
membrane anchor but expose the bulk of the
poly-peptide chain and the prosthetic group involved in
electron transfer to the lumen In higher plants, the
chloroplast and nuclear genomes both contribute to
the complement of thylakoid proteins [2]
Nucleus-encoded precursors to integral proteins may contain a
transit sequence only so that information for thylakoid
integration is contained in the mature protein: the cab protein family is thought to use an exclusive mechan-ism, the post-translational signal recognition particle (SRP) pathway [3] Others possess a bipartite pre-sequence consisting of a stroma-targeting peptide fol-lowed by a signal peptide-like hydrophobic domain Such proteins as CF0-II integrate via the spontaneous (unassisted) pathway without the need for stroma fac-tors recepfac-tors, ATP, and DpH [4] Members of the group of (predominately) lumenal proteins are passen-gers of the Sec and DpH-dependent (or Tat) pathways, corresponding to their relatively unfolded or folded state during translocation, respectively Identified components of the chloroplast Sec machinery are SecY⁄ SecE (forming the translocation pore) and the
Keywords
Cyanophora paradoxa; cyanelles;
conservative sorting; Sec translocase; Tat
translocase
Correspondence
W Lo¨ffelhardt, Max F Perutz Laboratories,
University Departments at the Vienna
Biocenter, Department of Biochemistry and
Molecular Cell Biology and Ludwig
Boltzmann Research Unit for Biochemistry,
Dr Bohrgasse 9, 1030 Vienna, Austria
Fax: +43 14277 9528
Tel: +43 14277 52811
E-mail: wolfgang.loeffelhardt@univie.ac.at
(Received 6 September 2004, revised 11
November 2004, accepted 17 December
2004)
doi:10.1111/j.1742-4658.2004.04533.x
Higher plant chloroplasts possess at least four different pathways for pro-tein translocation across and propro-tein integration into the thylakoid mem-branes It is of interest with respect to plastid evolution, which pathways have been retained as a relic from the cyanobacterial ancestor (‘conserva-tive sorting’), which ones have been kept but modified, and which ones were developed at the organelle stage, i.e are eukaryotic achievements as (largely) the Toc and Tic translocons for envelope import of cytosolic pre-cursor proteins In the absence of data on cyanobacterial protein transloca-tion, the cyanelles of the glaucocystophyte alga Cyanophora paradoxa for which in vitro systems for protein import and intraorganellar sorting were elaborated can serve as a model: the cyanelles are surrounded by a pepti-doglycan wall, their thylakoids are covered with phycobilisomes and the composition of their oxygen-evolving complex is another feature shared with cyanobacteria We demonstrate the operation of the Sec and Tat pathways in cyanelles and show for the first time in vitro protein import across cyanobacteria-like thylakoid membranes and protease protection of the mature protein
Abbreviations
GFP, green fluorescent protein; SRP, signal recognition particle.
Trang 2translocation ATPase SecA which is a stromal protein
that binds to the pore during action Energy source:
ATP Inhibitor: sodium azide, acting on SecA Proteins
that fold rather quickly or must acquire their
pros-thetic groups in the stroma prior to translocation enter
the Tat pathway, in most cases indicated by a
‘twin-arginine’ motif preceding the hydrophobic core of their
signal sequences The known components Tat A
(Hcf106), TatB (Tha4) and TatC are all
membrane-bound Energy source is DpH at the thylakoid
mem-brane Inhibitor is nigericin, dissipating DpH [5] All
these data were collected using higher plant
chloro-plasts, with the exception of fucoxanthin⁄
chloro-phyll c-binding protein and the secondary plastids
(derived from endosymbiotic red algae) of the diatom
Odontella sinensis where indications for a SRP
path-way were obtained [6]
Cyanelles are the peptidoglycan-surrounded plastids
of glaucocystophyte algae [7], assumed to be the first
phototrophic eukaryotes [8] The protein import
appar-atus of the cyanelle envelope seems to function in an
analogous way as for rhodoplasts and chloroplasts [9]
Are all four pathways for further protein routing
inside the chloroplast also operative in a primitive
plastid? The meaning of ‘conservative sorting’ [10], i.e
the retainment of prokaryotic translocons in organelles
of endosymbiotic origin as first exemplified by the Sec
pathway, has changed during the past decade: the Tat
pathway was considered as an achievement of higher
plants until its occurrence in (cyano)bacteria was
dem-onstrated [5] On the other hand, the spontaneous
pathway now seems to be restricted to chloroplasts
since related insertion processes in bacteria were found
to depend on the novel translocon component YidC
[11,12]
In cyanobacteria, there are dual Sec translocons, in
the thylakoid membrane as well as in the inner
envel-ope membrane [13] Tat translocase is assumed to be
located in the inner envelope membrane of
cyanobac-teria: numerous periplasmic proteins were found where
the corresponding genes contained signal sequences
with the twin-arginine motif [14] and Tat signal
pep-tides directed green fluorescent protein (GFP) to the
periplasmic space of transgenic cyanobacteria [15] On
the other hand, the Tat passengers known from
chlo-roplasts are largely absent from several completely
sequenced cyanobacterial genomes: the two extrinsic
proteins from the oxygen evolving complex, PsbP and
PsbQ, are replaced in cyanobacteria by the unrelated
proteins PsbV and PsbU [16], respectively, that both
lack the twin arginine motif in the precursors Even
when a conserved lumenal protein like Hcf136 was
considered, the sorting signal appeared to have
changed after gene transfer to the nuclear genome: the precursor⁄ intermediate was shown to use the Sec pathway in cyanobacteria and the Tat pathway in higher plants [17]
