The Transit Sequence Mediates the Specific Interaction of the. Precursor of Ferredoxin with Chloroplast Envelope Membrane Lipids*. (Received for publication ...
Vol. 268, No. 6, Issue of February 25, pp. 4037-4042, 1993 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
The Transit Sequence Mediatesthe Specific Interaction of the Precursor of Ferredoxin withChloroplast Envelope MembraneLipids* (Received for publication, September 11, 1992)
Ron van’t Hof$$, Wim van KlompenburgS, Marinus PilonST, Arkadiusz KozubekS(1, Gerda deKorte-KoolS, RudyA. DemelS, Peter J. Weisbeekn, and Ben de Kruijff+** From the $Centerfor Biomembranes and Lipid Enzymology, Department of Biochemistry of Membranes, the VDepartment of Molecular Cell Biology, the **Instituteof Molecular Biology, University of Utrecht, Padualaan8, 3584 CH Utrecht, The Netherlands
The interaction of the precursor of the chloroplast transit sequences of stromal proteins revealed that there is protein ferredoxin with membrane lipids was studied no similarity between their amino acid sequences except for in monolayer experiments in order to investigate the the N-terminal methionine-alanine (6), although transit sepossible involvementof membrane lipids in the proteinquences are characterizedby a positive charge, dueto thelack translocation process. The precursor efficiently and of acidic amino acids and an enrichment of hydroxylated specifically inserts intoa total lipid extract of its bio- amino acids. It is generally accepted that the import process logical target the outer envelope membrane of chloro- is initiatedby the bindingof the precursor proteinsby means plasts. This interaction is mediated by the transit se- of the transit sequence to the outer envelope membrane (7). quence as it can also be observed for the chemically This binding step depends on the utilizationof low amounts prepared transit peptideof ferredoxin but neither for of ATP (5-100 PM) in the intermembrane space (8). The the ferredoxin apoprotein nor holoprotein. Interactions withthe individual chloroplast lipids, monogalac-result that binding can be observed also in the absence of tosyl-diacylglycerol, sulfoquinovosyl-diacylglycerol, ATP (9), and that binding isonly reduced but not abolished and phosphatidylglycerol are predominantly involved after protease pretreatment of chloroplasts (10, 11) suggests which corresponds to the results obtained for transit that different binding typesexist. The precursor proteins are peptide fragmentsof the small subunitof ribulose-1,5- translocated across the envelope membranes which requires bisphosphatecarboxylase/oxygenase(van’t Hof, R., a 10-fold higher ATP concentration as compared to binding Demel, R. A., Keegstra, K., and De Kruijff, B. (1991) (12, 13). After translocation, the transit sequence is cleaved off by a stromal protease (14). Finally the proteins are routed FEBS Lett. 291, 350-354). No efficient interaction was obtained with digalactosyl-diacylglycerol to their properlocalization and assembled into holoenzymes. and phosphatidylcholine, suggesting that a loose lipid Theexactmechanism of transport across the envelope headgroup packing due to small lipid headgroups membranes and/or is still unknown. Several studies (15-17) report electrostatic repulsions facilitates efficient insertion. on the involvement of different proteins but their functions The observed preferences for interaction of the pre- areeitherstillunknownordebatable (17, 18). Itcan be cursor and transit peptide of ferredoxin for the chlo- expected also that the membrane lipids are involved in the roplast outer envelope membrane lipid extract and the import process. This involvement can be indirect by providing presequence of cytochrome c oxidase subunitIV for the and maintaining the essential barrier function of the memmitochondrial outer membrane lipid extract indicate brane during the protein translocation process or by assemthat targeting sequence-lipid interactions contribute to bling the putative proteinacious insertion/translocation maorganelle-specific protein targeting. chinery correctly in the membranes and more directly, after precursor-lipidinteractions,directingtheprecursortothe import apparatus by two-dimensional diffusion. In addition, The biogenesis of chloroplasts is largely dependent on the membrane lipids alone or in combination with proteins could post-translational import of nuclear encoded proteins (for a perform more direct roles in the binding, translocation, and recent review, see Ref. 1). These proteins are synthesized in release of proteins during the protein import process. This the cytosol as precursors generally containing an N-terminal hypothesis is particularly attractive because the chloroplast extension, the transit sequence (2). The transit sequence is membranes have an unique lipid composition (19). For innecessary (3,4) andsufficient (5) for the translocation across stance, the chloroplast membranes contain the lipid classes digalactosyl-dithe chloroplastenvelope membranes. A comparison of various monogalactosyl-diacylglycerol (MGalDG),’ acylglycerol (DGalDG),and sulfoquinovosyl-diacylglycerol * This workwas supported by The Netherlands Foundation for (SQDG) which are exclusively found in plasmid membranes. Biological Research (BION) and The Netherlands Foundation for Biophysics and with financial aid from the Netherlands Organization for Scientific Research (NWO). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. 8 TOwhom correspondence should be addressed Center for Biomembranes and Lipid Enzymology, Dept. of Biochemistry of Membranes, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands. Tel.: 31-30-535512;Fax: 31-30-522478. )I Present address: Institute of Biochemistry, University of Wroclaw, Przybyszewskiego 63,PL 51-148W, Wroclaw, Poland.
