A chloroplast RNA binding protein from stromal thylakoid membranesFriedrich Ossenbu¨hl*, Kristina Hartmann and Jo¨rg Nickelsen Lehrstuhl fu¨r Allgemeine und Molekulare Botanik, Ruhr-Univ
Trang 1A chloroplast RNA binding protein from stromal thylakoid membranes
Friedrich Ossenbu¨hl*, Kristina Hartmann and Jo¨rg Nickelsen
Lehrstuhl fu¨r Allgemeine und Molekulare Botanik, Ruhr-Universita¨t Bochum, Bochum, Germany
The intrachloroplastic localization of post-transcriptional
gene expression steps represents one key determinant for the
regulation of chloroplast development We have
character-ized an RNA binding protein of 63kDa (RBP63) from
Chlamydomonas reinhardtiichloroplasts, which
cofraction-ates with stromal thylakoid membranes Solubility
proper-ties suggest that RBP63is a peripheral membrane protein
Among RNA probes from different 5¢ untranslated regions
of chloroplast transcripts, RBP63preferentially binds to the
psbAleader This binding is dependent on a region com-prising seven consecutive A residues, which is required for D1 protein synthesis A possible role for this newly discov-ered RNA binding protein in membrane targeting of psbA gene expression is discussed
Keywords: chloroplast gene expression; D1 synthesis; mem-brane targeting; RNA binding; thylakoid
Chloroplast gene expression within plant or algal cells has
been shown to be dependent upon nuclear gene products,
which are translated in the cytoplasm and, subsequently, are
imported by the organelle Herein, they fulfil their function
by interacting with distinct elements and/or factors
associ-ated with the chloroplast gene expression machinery [1,2]
While the molecular mechanisms of regulatory interaction
during these processes are being pieced together, relatively
little is known about the intrachloroplast localization of
different steps of gene expression
For instance, the chloroplast DNA is organized in
nucleoids In developing higher plants, these are associated
with the inner plastid envelope through the PEND protein
Upon full chloroplast maturation, the cpDNA is localized
to thylakoid membranes by an undetermined mechanism
[3] This suggests that the plastid transcription machinery is
distributed in a similar way Further evidence for
subcom-partmentalization of chloroplast gene expression has been
obtained by the recent cloning of genetically defined loci,
which are required for distinct post-transcriptional steps of
chloroplast gene expression These factors could be detected
in the stromal compartment like Crp1 and Crs2 in maize
[4,5], or Maa3, Mbb1 and Nac2 in Chlamydomonas
reinhardtii[6,7,8], which are involved in processing/splicing
or stabilization of specific chloroplast transcripts,
respec-tively Conversely, association with the inner plastid
enve-lope and/or the so-called low density membranes (LDM),
which resemble the inner envelope membrane with regard to
their lipid composition [9], has been observed for the
translation termination factor RF4 from spinach and the RNA splicing factor Maa2 from C reinhardtii [10,11]
By application of in vitro run-on translation systems, a cotranslational insertion of thylakoid membrane proteins has been reported [12] This is consistent with the finding that chloroplast psbA and psbD transcripts are associated with thylakoids [13,14] Further evidence for an essential role of the thylakoid membrane for chloroplast gene expression was deduced from the analysis of a maize mutant lacking the chloroplast SecY homologue of the thylakoid protein translocation machinery In this mutant, chloroplast translation is defective [15]
Moreover, in vitro assays revealed a still growing number
of various RNA binding proteins (RBPs), which have been implicated in the control of post-transcriptional gene expression steps Some of these RBPs appear to mediate their function via distinct cis-acting elements within the 5¢ untranslated regions (UTRs) of plastid mRNAs, which are essential for mRNA maturation/stabilization and/or trans-lation initiation [16–20] Whereas some of these RNA binding activities are localized to the chloroplast stroma [18,21], recent work suggests that many other RBPs are associated with the abovementioned LDM system [9]
In C reinhardtii, the 5¢ UTR of the psbA mRNA encoding the D1 protein of the photosystem (PS) II reaction centre was shown to interact with RB47, a member of the polyA-binding protein family, which forms a complex with major proteins of 38, 55 and 60 kDa [22] RB60 represents a protein disulfide isomerase that exhibits no RNA binding activity, but is involved in the light and/or redox regulation
of D1 synthesis RB47 was localized to the LDM system [9] and, in addition, RB60 was shown to be partitioned between the stroma and the membrane phase following chloroplast fractionation experiments [23]
We have previously reported on a set of chloroplast RBPs, which interact with the 5¢ UTR of the psbD mRNA encoding the D2 protein of PS II of C reinhardtii Amongst those, a protein of 63kDa (RBP63) cofractionated with thylakoid membranes during separation of chloroplast lysates by sucrose step gradient centrifugation [18] In this
Correspondence to J Nickelsen, Lehrstuhl fu¨r Allgemeine und
Mole-kulare Botanik, Ruhr-Universita¨t Bochum, 44780 Bochum, Germany.
Fax: + 49 2343214184, Tel.: + 49 2343225539,
E-mail: joerg.nickelsen@ruhr-uni-bochum.de
Abbreviations: LDM, low density membranes; RBP, RNA binding
protein; UTR, untranslated region; PS, photosystem.
*Present address: Department fu¨r Biologie I, Ludwig-Maximilians
Universita¨t, Menzinger Str 67, 80638 Mu¨nchen, Germany.