One reason to investigate thylakoid transloca-tion⁄ integration in cyanelles is the bridge position of these organelles between chloroplasts and free-living cyanobacteria Cyanelle thylakoids resemble the cyano-bacterial ones in the composition of the OEC, the presence of phycobilisomes [18] as antenna system and the possibility of connections to the inner envelope membrane [19] In freeze-fracture experiments, cyanelle thylakoids also behaved cyanobacteria-like and upon isolation did not readily form closed vesicles as chloro-plast thylakoids do [20] In vitro experiments with cyanobacterial thylakoids are hampered through this problem: successful translocation is evidenced through protease protection of the mature (processed) protein which is not feasible when no tight vesicles can be pre-pared (C Robinson, personal communication) Assays that cannot be done ‘in thylakoido’ with cyanobacteria can be performed ‘in organello’ using intact, isolated cyanelles So phycobilisome assembly could be monit-ored via integration of imported, labeled core linker protein [18] The Sec pathway in cyanelles, which was made likely by the first demonstration of a functional organellar-encoded secY gene [21], was corroborated
by determining the energetic requirements for cyto-chrome c6, cytochrome c550 and PsbO import In this paper, we demonstrate Rieske Fe⁄ S protein as a Tat passenger, and, for the first time with phycobilisome-bearing thylakoids, we show protease protection of translocated, processed thylakoid lumenal proteins Another advantage of the cyanelle system is that pas-senger proteins can be studied that are different from the established chloroplast import systems (i.e that are not imported in vivo into chloroplasts) as AtpI
Results
Cytochrome c6uses the Sec pathway Using a modified cyanelle isolation procedure, the effi-ciency of homologous (envelope) import could be greatly increased [9,19] Import-competent cyanelles from Cyanophora paradoxa efficiently took up the
15 kDa pre-apocytochrome c6 and converted it into the protease-protected mature form of 9.2 kDa, comi-grating with the holoprotein (Fig 1, left panel) The time-course showed that the import was largely com-pleted after 3 min Prolonged incubation resulted in eventual degradation of the mature protein, presuma-bly through lumenal proteases [22] No intermediate
Trang 3was observed under these assay conditions, indicating
that envelope import is the rate-determining step
However, two-step processing, as reported for the
homologue from Chlamydomonas reinhardtii [23], could
be demonstrated when intermediate accumulated due
to the addition of the SecA inhibitor, sodium azide
(Fig 1, right panel), whereas nigericin, the inhibitor of
the DpH-dependent Tat pathway had no effect (data
not shown) The effect of azide was most pronounced
after 3 min compared with the control assay After
that time the intermediate is slowly imported and
pro-cessed, maybe because of intracyanellar ATP
genera-tion, as the azide-inhibition of SecA can be reverted by
a higher amount of ATP competing for the same
bind-ing sites [24] As sodium azide might inhibit
chloro-plast and cyanelle proteases (our own experiments and
K Cline, personal communication), it is somewhat
dif-ficult to compare the posterior time points Sodium
azide also appeared to impede the overall import
process of pre-apocytochrome c6 to some extent, since
a small amount of precursor protein was still bound to
the envelope (Fig 1, right panel) For a small
protein-like cytochrome c6 complete protease protection could
not be achieved [19] We therefore extended our
experiments to a heterologous system where tight
thylakoid vesicles can be obtained In vitro assays with
isolated spinach chloroplasts were performed including
competition experiments Saturating amounts of the
OEC23 (PsbP) precursor from spinach were used to
block the DpH-dependent Tat pathway [5] and – in
parallel – saturating amounts of the spinach OEC33
(PsbO) precursor, a well known Sec-passenger [24] to
inhibit the Sec-pathway (Fig 2) It could be clearly
shown that pre-apocytochrome c6 from C paradoxa
was readily imported into chloroplasts, processed to an intermediate form in the stroma and then translocated into the thylakoid lumen, where it was processed again
to its protease-protected mature form (Fig 2) In the heterologous system, the stroma-processing protease cleavage site obviously was not properly recognized leading to an intermediate of higher MW than that observed in the cyanelle system (Fig 1, right panel) When the OEC23 precursor was used as a competitor, the amount of mature protein was almost unchanged and its location in the thylakoid lumen could be pro-ven by protease protection (Fig 2) In competition experiments with the OEC33 precursor, the intermedi-ate accumulintermedi-ated in the stroma fraction by a factor of two (ImageQuant) and the amount of mature protein was reduced by a factor of two compared to the con-trol assay (Fig 2) The results of homologous and heterologous import experiments indicate that apocyto-chrome c6 is a Sec passenger in cyanelles (as its func-tional homologue plastocyanin in higher plants) in spite of the lack of protease protection due to system-inherent problems
Protease protection of lumenal proteins in cyanobacterial-type thylakoid vesicles
In order to identify additional Sec passengers and, eventually, to prove protease protection of a larger lumenal protein in cyanelle thylakoids, we cloned the psbO gene (GenBank accession number AJ784854) via
a PCR approach based on N-terminal sequence infor-mation [25] and used the labeled precursor to study the function of the cyanelle thylakoid translocons Figure 3 shows a typical import experiment in a time Fig 1 Time-course of import of35S-labeled pre-apocytochrome c6 into isolated cyanelles T, translation mix; –⁄ +, without ⁄ with addition of thermolysin; p, precursor; i, intermediate; m, mature protein Left panel: control; right panel: plus 10 m M sodium azide.