The abbreviations used are: MGalDG, monogalactosyl-diacylglycerol; prefd, preferredoxin; trfd, transit peptide of ferredoxin; apofd, apoferredoxin; holofd, holoferredoxin; pSSU, precursor of ribulose1,5-biphosphate carboxylase/oxygenase; MGluDG, monoglucosyl-diacylglycerol;DGalDG, digalactosyl-diacylglycerol;SQDG, sulfoquinovosyl-diacylglycerol;DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPG, 1,2-dioleoyl-sn-glycero-3-phosphoglycerol; DOPE, 1,2dioleoyl-sn-glycero-3-phosphoethanolamine;PC, phosphatidylcholine; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; PI, phosphatidylinositol; Pipes, piperazine-N,N’-bis(2-ethansulfonic acid); mN, millinewton.
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Precursor Ferredoxin-Chloroplast Membrane
Lipid Interactions
by the bicinchoninic acid protein assay (Pierce Chemical Co.) using Moreover, chloroplast membrane lipids contain a very high it bovine serum albumin as reference. The purity of the peptides was content of polyunsaturated fatty acid chains. Furthermore, was shownthat fragments of the transitsequence of the small estimated to be over 98% as determined by analytical high performance liquid chromatography runs. The identity of the peptides was subunit of ribulose-1,5-bisphosphatecarboxylase/oxygenase confirmed by N-terminal sequencing of 20 amino acids by Edman (pSSU) which were able to inhibitthe binding and importof degradation (251,by quantitative amino acid analysis (29), and for p S S U (20), were able to insert in lipid monolayers and have the presequence also by mass spectroscopy. Proteins and Polypeptides-Prefd from S. pratensis was purified a preference for the specific chloroplast lipids MGalDG and SQDG (21). From these results it was suggested that specific from an E. coli strain overexpressing the precursor protein as deby Pilon et al. (25). The protein was stored in 200-pl aliquots interactions between the transit sequence and chloroplast scribed in 25 mM Tris/HCl, pH 7.5, 8 M urea, and 0.02% (v/v) P-mercaptolipids could fulfill a role in the membrane translocation of ethanol at -20 "C. Holofd was isolated from leaves of S. pratensis chloroplast precursor protein. according to Pilon et al. (29) and stored in 25 mM Tris/HCl, pH 7.5, To test this hypothesis we analyzed the interaction of a full buffer at -20 "C in small aliquots. Apofd was prepared out of holofd as described (28) by removal of the 2Fe-2S cluster with trichloroacetic length translocation-competent precursor protein with differe n t lipids in monolayer experiments. We made use of t h e acid. The protein was stored in 1-ml aliquots in 150 mM Tris/HCl, 7.5, buffer. Polypeptide and protein concentrations were deterSilene pratensis. The pH precursor of ferredoxin (prefd) from mined by the bicinchoninic acid protein assay (Pierce) and according advantages of this protein are t h a t i t follows the general to Bradford (31) with bovine serum albumin as reference with idenimport pathway (22) and its import process is well studied. tical results. Lipids-l,2-Dioleoyl-sn-glycerol was obtained from Sigma. 1,2T h e ferredoxin transit sequence enables foreign proteins to enter chloroplasts and targets them to the stroma (23, 24). Dioleoyl-sn-glycero-3-phosphocholine(DOPC), 1,2-dioleoyl-sn-glycPrefd import (26) isonly ATP-dependent (25), does not ero-3-phosphoglycerol (DOPG), and 1,2-dioleoyl-sn-glycero-3-phos(DOPE) were synthesized according to established require cytosolic factors most likely due to its unfolded struc- phoethanolamine methods (32, 33). ture, and takes place independently form cofactor assembly Monoglucosyl-diacylglycerol (MGluDG) was isolated from Acho(29). Furthermore, prefd can be purified on milligram scale leplasma laidlawii grown on oleic acid (34) and was a kind gift from from Escherichia coli (25). By analyzingin a comparative way Dr. A. Wieslander. MGalDG, DGalDG, and SQDG wereisolated from the lipid-protein interactions for the apoprotein (apofd) and a total pea thylakoid membrane lipid preparation (21) obtained holoprotein (holofd) of ferredoxin and the chemically synthe- according to Bligh and Dyer (35). Removal of the majority of the sized transit peptide (trfd) we are ableto analyze which part pigments and a first crude separation in lipid classes was performed by chromatography on a 75-ml (2.5 X 15-cm) silica column (Baker, of the precursor protein is involved in the interactions with silica gel, 30-60 pm, Deventer, The Netherlands). The column was membrane lipids. To study the possibility that protein-lipid stepwise eluted with 750 ml of acetone:chloroform (l:l, by volume), in correct targetingof precursor eluting the majority of the pigments, 750 ml of chloroform, eluting interactions may be involved proteins to either chloroplastsor mitochondria, we compared mainly MGalDG, 750 ml of ch1oroform:methanol (9:1, by volume), the interaction of