(Received 13March 2002, revised 7 June 2002, accepted 19 June 2002)
Trang 2report, this particular protein is characterized further We
were able to show that RBP63is a stromal thylakoid
membrane protein It preferentially binds to the 5¢ UTR of
the psbA message determined by an A-rich region eight
nucleotides upstream of the AUG start codon To the best
of our knowledge, this is the first RNA binding activity
found exclusively within thylakoid membranes
M A T E R I A L S A N D M E T H O D S
Algal strains, preparation of protein fractions
and western analysis
The C reinhardtii strain used harboured the cw15 mutation,
which facilitates chloroplast isolation It was maintained on
Tris/acetate/phosphate medium [24] at 25C and cultures
were grown to a density of 2· 106cells Æ mL)1 in this
medium containing 1% sorbitol Cells were harvested by
centrifugation and chloroplasts and chloroplast
subfrac-tions were prepared exactly as described previously [18]
For the separation of stroma and grana thylakoids,
isolated unstacked thylakoid membranes were resuspended
at 0.4 mgÆmL)1 chlorophyll in buffer B (15 mM tricine/
KOH pH 7.9, 0.1Msorbitol, 10 mMNaCl, 5 mMMgCl2,
10 mMNaF) and incubated for 30 min at 4C to allow for
restacking [25] Subsequent fractionation was carried out as
described previously [26] In brief, restacked thylakoid
membranes were incubated with 0.4% digitonin in buffer B
for 2 min at room temperature The incubation was stopped
by adding 10 vol buffer B The suspension was centrifuged
four times at 4C Each supernatant was used for the next
centrifugation step The relative acceleration rates were
1000 g for 10 min, 10 000 g for 3 0 min, 40 000 g for 30 min
and 150 000 g for 1 h The different pellets corresponding
to thylakoid membranes (P1000g), grana thylakoids (P10000g),
intermediate membranes (P40000g) and stroma thylakoids
(P150000g) were resuspended in 2· lysis buffer, diluted with
75% glycerol and stored at)20 C [18]
Western analyses were carried out as described previously
[18] Protein and chlorophyll concentrations were
deter-mined as described previously [27,28]
Membrane solubilization analysis
For membrane solubilization analysis, isolated thylakoid
membranes corresponding to 1 mg chlorophyll were
incu-bated as indicated in Fig 2 at 4C for 30 min and
centrifuged at 100 000 g for 1 h The pellets were
resus-pended in 2· lysis buffer NaCl in the supernatants of the
salt washes was removed by centrifugation through ultrafree
centrifugal filter (Millipore Corporation) All fractions were
diluted with 75% glycerol and stored at )20 C Equal
amounts of the soluble and the membrane fractions
corresponding to 1 lg chlorophyll of untreated thylakoid
membranes were analysed by UV cross-linking assay
Sedimentation analysis
For sedimentation analysis of RBP63activity, isolated
chloroplasts were hypotonically lysed in buffer containing
5 mM 6-amino hexanoic acid, 25 lgÆmL)1 pepstatin A,
10 lgÆmL)1leupeptin, 1 mMbenzamidine HCl and 1 mM
phenylmethanesulfonyl fluoride After centrifugation for
1 h at 100 000 g the sedimented membranes were solubi-lized in the same buffer containing 0.5% Triton X-100, loaded on a 15–80% linear glycerol gradient and centrifuged for 18 h at 180 000 g The gradient was fractionated into 22 fractions of 500 lL; 10 lL of these fractions were used for
UV cross-linking experiments
In vitro synthesis of RNA and UV cross-linking of RNA with proteins
Templates for the in vitro synthesis of psbD leader RNA probes, KS-RNA, and psbC-RNA were generated as described [9,18] A PCR fragment comprising positions +1041 to +1157 relative to the AUG of the psbA mRNA (corresponding to the coding region of the C-terminal amino acids of D1 and the 3¢ UTR of the psbA mRNA) was amplified with the oligonucleotides psbA3/1 (5¢-CTCTAGC TCAAACAACT-3¢) and psbA3/2 (5¢-GCCTATGGTAGC TATTA)3¢) and cloned into the pBluescriptII KS+vector The resulting clone, p41.a9, was sequenced (MWG-Biotech
AG, Ebersberg, Germany) For in vitro synthesis of the psbA 3¢ UTR RNA (psbA3¢ RNA), p41.a9 was digested with EcoRI Other templates for in vitro synthesis of the different 5¢ UTR RNAs were generated by PCR with the following oligonucleotides: psbA RNA (wild-type sequence
of the psbA mRNA corresponding to positions)91 to +13 relative to the AUG); T7-psbA5¢ (5¢-GTAATACGACTCA CTATAGGGTACCATGCTTTTAATAGAAG-3¢) and
2054 (5¢-GATCCATGGTCATATGTTAATTTTTTTAA AG-3¢);)36-RNA (wild-type sequence of the psbA mRNA corresponding to positions )36 to +13 relative to the AUG); T7–36ntA5¢ (5¢-GTAATACGACTCACTATAGG GTTTACGGAGAAATTAAAAC-3¢) and 2054; M1-RNA (sequence of the psbA mM1-RNA corresponding to positions)36 to +13 relative to the AUG with an exchange
at positions)27 to )19 to C residues); psbA-T7mut1 (5¢-GT AATACGACTCACTATAGGGTTTACGGAGCCCCC CCCCC-3¢) and psbA3¢mut1 (5¢-GATCCATGGTCATAT
M2-RNA (sequence of the psbA mM2-RNA corresponding to positions)36 to +13 relative to the AUG with an exchange
at positions )14 to )4 to C residues): T7-36ntA5¢ and psbA3¢mut2 (5¢-GATCCATGGTCATATGGGGGGGG GGGGAAAGTTTTAATTTC-3¢); M2a-RNA (sequence