Trang 4course from 3 to 15 min Compared to
pre-apocyto-chrome c6, thylakoid import was retarded in the
stroma, whereas envelope translocation was almost
completed after 3 min The resulting intermediate form
of the OEC33 protein (iOEC33) appeared to be
trans-ported into the thylakoid lumen in a time-dependent
manner (Fig 3) When sodium azide was added,
mat-uration of iOEC33 was completely stopped (Fig 3)
These data do not unequivocally show if the mature
protein is internalized into the thylakoid lumen and
thus we wanted to demonstrate protease protection, at
least for larger proteins as OEC33 We established a
method to isolate tight thylakoid vesicles, which was
hitherto not achieved for phycobilisome-bearing,
cyanobacterial-type membranes When the novel
cyanelle fractionation procedure was applied after an
import experiment, the mature OEC33 localized to the thylakoid fraction (Fig 4) Thermolysin treatment digested the residual amount of precursor bound to the peptidoglycan-containing envelope membranes, which co-sedimented with the thylakoids and therefore served as internal controls for protease activity, whereas the internalized mature protein was protected
in the thylakoid lumen Sodium azide decreased the amount of precursor imported into the cyanelles (Fig 4), possibly due to inhibition of other ATP-dependent processes [26] iOEC33 accumulated in the stroma and even a small proportion of mature PsbO fractionated with the thylakoid membranes, possibly due to the longer incubation time (25min.) When this membrane pellet (containing also envelope membranes) was treated with thermolysin, the bound precursor as
Fig 2 Import of 35 S-labeled pre-apocytochrome c6of C paradoxa into isolated spinach chloroplasts Tr, translation mix; S, stroma; T–, thyl-akoid membranes; T+, thylthyl-akoid membranes treated with thermolysin; p, precursor; i, intermediate; m, mature protein Left panel, control; middle panel, saturation of the Tat-pathway by the 23-kDa protein; right panel, saturation of the Sec-pathway by the 33-kDa protein.
Fig 3 Time-course of import of 35 S-labeled pre-OEC33 into isolated cyanelles ivT, translation mix; p, precursor; i, intermediate;
m, mature protein (A) control; (B) plus
10 m M sodium azide; control + TL, plus thermolysin.