the transit sequence of ferredoxin and the eluting mainly DGalDG, and finally 750 ml of ch1oroform:methanol by volume), eluting SQDG and phospholipids. MGalDG, c (1:1, presequence of themitochondrialprecursorcytochrome DGalDG, and SQDG were further purified by chromatography on a oxidase subunit IV (P25L18W)by making use of monolayers carboxymethyl cellulose column (21). Remaining traces of pigment of lipid extracts from both chloroplasts and mitochondrial were removed by chromatography on a 8-ml silica column (1 X 10 outer membranes. cm) (Baker, silica gel, 30-60 pm, Deventer, The Netherlands) for of hexane:isopropyl alcohokwater The results show that the interaction of the precursor of MGalDG eluted with 160ml (85:15:0.4, by volume), for DGalDG with 160 ml of hexane:isopropyl ferredoxin is specificforlipidextracts of thechloroplast alcohokwater (70:302, by volume), and for SQDG with 160 ml of membranes and is mediated by its transit sequence. hexane:isopropyl alcohokwater (70:302, by volume) followed by 30 ml of ch1oroform:methanol (l:l, by volume). MATERIALS AND METHODS Membrane Lipid Extracts-Total chloroplast outer envelope membrane lipids were obtained by extraction according to Bligh and Dyer Peptides-A 47-mer corresponding to the transit sequence of fer- (35) of outer envelope membranes isolated according to Keegstra and redoxin from S. pratensis was synthesized on an Excell Pepsynthes- Jousif (36) out of 14-day-old pea seedlings cu. Feltham First (37). izer by Millipore, Watford, United Kingdom. Trfd, with the sequence Total mitochondrial outer membrane lipids were obtained from rat ASTLSTLSVSASLLPKQQPMVASSLPTNMGQALFGLKAGSRG liver mitochondria according to Hovius et al. (38). Proteins were RVTAM, did not contain the N-terminal methionine which in vivo removed from the lipid extracts by silica column chromatography. is removed in the cytosol (27). The purified transit peptide is biolog- An 8-ml (10 X 1-cm) silica column (polygosyl60-4063 Machereyically active because it efficiently inhibits the import of prefd into Nagel, Duren, Germany) was pretreated with 80 mlof 1% (v/v) isolated chloroplasts (29). A 25-mer resembling the presequence of ammonia in ch1oroform:methanol (l:l, by volume) and pre-eluted cytochrome c oxidase subunit IVof Saccharomycescereuisiaewas with 1.5 pmol of egg PC in order to block the most active sites. The synthesized on an Excell Pepsynthesizer by the Hubrecht Laborato- pretreatment of the silica columns prevents specific removal of PC ries, Utrecht, The Netherlands. In its sequence, MLSLRQSIRFFKP out of the lipid extracts. The lipid extracts were eluted with 40 ml of W C S S R Y L L (P,L18W), the tryptophan W18 replaces chloroform followed by 80 ml of ch1oroform:methanol (l:l,by volAT RT leucine L18 of the wild type sequence. It was shown that P&IBW ume). The lipid composition of the chloroplast outer envelope lipid was able to inhibit the import of a fusion protein of the presequence extract was determined by two-dimensional thin layer chromatograof cytochrome c subunit IV fused to themature sequence of dihydro- phy (TLC) according to Ref. 21 and was found to be MGalDG 5%, folate reductase into yeast mitochondria with similar efficiency as DGalDG 32%, SQDG 6%, PC 46%, PG 5%, and PI 5%. The mitothe wild type presequence.' Both peptides contain an amide group on chondrial outer membrane lipid extract was determined according to their C terminus to avoid a negative charge at thisposition. Ref. 38 and was found to he PC 48%, PE 31%, cardiolipin 9%, PI The peptides were purified by reversed phase high pressure liquid IO%, and phosphatidylserine 1%.These compositions are in good chromatography on a 25 X 2.5-cm column packed with C,, nucleosyl agreement with previously published data (38, 40, 41). 120-10 (Macherey-Nagel, Duren, Germany) using linear water-aceMonolayer Experiments-The Wilhelmy plate method was used to tonitrile gradients containing 0.1% (v/v) trifluoroacetic acid as eluent. measure peptide and protein-induced changes in the surface pressure The transit peptide eluted at 35% acetonitrile and the presequence of monolayers at constant surface area (39). Two ml of 10 mM Pipes, at 42% acetonitrile. After lyophilization, the peptides were stored as 50 mM NaC1, pH 7.4 (unless indicated otherwise), was placed in a dry material under nitrogen at -20 "C. Stock solutions were prepared Teflon trough with a diameter of 2 cm and a depth of 0.6 cm and by dissolving the peptides in degassed distilled water and were stored continuously stirred. The monomolecular lipid layer was formed by under nitrogen at -20 "C. Peptide concentrations were determined spreading a lipid containing chloroform solution on the airbuffer interface to initial surface pressure between 18 and 35 mN/m. Peptides and proteins were added to the subphase through a small hole * K. Nicolay, unpublished results.