of the psbA mRNA corresponding to positions)36 to +13 relative to the AUG with an exchange at positions)17 to )12 to C residues); T7-36ntA5¢ and psbA3¢mut2a (5¢-GATCCATGGTCATATGTTAATTTTGGGGGGG TTTTAATTTC-3¢); M2b-RNA (sequence of the psbA mRNA corresponding to positions)36 to +13 relative to the AUG with an exchange at positions)11 to )8 to C residues); T7-36ntA5¢ and psbA3¢mut2b (5¢-GAT CCATGGTCATATGTTAAGGGGTTTAAAGTTTTA ATTTC-3¢); M2c-RNA (sequence of the psbA mRNA corresponding to positions)36 to +13 relative to the AUG with an exchange at positions )7 to )4 to C residues); T7-36ntA5¢ and psbA3¢mut2c (5¢-GATCCATGGTCATA TGGGGGTTTT TTTAAAGTTTTAATTTC-3¢); M2d-RNA (sequence of the psbA mM2d-RNA corresponding to positions)36 to +13 relative to the AUG with an exchange
at positions )17 to )15 to C residues); T7-36ntA5¢ and psbA3¢mut3(5¢-GA TCCATGGTCATATGTTAATTTT TTTGGGGTTTTAATTTC-3¢); psbB-RNA (wild-type
Trang 3sequence of the psbB mRNA corresponding to positions
)147 to +24 relative to the AUG): T7-psbB5¢
(5¢-GTAATACGACTCACTATAGGGTAAATTAATT
TAATTTAAAATC-3¢) and psbB3¢ (5¢-TACACGATA
CCAAGGTAAACC-3¢) Each template contained the
pro-motor of the T7 RNA polymerase fused to the 5¢ end of the
described fragments In vitro transcription of RNA, UV
cross-linking of RNAs with proteins and quantification of
binding signals was carried out as described [18] For
competition experiments radiolabelled RNA and
non-labelled competitors were mixed prior to the addition of
proteins Quantification of competitor RNA amounts was
performed by measuring the incorporation of low levels of
radioactivity into transcripts [18] Signal intensities in
com-petition binding experiments were quantified
densitometri-cally by using an ICU-1 unit and theIMAGE DOC/EASY WIN2
software from Herolab
R E S U L T S
RBP63 cofractionates with stromal thylakoids
Previously, we have analysed interactions of chloroplast
proteins with the 5¢ UTR of the psbD mRNA in C
rein-hardtii [18] Chloroplast lysates were fractionated by
centrifugation through a 1.0M sucrose cushion in the
absence of MgCl2 Under these experimental conditions,
neither stroma nor LDMs entered the cushion [9,18], and
could be collected together in one fraction, which contained
the majority of RBPs (Fig 1A, lane 3)
By using the UV cross-linking technique, RNA binding
activities of 40, 63and 90 kDa were detected in cT fractions
representing crude thylakoid membranes, which sedimented
through the sucrose cushion (Fig 1A, lane 4) After further
purification of these membranes by flotation in a second
sucrose step gradient (1.3M/1.8Msucrose) and subsequent
washing by sedimentation [18], only the 63kDa (RBP63) and trace amounts of the 90 kDa species were detectable (Fig 1A, lane 5) This suggests that RBP63is associated with thylakoid membranes, while RBP40 and RBP90 represent stromal contamination of the cT fraction, which still contained substantial amounts of the stromal Rubisco enzyme (Fig 1C)
However, a signal in the range of 63kDa was also detected in the stromal fraction (Fig 1A, lane 3) To test, whether this activity represents stroma-localized RBP63, high resolution SDS/PAGE was performed As shown in Fig 1B, the stromal component is approximately 61 kDa (RBP61) in size and, thus, distinctly different from RBP63 Further evidence supporting this finding was obtained from sedimentation analyses, which demonstrated that RBP61 and RBP63are part of two different high molecular weight complexes of 450 kDa and 700 kDa, respectively (Fig 1E, fractions 5 and 9–15, respectively) From these data, it can be concluded that active RBP63is associated exclusively with the thylakoid membrane and is not partitioned between the stroma and the thylakoids Thylakoid membranes can be divided into stroma lamellae and stacked grana regions To test whether RBP63shows any selective accumulation within these thylakoid membrane subfractions, appressed grana and unappressed intermediate and stroma thylakoids were separated by differential centrifugation following digitonin treatment as described earlier [26] As shown in Fig 1D, RBP63activity was found to be significantly enriched in the stromal thylakoid membrane fraction together with the CF1 subunit of the chloroplast ATPase and the PsaD subunit of
PS I, which serve as marker molecules for stromal thylak-oids [29] Low amounts of RBP63were detectable in the intermediate fraction, whereas PS I and ATPase already showed a significant enrichment In view of actual models of the domain structure of the photosynthetic membrane it
Fig 1 Fractionation pattern of RBP63 Chloroplast lysate (A) and -subfractions (C) were analysed by UV cross-linking to the psbD 5¢ UTR-RNA (B) The stromal (S) and the crude thylakoid membrane fraction (cT) were analysed as in (A), but proteins were separated on an 8% instead of a 10% acrylamide/SDS gel The sizes of marker proteins are indicated in kDa P, Protein-free control; T, thylakoid membrane fraction (C) Chloroplast fractionation was controlled by Western analysis with antibodies against Rubisco (RbcL), PS I (PsaD) and ATP-synthase (CF1) (D) Floated thylakoid membranes (T) were isolated and separated into grana (GT), intermediate (IT), and stroma (ST) thylakoids Identical amounts of chlorophyll were either analysed by UV cross-linking using the psbD-RNA (2 lg chlorophyll) or by Western analysis (20 lg chlorophyll) with antibodies against PS I (PsaD) and ATP-synthase (CF1) (E) Chloroplast lysates were centrifuged on a linear 15–80% glycerol gradient Given fractions were analysed by UV cross-linking with the psbD RNA Sedimentation of marker proteins (in kDa) is indicated at the top The arrows point to RBP63, and RBP61 is marked by asterisks.