Trang 5well as the putative mature protein band disappeared,
indicating that PsbO protein generated in the presence
of azide is only bound to the thylakoid surface (or
rather arrested in a preliminary insertion stage that
allowed processing) but not internalized and thus not
protease-protected Nigericin also caused a drop in
over-all import efficiency, but did not lead to accumulation
of the intermediate in the stroma Furthermore, in
con-trast to the azide experiment, mature protein
fractionat-ing with the thylakoids was protease-protected (Fig 4)
All these data point towards a functional Sec translocase
with an azide-sensitive SecA homologue in the cyanelle
thylakoid membrane and that lumenal cytochrome c6as
well as OEC33 protein had used this pathway
The Rieske Fe/S protein is inserted into cyanelle
thylakoids via theDpH-dependent Tat pathway
In chloroplasts, Rieske Fe⁄ S protein, a
nuclear-enco-ded subunit of the cytochrome b6⁄ f complex, appeared
in the stroma after in vitro import and only slowly
translocated further into the thylakoid membrane
sys-tem It could also be shown, via competition
experi-ments and the sensitivity to nigericin, that thylakoid
translocation⁄ integration of this protein in chloroplasts
takes place through the DpH-dependent Tat pathway
[27] As the Rieske protein lacks a cleavable signal
peptide, its transport is mediated by the N-terminal
membrane anchor which does not contain the
twin-arginine motif typical of Tat transport signals Instead,
higher plant as well as cyanelle Rieske proteins contain
a lysin-arginine sequence at this position, whereas
cyanobacterial Rieske proteins do possess the
twin-arginine motif This renders the Rieske protein an
unusual Tat substrate and, concerning the evolutionary
position of the cyanelles, it was interesting which
pathway the authentic Rieske protein might use in the
cyanelle system For that reason we cloned two petC genes from C paradoxa via a PCR approach (petC1, GenBank accession number AJ784852 and petC2, GenBank accession number AJ784853) We performed
in organello experiments with isolated intact cyanelles and the precursor corresponding to petC1 in a time-course manner in the presence⁄ absence of specific translocase inhibitors (Fig 5) A striking feature in the targeting process of Rieske protein is the remarkably slow sorting of the protein within the cyanelles to its final destination, the thylakoid membrane system While the import of the Rieske precursor into the stroma proceeded within 5 min (Fig 5), only a minor fraction (20%) reached the thylakoids and was cor-rectly integrated during a total incubation time of
25 min (Fig 6) The majority of the processed mature protein of approximately 20 kDa accumulated in the
Fig 4 Fractionation of isolated cyanelles after import of 35 S-labeled pre-OEC33 ivT, translation mix; C, intact cyanelles; S, stroma; T–, thyla-koid membranes; T+, thylathyla-koid membranes treated with thermolysin; p, precursor; i, intermediate; m, mature protein; azide, plus 10 m M
sodium azide; nigericin, plus 2 l M nigericin.
Fig 5 Import of 35 S-labeled pre-Rieske-protein into isolated cya-nelles ivT, translation mix; p, precursor; m, mature protein; azide, plus 10 m M sodium azide; nig, plus 2 l M nigericin.
Trang 6cyanelle stroma Once the Rieske protein had
reached the thylakoids, it was to a large extent protected
against the activity of proteases that were added
exter-nally to the thylakoid vesicles, indicating that the
C-terminal hydrophilic domain had been completely
translocated into the lumenal space (Fig 6) Only the
utmost N-terminal residues preceding the membrane
anchor domain remained accessible to thermolysin
resulting in a decrease in apparent molecular mass of
approximately 0.5 kDa In the presence of nigericin,
thylakoid translocation of the Rieske protein was
com-pletely abolished, and the bulk of intracyanellar mature
protein localized with the stroma, while the thylakoid
membrane fraction contained only a minor amount of
loosely bound Rieske protein which disappeared after
thermolysin treatment Sodium azide reduced the
amount of membrane-integrated Rieske protein to
about 30% of the control assay This fraction of
mature protein was protease-protected and fully
integ-rated into the thylakoid membrane system through its
single membrane span A slight downward shift in
polypeptide mobility was best noticeable here due to
the removal of about four amino acids from the
stroma-exposed N terminus by thermolysin treatment,
indicating correct integration into the cytochrome b6f
complex In evaluating the azide effect on thylakoid
translocation of Rieske protein one should consider its
reported inhibitory action on numerous
nucleotide-binding proteins [28]: the azide-sensitive steps occur
very likely after the import of the apoprotein and prior
to membrane integration of the holoprotein [27] Thus
our interpretation of the experiments shown in Fig 6
is to name Rieske a bona fide Tat passenger: a similar conclusion was made in the chloroplast system [27]
The first precursor to a cyanelle lumenal protein (PsbU) containing the twin-arginine motif PrePsbU from the red alga Cyanidium caldarium was found to contain the twin-arginine motif in the thyla-koid transfer domain of the presequence [29]: this was the first incidence in algae containing ‘primitive’ plast-ids and prompted us to clone the counterpart from
C paradoxa based on sequence information from an EST collection (S Burey and H Bohnert, unpublished data) This completed the collection of cyanelle OEC component genes (GenBank accession number AJ784849) and presented another Tat passenger candi-date (Fig 7) Cyanelle import of prePsbU occurred as readily as that of the other small cyanelle protein, pre-cytochrome c6, with almost no envelope-bound precur-sor or intermediate (Fig 8) However, azide addition did not result in the accumulation of intermediate, excluding the Sec pathway Nigericin did not produce
a clear-cut effect either (except also increasing the amount of envelope-bound precursor) Small proteins obviously can escape from cyanelle thylakoid vesicles, thus mature PsbU localized to the stroma fraction in comparable amounts irrespective of any inhibitor used (Fig 8) Considering these experimental difficulties with regard to protease protection and the reported DpH-independence of Tat translocation in C reinhardtii chloroplasts [30] we propose that PsbU, i.e one of the three cyanelle OEC proteins, is a Tat passenger
Fig 6 Fractionation of isolated cyanelles after import of the 35S-labeled pre-Rieske-protein ivT, translation mix; C, intact cyanelles; S, stroma; T–, thylakoid membranes; T+, thylakoid membranes treated with thermolysin; p, precursor; m, mature protein; azide, plus 10 m M
sodium azide; nigericin, plus 2 lm nigericin.