Trang 4appears likely that the intermediate fraction might be
enriched in thylakoid margin regions which constitute a
distinct subdomain and contain amounts of PS I and
ATPase similar to stromal thylakoids [29] However, the
data clearly indicate that RBP63does not cofractionate with
the grana thylakoid membranes
To test the nature of the association between RBP63and
thylakoids, membranes were washed with high salt (0.1–
2.0M NaCl) or with buffer containing 0.1–2.0% of either
detergent Brij 35 or Triton X-100 Salt treatment did not
induce any release of RBP63into the soluble phase,
although some activity was lost in the membrane phase,
probably due to degradative processes during extensive
washing of membranes which was required to completely
remove NaCl (Fig 2A) In contrast, complete release was
observed with high concentrations (2.0%) of Brij 35 This
nonionic detergent preferentially dissolves
peripheral/ex-trinsic membrane proteins because of its high
hydrophilic-lipophile balance number [30,31] Under these conditions,
chlorophyll could not be measured in the supernatants
(Fig 2B) demonstrating that intrinsic membrane proteins
are not dissolved [32] By using Triton X-100, both
chlorophyll and RBP63were readily detected in the
supernatant fraction Complete release of chlorophyll was
achieved with high concentrations (1.0%) of Triton X-100,
whereas only low concentrations (0.1%) were required to
release all RBP63activity (Fig 2C) Taken together, these
results indicate that RBP63is a membrane protein of
thylakoids It might be peripherally associated with the
membrane via a hydrophobic polypeptide anchor, as has
been suggested for thylakoid phosphatases from
C reinhardtii[33]
RBP63 preferentially binds to thepsbA 5¢ UTR Initially, RBP63was detected by using a radiolabelled RNA probe containing the psbD 5¢ UTR However, other tested radiolabelled 5¢ UTR-probes from the psbA mRNA (Fig 3A) or psbB, psbC, rbcL and rps4 mRNAs (data not shown) were also able to generate a similar RBP63signal under noncompetitive and, hence, unphysiological condi-tions To distinguish between different affinities of RBP63
to the various RNAs, comparative competition experi-ments were performed These used radiolabelled psbD-RNA and an excess of unlabeled 5¢ UTR psbD-RNA probes from other chloroplast mRNAs encoding subunits of the
PS II core, including psbA, psbB, and psbC The psbB, psbC and even the psbD RNA exhibited only moderate and nearly the same competition effects However, surprisingly, a very strong reduction of the RBP63signal was obtained when the psbA RNA was used as a competitor (Fig 3B), thus suggesting a high affinity of RBP63for the psbA 5¢ UTR RNA Consequently, competition experiments similar to those described in Fig 3B were performed, except that psbA 5¢ UTR was used in place of psbD as the radiolabelled probe Again, the homologous psbA RNA led to the most significant competition effect, whereas the addition of an excess of psbB, psbC, and psbD RNAs resulted in only minor competition (Fig 3C) Additional RNAs were tested and included the 5¢ UTRs of rbcL and rps4 mRNAs as well as
an unrelated in vitro transcript comprising the polylinker region of the pBluescript KS+vector, which competed at low levels in the same range (data not shown) Similar to several other chloroplast RNA binding proteins, RBP63 exhibited a high affinity for the ribohomopolymers polyA and polyU, whereas polyG and polyC were not recog-nized (data not shown) Also the addition of a 500-fold excess of dsDNA from the psbA 5¢ region had no effect
on RBP63-binding (data not shown) In conclusion, these data confirm the high affinity of RBP63for the psbA leader
Analysis of thecis-acting determinants for RBP63-binding
Within chloroplasts of C reinhardtii, two forms of the psbA mRNA exist: a larger form with a 5¢ UTR of 91 nucleotides (which had been used in the binding assays shown in Fig 3C) and the predominant, shorter form with a leader of
36 nucleotides (Fig 5A) [34,35] It has been hypothesized that the larger form represents the precursor to the shorter mRNA which is generated by a 5¢ processing event [36] Similar to the situation found for psbD and psbB gene expression [37,38], a tight molecular connection between processes of 5¢ RNA maturation and translation initiation had been postulated for the psbA gene [36] In order to distinguish whether RBP63also binds to the shorter psbA message, further comparative competition experiments were carried out by using the two different psbA 5¢ UTR forms as unlabeled competitors The two RNAs reduced the RBP63 signal with almost the same efficiency indicating that RBP63 recognizes the psbA mRNA via an element located between position)36 and +1 of its leader (Fig 4) In contrast, an RNA probe covering the psbA 3¢ UTR competed only to a low level (Fig 4)
Fig 2 Association of RBP63 with thylakoid membranes Thylakoid
membranes were incubated with NaCl (A), Brij35 (B) and Triton
X-100 (C) as indicated The soluble (S) and the membrane phases (M)
were separated and analysed by UV cross-linking assays with the psbD
RNA The relative amounts of chlorophyll released into the soluble
phases during the treatments are indicated (Chl).