Trang 7AtpI: SRP-dependent or spontaneous thylakoid
integration?
As other primitive plastids, cyanelles encode a higher
number of ATP synthase subunits than higher plant
chloroplasts, e.g atpG and atpD [32] However, in
con-trast to all sequenced plastid genomes, atpI is a nuclear
gene in C paradoxa [7] This means that a precursor
exceeding Cab protein with respect to hydrophobicity
has to be imported into cyanelles, must cross the
stroma and insert into the thylakoid membranes In the
case of Cab, the solution is the post-translational SRP
pathway involving a transit complex consisting of Cab,
SRP54 and SRP43 [3] We cloned two atpI genes via a
PCR approach based on highly conserved domains of
the protein They are listed in under the GenBank
accession numbers AJ784850 and AJ784851,
respect-ively The closely related sequences comprise four to
five putative transmembrane regions as candidates for
binding to SRP54 [33] and a hydrophilic loop with some resemblance to the ‘L18’ domain of Cab protein (Fig 9) which was shown to interact with SRP 43 [3] Highly hydrophobic precursor proteins pose problems upon in vitro import into isolated chloroplasts [34] This also applies for cyanelle envelope translocation: only a small fraction of AtpI is processed and internal-ized (Fig 10A) though a time course is noticeable Cya-nelle fractionation after incubation resulted in recovery
of substantial amounts of preAtpI in the thylakoid (and envelope) fraction Low amounts of mature pro-tein were detected in the thylakoid fraction: here no influence of added azide or nigericin became apparent Due to cleavage sites in the stromal loops thylakoid-inserted AtpI was degraded upon thermolysin treat-ment (Fig 10B) With regard to the insertion pathway, Sec and Tat do not seem to be involved In order to
Nucleus-encoded:
pre-cytochrome c6
KKGRREFVAAAGALFAAFAASPAAFA Plastid-encoded:
pre-cytochrome c550
MRKLFLLMFCLSGLILTTDIRPVRA Fig 7 Thylakoidal signal peptides of intermediates (precursors) to
cyanelle proteins that are imported into or synthesized within the
organelle, respectively Charged residues are underlined C,
C-ter-minal domain; H, hydrophobic core (bold); N, N-terC-ter-minal domain.
The signal sequences of nucleus-encoded precursors start with the
first amino acid after the putative SPP cleavage sites taken from
predictions of the CHLOROP program [31].
Fig 8 Fractionation of isolated cyanelles after import of the
35
S-labeled prepsbU-protein ivT, translation mix; S, stroma; T–,
thylakoid membranes; T+, thylakoid membranes treated with
thermolysin; p, precursor; m, mature protein; azide, plus 10 m M
sodium azide; nigericin, plus 2 lm nigericin.
Fig 9 Sequence comparison of a hydrophilic loop between the putative TM helices 3 and 4 of AtpI from C paradoxa to the ‘L18’ domain of pea LHCP Similarities are indicated by bold letters.
A
B
Fig 10 (A) Time-course of import of the35S-labeled preatpI-protein into isolated cyanelles T, translation mix; p, precursor; m, mature protein; 0, incubation for 20 min on ice; –, control; +, plus thermo-lysin (B) Fractionation of isolated cyanelles after import of the
35 S-labeled preatpI-protein ivT, translation mix; S, stroma; T–, thylakoid membranes; T+, thylakoid membranes treated with thermolysin; p, precursor; m, mature protein; azide, plus 10 m M
sodium azide; nigericin, plus 2 l M nigericin.