Trang 5For further examination of the cis-acting determinants,
which are essential for RBP63binding, we generated several
mutant versions of the shorter psbA 5¢ UTR Based on the
observation that RBP63exhibits high affinity to stretches of
A and U residues, the two A-rich regions at position)27 to )19 (A-tract 1) and position )14 to )4 (A-tract 2) relative
to the AUG start codon of the psbA leader were changed into C-tracts, resulting in the mutant RNAs M1 and M2, respectively (Fig 5A) When these mutant RNAs were used
as competitor RNAs in competition experiments, M1-RNA still reduced the RBP63signal as efficiently as did)36-RNA (Fig 5B) suggesting that A-tract 1 is not essential for RBP63binding In contrast, a significantly weaker compe-tition effect was observed with M2 RNA, indicating that crucial cis-acting determinants for RBP63-binding activity are located within A-tract 2
To analyse these in more detail, four additional leader mutants of A-tract 2 were generated, which contained C-tracts localized at positions)17 to )12 (M2a RNA), )11
to)8 (M2b RNA), )7 to )4 (M2c RNA) and )17 to )15 (M2d RNA) (Fig 5A) These new mutant RNAs were used
as competitor RNAs as described above As shown in Fig 5C, both M2c RNA and M2d RNA were able to reduce the RBP63 signal to the same degree as wild-type )36-RNA Addition of either M2a RNA or M2b RNA, however, caused only weak competition effects in the range
of those obtained with M2 RNA This indicated that RBP63binding to the psbA leader is determined mainly by the tract of seven A residues located between position)14 and)8 relative to the AUG start codon
D I S C U S S I O N
In this study, we report on the identification and character-ization of the chloroplast RNA binding protein RBP63, which is part of a high molecular weight complex of
700 kDa RBP63exhibits a high affinity for the 5¢ UTR of the psbA message when compared to various different 5¢ UTRs of chloroplast genes Moreover, its binding activity in vitrois dependent on a tract of seven consecutive A-residues located 14–8 nucleotides upstream of the psbA AUG start codon Based on the fact that chloroplast mRNAs often contain long stretches of A residues, it appears unlikely that
Fig 3 RBP63 binds with high affinity to the psbA 5¢ UTR (A) Floated thylakoid membranes were analysed by UV cross-linking assays with radiolabelled 5¢ UTR probes of either the psbD or the psbA mRNA (psbD- and psbA-RNA, respectively) (B) cT-fractions were incubated with radiolabelled psbD-RNA and a 5-, 50-, or 500-fold molar excess of the indicated competitor RNAs representing the 5¢ UTR RNAs of the psbA, psbB, psbC and psbD mRNA (C) As in (B) except that psbA RNA instead of psbD RNA was radiolabelled Each diagram displays the intensities of RBP63signals in relation to the RBP63signal without competitor from one representative experiment, in which the exposure time was the same for all lanes.
Fig 4 RBP63 binds to the short psbA leader form Competition
experiments similar to those described in Fig 3C were performed by
using the larger (psbA) and the shorter ( )36) psbA 5¢ UTR (Fig 5A) as
well as the psbA 3¢ UTR.
Trang 6this A-stretch on its own is already sufficient to mediate high
affinity binding of RBP63 Rather, additional cis-acting
determinants, such as the secondary structure of the leader,
for example, might facilitate site-specific RNA recognition
by RBP63, similar to RNA–protein complex formation in
other systems [20,39] Nevertheless, the A-rich region had
previously been shown to be required for D1 synthesis in
C reinhardtii In the chloroplast transformant RBS11,
deletion of the sequence between position)26 and )11 led
to the elimination of psbA mRNA translation in vivo,
whereas the stability and 5¢ maturation of the message were
unaffected (Fig 5A) [36] As the deletion in the translational
RBS11 mutant covers four of the seven A residues of the
psbAleader that are essential for RBP63-binding, a
corre-lation between RNA binding activity in vitro and
transla-tional activity in vivo becomes obvious Thus, we speculate
that RBP63might be involved in the translational control of
psbAgene expression in C reinhardtii This resembles the
situation found for the regulation of translation initiation of
the psbD gene in chloroplasts of C reinhardtii In the case of
the psbD 5¢ UTR, a U-rich element located 25 to 14
nucleotides upstream of its AUG start codon was shown to
be required for translation but not for RNA stabilization or
5¢ maturation [38] This region is recognized by a stromally
localized 40 kDa protein (RBP40), which has been
postu-lated to be involved in D2 synthesis [18]
In spinach, the ribosomal protein S1 has been shown to
interact with A- or U-rich sequence elements within the
psbA5¢ UTR [40] In contrast, detailed competition binding
experiments revealed that several other chloroplast RNA
probes are also bound by S1 with the same affinity [41] In
C reinhardtii, similar to Escherichia coli, the chloroplast S1
protein has a size in the range of 60 kDa [18], thus
resembling RBP63 However, when thylakoid membrane
proteins were analysed with a polyclonal antiserum against
the E coli S1 protein, no immunoreactive material was
detected (data not shown) These data, as well as the fact that RBP63binds with high affinity solely to the psbA leader strongly support the idea that these two factors are distinct, though, formally, we cannot exclude that RBP63represents
a membrane-bound version of the S1 protein
However, one of the most intriguing features of RBP63is its association with stromal thylakoid membranes To our knowledge, this represents the first example of a chloroplast RNA binding protein within thylakoids While analyses of such proteins were initiated mainly from the soluble stromal phase, more recently a partitioning of various RBPs between the soluble and the membrane fraction was reported following the use of a radiolabelled RNA probe
of the psbC 5¢ UTR in UV cross-linking experiments [9] Further fractionation of the membrane phase revealed that the bulk of RBPs is specifically enriched (over 100-fold) within the above mentioned LDM, which can easily be separated from thylakoids by sucrose density centrifugation
in the absence of MgCl2[9] During the course of this work, stroma and LDM phase were not separated and, except for RBP63, all RBPs were detected in the stroma/LDM fraction
by using either a psbD or a psbC 5¢ UTR probe (Fig 1A; data not shown), indicating that the floated thylakoid membranes were not contaminated by LDM membranes (Fig 1A, lane 5)
It has been hypothesized that LDMs, which resemble the inner envelope with regard to their lipid composition, represent the sites of thylakoid membrane protein synthesis and that de novo formed photosynthetic complexes are transported via vesicles from the inner envelope to the thylakoids [42,43] This appears to be consistent with the finding that many RBPs, which are likely to be involved in post-trancriptional gene expression steps in the chloroplast, cofractionate with LDMs On the other hand, the repair mechanism of PS II in mature chloroplasts mainly involves the exchange of photo-damaged D1 protein by a newly
Fig 5 cis-acting determinants of RBP63
binding to the short psbA leader (A) Sequence
alignment of the larger (psbA) and the shorter
( )36) psbA 5¢ UTRs from the wild-type and
different mutated 5¢ UTR versions Asterisks
represent conserved residues Positions
rela-tive to the initiation codon and the 5¢
pro-cessing site (vertical arrow) are marked above
the sequence The region that has previously
been deleted in the chloroplast mutant RBS11
[34] is boxed (B and C) Competition
experi-ments were carried out similar to those
described in Fig 3C by using the indicated
RNAs Each diagram displays the intensities
of RBP63 signals in relation to the RBP63
signal without competitor from one
represen-tative experiment, in which the exposure time
was the same for all lanes Competition with
M2d-RNA was performed independently and,
thus, RBP63signals were quantified in
rela-tion to the 0x value given in the respective
M2d-RNA lane.
Trang 7synthesized one and has been localized to the stroma
lamellae of thylakoid membranes Subsequently, intact
PS II is moving to its functional localization in the grana
regions [44] Assuming a cotranslational insertion of D1
[45], it appears likely that gene expression for this repair
mechanism is restricted to the subfraction of the stromal
thylakoid membranes, especially, as the tightly stacked
grana regions would not allow access of the RNA
polymerase, ribosomes or other soluble complexes involved
in chloroplast gene expression to the membrane In
conjunction with data obtained from yeast mitochondria
[46], this leads to the model of a molecular tether, which
localizes chloroplast transcripts encoding integral
mem-brane proteins to stromal thylakoids [47,48] RBP63
appears to be a good candidate for a molecular tether of
chloroplast mRNAs It combines properties, which have to
be postulated for such a factor, namely, it is associated with
stromal thylakoids and binds to RNA In particular, the
fact that RBP63binds with high affinity to the psbA 5¢ UTR
and might thereby target the mRNA at stromal thylakoid
membranes is striking, as most translational activity in
mature chloroplasts is restricted to D1 synthesis due to
constraints of PS II repair [44]
In conclusion, two different processes of PS II generation
have to be considered, which might overlap in time: the
de novoassembly in premature developing chloroplasts and
the mechanisms, which are involved in PS II maintenance in
mature chloroplasts [49] Based on available data and actual
hypotheses, we hence propose the following scenario for the
control of psbA gene expression in C reinhardtii In
devel-oping chloroplasts, psbA mRNA translation is regulated via
its 5¢ UTR in a light and/or redox-controlled manner by the
previously described complex of RB47, RB60, RB38 and
RB55 This process may take place at the LDM system, since
it has been shown immunologically that RB47 is localized to
this chloroplast subfraction [9] Furthermore, RB60 has also
been demonstrated to be partitioned between the soluble and
membrane phase during chloroplast fractionation
experi-ments, although no distinction between LDMs and
thylak-oid membranes has been made during this analysis [23] As
RB60 exhibits no RNA binding activity in UV cross-linking
experiments, it is clearly distinct from RBP63which is
described here [16] If new D1 protein is required for the
repair of PS II in mature chloroplasts, then psbA translation
is targeted at the stromal thylakoid region by RBP63, which
might interact with or replace the RB47/RB60 complex and
promote the first assembly of ribosomes on the 5¢ UTR In
contrast with RB47 [22], the RNA binding activity of RBP63
is not significantly altered by the energy and/or redox status
of the chloroplast (data not shown) However, the
originat-ing nascent polypeptide chain compounded with ribosomes
(the so-called ribosome nascent chain complexes) has then to
be targeted at the D1 insertion point within the stromal
thylakoids, a process which appears to be mediated by the
chloroplast homologue of the 54 kDa signal recognition
particle protein [50]
Although this model has still to be considered speculative,
it is based on data that show for the first time that putative
trans-acting regulatory factors of psbA mRNA translation
initiation are localized in different membrane
subcompart-ments of the chloroplast Furthermore, it takes into account
that two different pathways for D1 synthesis might exist
strongly depending on the developmental stage of the chloroplast
A C K N O W L E D G E M E N T S
We thank T Stratmann and T Arndt for excellent technical assistance and U Ku¨ck for providing laboratory space Antisera against the Rubisco holoenzyme, the CF1 subunit of the chloroplast ATPase and PsaD were kindly provided by G Wildner, R Berzborn and J.-D Rochaix, respectively Plasmid pDH245 was a generous gift of
W Zerges This work was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 480-TP B8).
R E F E R E N C E S
1 Leon, P., Arroyo, A & Mackenzie, S (1998) Nuclear control of plastid and mitochondrial development in higher plants Ann Rev Plant Physiol Plant Mol Biol 49, 453–480.