Trang 8assess an SRP-based mechanism, the import efficiency
will have to be increased first Spontaneous insertion is
less likely for a polytopic membrane protein as AtpI
but cannot be excluded at present
Discussion
Two of the four pathways operating in thylakoid
translocation⁄ integration in higher plant chloroplasts,
i.e the Sec and the Tat translocase could be
demon-strated to function in the cyanelles of C paradoxa
With respect to the hydrophobicity of the core
domains signal sequences from cyanelle genes surpass
those from nuclear genes (Fig 7) This has also been
observed with higher plants: the replacement (after
gene transfer) of leucine and phenylalanine by alanine
was interpreted as a measure to avoid interactions with
cytosolic SRP [35]
Interestingly, there seem to be relatively more
known Sec passengers in these primitive plastids than
Tat passengers, whereas the opposite was found for
chloroplasts [5] There are several reasons for the
observed prevalence of the Sec pathway in cyanelles:
one of them is the replacement of evolutionary ancient,
i.e cyanobacterial proteins by unrelated counterparts
in higher plants The cyanelle-encoded cytochrome c550
fulfills the function of the Tat passenger PsbP in the
OEC By analogy to the other c-type cytochromes it
should use the Sec pathway This was proven by
homologous and heterologous import experiments of a
construct containing the FNR transit sequence from
C paradoxa at the N terminus (T Ko¨cher and
J Steiner, unpublished data) Second, Tat passengers
like polyphenol oxidase, PsbT, PsaN and others
(with-out a cyanobacterial homologue) might be absent from
cyanelles Third, C paradoxa with its peculiar gene
distribution between the nuclear and cyanelle genomes
allows to test the hypothesis that rapidly folding
(small) polypeptides without prosthetic groups can be
Sec passengers in cyanobacteria and in primitive
plast-ids (when they are plastom-encoded) but should be
Tat passengers as nuclear gene products in higher
plant chloroplasts In the latter case, protein targeting
to the thylakoid lumen is much more time-consuming
and the intermediate should be rather tightly folded
upon arrival at the membrane [17] The
cyanelle-enco-ded Hcf136 homolog resembles its cyanobacterial
counterpart in the absence of the twin arginine motive,
i.e is a putative Sec passenger PsbU, on the other
hand, an OEC component of cyanobacteria and
chlo-rophyll b-less algae, is nucleus-encoded in the latter
Consequently, PsbU contains a twin-arginine motif in
its bipartite presequence and its translocation therefore
most likely is Tat-dependent In this case, evolutionary replacement through PsbQ in higher plants is not accompanied by a change in the translocase used Isolated cyanobacterial thylakoid vesicles do not allow in thylakoido import experiments or protease protection of luminal proteins, in contrast to spinach thylakoids [5,34] Therefore it was a considerable pro-gress to show protease protection at least for the
33 kDa PsbO protein after internalization into cyanelle thylakoids It is unknown, why these membranes are still leaky for smaller proteins as cytochrome c6 and PsbU Sodium azide appeared to be the diagnostic inhibitor of choice for in cyanello experiments Thyla-koid import was retarded for small Sec passenger proteins and completely blocked for larger ones, respectively This indicates high azide-sensitivity of cyanelle SecA Thus the Sec pathway can be excluded when no significant azide effect is observed, e.g in the case of PsbU
Nigericin completely abolished protease protection
of membrane-inserted Rieske protein However, azide addition resulted in a reduction of the amount of correctly assembled cyanelle Rieske protein as was observed with the chloroplast in organello system [27] There it was shown that although the Rieske protein is targeted exclusively by the DpH ⁄ Tat pathway, some azide-sensitive stromal factors, such as the Cpn60 chaperonin [36] might play a role in correct folding and⁄ or attachment of the Fe ⁄ S cluster to the Rieske mature (apo)protein Recently, an interaction and⁄ or regulation partner for the Rieske protein has been identified in Arabidopsis thaliana [37] A thylakoid lum-enal FKBP (immuno-suppressant FK506 binding pro-tein) was isolated, whereof only the precursor, but not the mature form, interacted with Rieske protein AtFKBP13 might serve as an ‘anchor’ chaperone that holds the Rieske protein in the cytoplasm or in the stroma so that excessive Rieske protein is not targeted
to the thylakoid, since its integration into the cyto-chrome b6f complex underlies complex regulation and coordination events It might well be that sodium azide blocks either one of those chaperones and⁄ or other factors necessary for the build-up of a functional cyto-chrome b6f complex, and the resulting malfunctioning