2 Goldschmidt-Clermont, M (1998) Coordination of nuclear and chloroplast gene expression in plant cells Internat Rev Cytol 177, 115–180.
3 Sato, N., Rolland, N., Block, M.A & Joyard, J (1999) Do plastid envelope membranes play a role in the expression of the plastid genome? Biochimie 81, 619–629.
4 Fisk, D.G., Walker, M.B & Barkan, A (1999) Molecular cloning
of the maize gene crp1 reveals similarity between regulators of mitochondrial and chloroplast gene expression EMBO J 18, 2621–2630.
5 Jenkins, B.D & Barkan, A (2001) Recruitment of a peptidyl-tRNA hydrolase as a facilitator of group II intron splicing in chloroplasts EMBO J 20, 872–879.
6 Rivier, C., Goldschmidt-Clermont, M & Rochaix, J.-D (2001) Identification of an RNA-protein complex involved in chloroplast group II intron trans-splicing in Chlamydomonas reinhardtii EMBO J 20, 1765–1773.
7 Vaistij, F.E., Boudreau, E., Lemaire, S.D., Goldschmidt-Cler-mont, M & Rochaix, J.D (2000) Characterization of Mbb1, a nucleus-encoded tetratricopeptide repeat-like protein required for expression of the chloroplast psbB/psbT/psbH gene cluster in Chlamydomonas reinhardtii Proc Natl Acad Sci USA 97, 14813– 14818.
8 Boudreau, E., Nickelsen, J., Lemaire, S.D., Ossenbu¨hl, F & Rochaix, J.D (2000) The Nac2 gene of Chlamydomonas reinhardtii encodes a chloroplast TPR-like protein involved in psbD mRNA stability EMBO J 19, 3366–3376.
9 Zerges, W & Rochaix, J.D (1998) Low density membranes are associated with RNA-binding proteins and thylakoids in the chloroplast of Chlamydomonas reinhardtii J Cell Biol 140, 101–110.
10 Rolland, N., Janosi, L., Block, M.A., Shuda, M., Teyssier, E., Miege, C., Cheniclet, C., Carde, J.P., Kaji, A & Joyard, J (1999) Plant ribosome recycling factor homologue is a chloroplastic protein and is bactericidal in Escherichia coli carrying temperature-sensitive ribosome recycling factor Proc Natl Acad Sci USA 96, 5464–5469.
11 Perron, K., Goldschmidt-Clermont, M & Rochaix, J.D (1999) A factor related to pseudouridine synthases is required for chlor-oplast group II intron trans-splicing in Chlamydomonas reinhardtii EMBO J 18, 6481–6490.
12 Wollman, F.A., Minai, L & Nechushtai, R (1999) The biogenesis and assembly of photosynthetic proteins in thylakoid membranes Biochim Biophys Acta 1411, 21–85.
13 Breidenbach, E., Jenni, E & Boschetti, A (1988) Synthesis of two proteins in chloroplasts and mRNA distribution between thyla-koids and stroma during the cell cycle of Chlamydomonas reinhardtii Eur J Biochem 177, 225–232.
Trang 814 Herrin, D & Michaels, A (1985) The chloroplast 32 kDa protein
is synthesized on thylakoid-bound ribosomes in Chlamydomonas
reinhardtii FEBS Lett 184, 90–95.
15 Roy, L.M & Barkan, A (1998) A SecY homologue is required
for the elaboration of the chloroplast thylakoid membrane and
for normal chloroplast gene expression J Cell Biol 141,
385–395.
16 Danon, A & Mayfield, S.P (1991) Light-regulated translational
activators: identification of chloroplast gene specific
mRNA-binding proteins EMBO J 10, 3993–4001.
17 Zerges, W & Rochaix, J.-D (1994) The 5¢ leader of a chloroplast
mRNA mediates the translational requirements for two
nucleus-encoded functions in Chlamydomonas reinhardtii Mol Cell Biol.
14, 5268–5277.
18 Ossenbu¨hl, F & Nickelsen, J (2000) Cis- and trans-acting
determinants for translation of psbD mRNA in Chlamydomonas
reinhardtii Mol Cell Biol 20, 8134–8142.
19 McCormac, D.J., Litz, H., Wang, J., Gollnick, P.D & Berry, J.O.
(2001) Light-associated and processing-dependent protein binding
to 5¢ regions of rbcL mRNA in the chloroplasts of a C4 plant.
J Biol Chem 276, 3476–3483.
20 Fargo, D.C., Boynton, J.E & Gillham, N.W (2001) Chloroplast
ribosomal protein S7 of Chlamydomonas binds to chloroplast
mRNA leader sequences and may be involved in translation
initiation Plant Cell 13, 207–218.
21 Nakamura, T., Ohta, M., Sugiura, M & Sugita, M (2001)
Chloroplast ribonucleoproteins function as a stabilizing factor of
ribosome-free mRNAs in the stroma J Biol Chem 276, 147–152.
22 Bruick, R.K & Mayfield, S.P (1999) Light-activated translation
of chloroplast mRNAs Trends Plant Sci 4, 190–195.
23 Trebitsh, T., Meiri, E., Ostersetzer, O., Adam, Z & Danon, A.
(2001) The protein disulfide isomerase-like RB60 is partitioned
between stroma and thylakoids in Chlamydomonas reinhardtii
chloroplasts J Biol Chem 276, 4564–4569.
24 Gorman, D.S & Levine, R.P (1965) Cytochrome f and
plasto-cyanin: their sequence in the photosynthetic electron transport
chain of Chlamydomonas reinhardtii Proc Natl Acad Sci USA
54, 1665–1669.