or only partially moulded unit fails to be translocated via the Tat-pathway The effects of nigericin on thyla-koid translocation of the other candidate Tat passen-ger, PsbU, were not clear-cut In this context it should be noted that the Tat translocase in the alga
C reinhardtii appeared to operate in the absence of a DpH [30], whereas proton efflux at the expense of the
pH gradient was a prerequisite for Tat-dependent translocation in higher plant chloroplasts [38]
Trang 9AtpI would be a candidate for the post-translational
SRP pathway in cyanelles In Escherichia coli, its
homologue, subunit a, needs the assistance of YidC for
integration into the inner membrane [39] Mitochondrial
Atp6 is also partially dependent upon Oxa1 in that
respect [40] In chloroplasts, by analogy, the
cotransla-tional SRP pathway and the Albino3 translocon
might be involved However, in the ac29 mutant of
C reinhardtii, where one out of the two albino3 genes is
inactivated, no effect on ATP synthase (at least on the a
subunit) could be observed [41] Thus far, there is no
evidence for ALB3 in the plastids of chlorophyll b-less
algae In C paradoxa, SecY is a component of two
different high molecular mass thylakoid-bound protein
complexes (F Yusa and W Lo¨ffelhardt, unpublished
data), a parallel to A thaliana where ALB3 is also
contained [42] Another crucial point will be to identify
SRP43 in nonchlorophytes, e.g diatoms that integrate
fucoxanthin chlorophyll a⁄ c-binding protein into their
thylakoid membranes [6] On the other hand, reports on
vesicle transport of cab protein from the inner envelope
membrane to the thylakoid membrane in C reinhardtii
[43] render the function of the transit complex and of
ALB3 questionable in this alga However this vesicle
transport must be different from that described for
higher plant chloroplasts that can be visualized upon
lowering the temperature and is sensitive to microcystin
[44]
The fourth pathway might also exist in cyanelles:
SecE from C paradoxa is not yet identified; in
chloro-plasts it was shown to use the spontaneous pathway
[11,12] Chloroplast and cyanelle thylakoids are both
rich in galactolipids which obviously support
unas-sisted integration of proteins in contrast to the
phos-pholipids of the E coli cytoplasmic membrane [12]
Experimental procedures
Materials
Cyanophora paradoxa LB555UTEX was grown as
previ-ously described [45] In general, cells were harvested in the
exponential growth phase Nucleic acids were isolated
according to published methods [45] Spinach (Spinacia
oleracea) was purchased from the local market and kept
overnight at 4C before isolating chloroplasts Pea
seed-lings (Pisum sativum) were grown for 8–10 days under a
16 h photoperiod
Protein import into isolated chloroplasts
Precursor proteins were synthesized by in vitro transcription
of the corresponding cDNA clones and subsequent in vitro
translation in cell-free wheat germ lysates in the presence of [35S]methionine Intact chloroplasts were isolated from pea
or spinach leaves by Percoll gradient centrifugation and were used in protein import experiments essentially as des-cribed [46] Competition experiments were performed with precursor proteins that were obtained by overexpression in Escherichia coli[47] and recovered from inclusion bodies by solubilization in a buffer containing 7 m urea, 30 mm Hepes,
pH 8.0 and 2 mm EDTA The solubilized proteins were included in the import assays at concentrations up to 4 lm, taking care that the concentration of urea in the assays never exceeded 300 mm Control assays contained the same amount of buffer lacking any such solubilized protein
Protein import into isolated cyanelles
Import-competent cyanelles were isolated as described in [19] They were used in protein transport experiments as described in [18]
Isolation of cyanelle thylakoid membranes
The import reaction was stopped by the addition of 1 mL ice-cold sorbitol resuspension medium (SRM) buffer (50 mm Hepes, 0.33 m sorbitol, pH 8.0) followed by centri-fugation at 800 g and 4C for 2 min The cyanelle pellet was washed in SRM and resuspended in 500 lL 2· SRM and incubated for 25 min at room temperature with 15 lL
of a 10 mgÆmL )1lysozyme stock solution in the presence of protease inhibitors (Complete, Roche), which led to diges-tion of the peptidoglycan wall, cyanelle lysis and release of the phycobilisomes from the thylakoid membrane After
centrifugation for 5 min at 9300 g in an Eppendorf
centri-fuge at 4C an aliquot of the deep-blue stromal superna-tant was precipitated with 100% (v⁄ v) acetone, the pellet containing the thylakoids and the peptidoglycan-linked outer envelope membrane was washed in 2· SRM and finally resuspended in 500 lL 2· SRM An aliquot (250 lL) was treated with thermolysin plus 10 mm CaCl2
for 30 min on ice After stopping the reaction with EDTA all aliquots were pelleted by centrifugation
Miscellaneous
Gel electrophoresis of proteins under denaturing conditions was carried out according to [48] Import data were ana-lyzed using a PhosphoImager and the molecular dynam-ics imagequant program (version 3.3), such that all the signals remained in the linear detection range