25 Ojakian, G.K & Satir, P (1974) Particle movements in
chlor-oplast membranes: quantitative measurements of membrane
fluidity by the freeze-fracture technique Proc Natl Acad Sci.
USA 71, 2052–2056.
26 Adir, N., Shochat, S & Ohad, I (1990) Light-dependent D1
protein synthesis and translocation is regulated by reaction center
II: reaction center II serves as an acceptor for the D1 precursor.
J Biol Chem 265, 12563–12568.
27 Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K.,
Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M.,
Olson, B.J & Klenk, D.C (1985) Measurement of protein using
bicinchoninic acid Anal Biochem 150, 76–85.
28 Arnon, D.J (1949) Copper enzymes in isolated chloroplast:
polyphenoloxidase in Beta vulgaris Plant Physiol 24, 1–15.
29 Albertson, P.A (2001) A quantitative model of the domain
structure of the photosynthetic membrane Trends Plant Sci 6,
349–354.
30 Bhairi, S.M (2001) A Guide to the Properties and Uses of
Deter-gents in Biology and Biochemistry Calbiochem-Novabiochem.
Corporation, San Diego, CA, USA.
31 Jagow, G & Scha¨gger, H (1994) Membrane Protein Purification.
Academic Press Inc, London.
3 2 Thornber, J.P., Morishige, D.T., Anandan, S & Peter, G.F.
(1991) Chlorophyll-carotenoid proteins of higher plant thylakoids.
In Chlorophylls (Scheer, H., ed.), pp 549–585 CRC Press, Boca
Raton.
33 Matika, A (1999) Die Regulation der Photosynthese Durch Pro-teinphosphatasen in Chlamydomonas Reinhardtii Dissertation, Ruhr University, Bochum.
34 Erickson, J.M., Rahire, M & Rochaix, J.-D (1984) Chlamydo-monas reinhardii gene for the 32,000 mol wt protein of photo-system II contains four large introns and is located entirely within the chloroplast inverted repeat EMBO J 3, 2753–2762.
35 Nickelsen, J., van Dillewijn, J., Rahire, M & Rochaix, J.-D (1994) Determinants for stability of the chloroplast psbD RNA are located within ist short leader region in Chlamydomonas reinhardtii EMBO J 13, 3182–3191.
36 Bruick, R.K & Mayfield, S.P (1998) Processing of the psbA 5¢ untranslated region in Chlamydomonas reinhardtii depends upon factors mediating ribosome association J Cell Biol 143, 1145– 1153.
37 Vaistij, F.E., Goldschmidt-Clermont, M., Wostrikoff, K & Rochaix, J.-D (2000) Stability determinants in the chloroplast psbB/T/H mRNAs of Chlamydomonas reinhardtii Plant J 21, 469–482.
38 Nickelsen, J., Fleischmann, M., Boudreau, E., Rahire, M & Rochaix, J.D (1999) Identification of cis-acting RNA leader ele-ments required for chloroplast psbD gene expression in Chlamy-domonas Plant Cell 11, 957–970.
39 Klaff, P., Mundt, S.M & Steger, G (1997) Complex formation of the spinach chloroplast psbA mRNA 5¢ untranslated region with proteins is dependent on the RNA structure RNA 3, 1468–1479.
40 Alexander, C., Faber, N & Klaff, P (1998) Characterization of protein-binding to the spinach chloroplast psbA mRNA 5¢ untranslated region Nucleic Acids Res 26, 2265–2272.
41 Shteiman-Kotler, A & Schuster, G (2000) RNA-binding char-acteristics of the chloroplast S1-like ribosomal protein CS1 Nucleic Acids Res 28, 3310–3315.
42 Zerges, W (2000) Translation in chloroplasts Biochimie 82, 583–601.
43 Kroll, D., Meierhoff, K., Bechtold, N., Kinoshita, M., Westphal, S., Vothknecht, U.C., Soll, J & Westhoff, P (2001) VIPP1, a nuclear gene of Arabidopsis thaliana essential for thylakoid membrane formation Proc Natl Acad Sci USA 98, 4243–4248.
44 Erickson, J.M (1998) Assembly of photosystem II In The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas (Rochaix, J.D., Goldschmidt-Clermont, M & Merchant, S., eds), pp 255–285 Kluwer Academic Publishers, Dordrecht.
45 Zhang, L., Paakkarinen, V., van Wijk, K.J & Aro, E.M (1999) Co-translational assembly of the D1 protein into photosystem II.
J Biol Chem 274, 16062–16067.
46 Fox, T.D (1996) Genetics of Mitochondrial Translation: Transla-tional Control, Monograph Series 30 pp 733–758 Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
47 Rochaix, J.D (1996) Post-transcriptional regulation of chloro-plast gene expression in Chlamydomonas reinhardtii Plant Mol Biol 32, 327–341.
48 Hauser, C.R., Gillham, N.W & Boynton, J.E (1998) Regulation
of chloroplast translation In The Molecular Biology of Chloro-plasts and Mitochondria in Chlamydomonas (Rochaix, J.D., Goldschmidt-Clermont, M & Merchant, S., eds), pp 197–217 Kluwer Academic Publishers, Dordrecht.
49 Choquet, Y & Vallon, O (2000) Synthesis, assembly and degradation of thylakoid membrane proteins Biochimie 82, 615–634.
50 Nilsson, R., Brunner, J., Hoffman, N.E & van Wijk, K.J (1999) Interactions of ribosome nascent chain complexes of the chloro-plast-encoded D1 thylakoid membrane protein with cpSRP54 EMBO J 18, 733–742.