PCR, gene isolation
The nucleotide sequences determined via reverse translation
of highly conserved regions of the ATP synthase subunit
Trang 10CF0-IV (AtpI) and of the Rieske iron–sulfur protein (PetC)
were used to design degenerate primers
AtpI: forward primer 5¢-GCNTAYTTYTAYGCNGG-3¢,
reverse primer 5¢-GGYTTNGTRAARTCYTC-3¢ (product
size: 111 bp); PetC: forward primer 5¢-CARGGNYTNAA
RGGNGAYCCNACNTA-3¢, reverse primer 5¢-TAYTGN
WSNCCRTGRCANGGRCA-3¢ (product size: 156 bp)
The PCR reaction mixture (50 lL) included 100 ng of
DNA from C paradoxa, 0.1 lm concentration of each
primer species, 10 mm Tris⁄ HCl (pH 8.3), 50 mm KCl,
1 mm MgCl2, 0.2 mm dNTPs, and 1 U of Taq DNA
polymerase (Dynazyme, Finnzymes Oy, Espoo, Finland)
The following thermal cycle was used: Step 1, 96C for
5 min; Step 2, 94C for 1 min; Step 3, 50 C for 2 min;
Step 4, 72C for 3 min; Step 5, repeat steps 2–4 35 times;
Step 6, 72C for 7 min The predominant PCR product
was cloned into pGEM-T and sequenced After
identifi-cation of the correct products the fragments were
labe-led with the Digoxigenin Labeling⁄ Detection System
(Boehringer Mannheim, Mannheim, Germany) and used
for screening of a C paradoxa cDNA library in the
vec-tor k-ZAP II (Stratagene, La Jolla, CA, USA) Plaque
hybridization was performed under high stringency
conditions [49]
Full-length cDNAs were cloned into the vector pBAT
[50] to allow sufficient translation efficiency psbO: forward
primer: 5¢-AANGGNACNCKYTCNCCNCC-3¢ The
for-ward primer was designed using a peptide sequence
(EGLTYDQ) obtained via Edman-sequencing [25] psbU:
forward primer: 5¢-AAGAATTCACGAGGCAGAAATG
GCGTTC-3¢, reverse primer: 5¢-AAGGATCCTGGGGAC
AGCAGAAACTTGG-3¢
The psbU gene was isolated by PCR with a proof-reading
polymerase (Pfu) using data from a C paradoxa EST
lib-rary (S Burey and H J Bohnert, unpublished data) and
directly cloned into the pBAT-vector via its EcoRI and
BamHI sites
Acknowledgements
We appreciate support from the Austrian Research
Fund (P15438-MOB, to W.L.) We thank Hans
Bohnert (Urbana, IL, USA) and Suzanne Burey for
providing EST data from Cyanophora paradoxa
References
1 Hankamer B, Morris E, Nield J, Carne A & Barber J
(2001) Subunit positioning and transmembrane helix
organisation in the core dimer of photosystem II FEBS
Lett 504, 142–151
2 Herrmann RG (1998) Thylakoid membranes A
para-digm for biogenetic and phylogenetic compexity In
Photosynthesis, Mechanisms and Effects(Garab G, ed), Vol IV, pp 3049–3056
3 Eichhacker LA & Henry R (2001) Function of a chloro-plast SRP in thylakoid protein export Biochim Biophys Acta 1541, 120–134
4 Schleiff E & Klo¨sgen RB (2001) Without a little help from ‘my’ friends: direct insertion of proteins into chloroplast membranes? Biochim Biophys Acta 1541, 22–33
5 Mori H & Cline K (2001) Post-translational protein translocation into thylakoids by the Sec and DeltapH-dependent pathways Biochim Biophys Acta 1541, 80–90
6 Lang M & Kroth P (2001) Diatom fucoxanthin chloro-phyll a⁄ c-binding protein (FCP) and land plant light-harvesting proteins use a similar pathway for thylakoid membrane insertion J Biol Chem 276, 7985–7991
7 Lo¨ffelhardt W & Bohnert HJ (2001) The cyanelle (muro-plast) of Cyanophora paradoxa: a paradigm for endosym-biotic organelle evolution In Symbiosis (Seckbach, J, ed.), pp 111–130 Kluwer Academic Publishers, Dordrecht
8 Martin WF, Stoebe B, Goremykin V, Hansmann S, Hasegawa M & Kowallik KV (1998) Gene transfer to the nucleus and the evolution of chloroplasts Nature
393, 162–165
9 Steiner JM & Lo¨ffelhardt W (2002) Protein import into cyanelles Trends Plant Sci 6, 72–77
10 Smeekens S, Weisbeek P & Robinson C (1990) Protein transport into and within chloroplasts Trends Biochem Sci 15, 73–76
11 Steiner JM, Ko¨cher T, Nagy C & Lo¨ffelhardt W (2002) Chloroplast SecE: evidence for spontaneous insertion into the thylakoid membrane Biochem Biophys Res Commun 293, 747–752
12 Jiang F, Yi L, Moore M, Chen M, Rohl T, van Wijk K-J,
de Gier J-WL, Henry R & Dalbey R (2002) Chloroplast YidC homolog Albino3 can functionally complement the bacterial YidC depletion strain and promote membrane insertion of both bacterial and chloroplast thylakoid proteins J Biol Chem 277, 19281–19288
13 Nakai M, Sugita D, Omata T & Endo T (1993) SecY protein is localized in both the cytoplasmic and thyla-koid membranes in the cyanobacterium Synechococcus PCC 7942 Biochem Biophys Res Commun 193, 228–234
14 Fulda S, Huang F, Nilsson F, Hagemann M & Norling B (2000) Proteomics of Synechocystis sp strain PCC 6803 – identification of periplasmic proteins in cells grown at low and high salt concentrations Eur J Biochem 277, 5900–5907
15 Spence E, Sarcina M, Ray N, Moller SG, Mullineaux CW
& Robinson C (2003) Membrane-specific targeting of green fluorescent protein by the Tat pathway in the cyanobacterium Synechocystis PCC6803 Mol Microbiol
48, 1481–1489