Precursor Ferredoxin-Chloroplast
Membrane Interactions Lipid
at the edge of the trough. The results shown were obtained using saturating amounts of (po1y)peptides.The surface pressure increase was measured in time until a stable surface pressure was reached. The values plotted in this study correspond to these equilibrium pressures and were obtained for final subphase (po1y)peptideconcentrations of 1.5 pg/ml. Experiments were performed at room temperature. By desalting prefd and the addition of urea up to 6 M to trfd, apofd, and holofd stock solutions, it was checked that differences in the interaction of the (po1y)peptideswith lipid monolayers were not a result of differences in buffer from which they were added. RESULTS
In this article the interaction between (po1y)peptides and lipids are studied in monolayer experiments. The changes in surface pressure after the addition of the (po1y)peptides to the aqueous subphase are measured and are interpreted as a result of the insertion of the (po1y)peptides into the lipid monolayer. The limiting surface pressure is defined as the pressure where the (po1y)peptides can no longer penetrate and subsequently, the change in surface pressure is zero. Fig. 1 shows that injection of prefd underneath a lipid 2 extract of its. target membrane the outerenvelope membrane of chloroplasts at an initial surface pressure of23 mN/m results in a rapid and large increase of the surface pressure. Direct prefd-lipid interactions must be involved because prefd at saturating concentrations(>1.25 pg/ml) in the absence of a lipid monolayer maximally increases the surface pressure up to 16 mN/m (data not shown).Injection of saturating concentrations of both the unfolded (29) apoprotein(>1.2 pg/ ml) and the folded holoprotein ( ~ 1 . pg/ml) 2 (29) underneath the monolayer results in virtually no increase in surface pressure under these conditions (Fig. 1). This demonstrates that the transit sequence in the precursorisessential for penetration into the lipid layer. That itis the transitsequence itself which is largely responsible for this effect is shown by the large increase in surface pressure induced by saturating amounts (>1.2 pg/ml) of trfd. The lower end values observed for trfd as compared to prefd suggest that the matureregion contributes to thelipid-protein interaction. The lipid extract of chloroplast outer envelope membranes is composed of manydifferent lipids (19). To investigate whether individual lipids mediate the insertion of prefd and trfd, both were injected underneath monolayers consisting of
i
4039
purified individual lipids. At different initial surface pressures the surface pressure increases were measured (Figs. 2 and 3). For example, prefd inserts efficiently in MGalDG monolayers, with an efficiency comparable to the interaction with the chloroplastmembrane lipid extract (compare Fig. 1). The surface pressure increase isless a t higher initial surface pressures due to the tighter lipid packing of the monolayer, but prefd even penetrates at initial surface pressures above 30 mN/m which is thought tobe the surface pressureof biological membranes(42).Similarresults are obtained with SQDG, DOPG, and DOPE. In contrast, the interaction with DGalDG and DOPC is significantly reduced, resulting in lower limiting insertionpressures of approximately 26 mN/m. Also, trfd significantly inserts inmonolayers of individual lipids to high initial surface pressures. Compared to prefd, trfd interacts stronger with DOPG and, especially a t lower initial surface pressures, weaker with MGalDG andDOPE.The lowest penetration is again found for DGalDG and DOPC. 1
18
22
26
30
initial surface pressure
34
(mNlm)
FIG.2. Surface pressure increases after the injection of prefd underneath monolayers of MGDG (a), DGDG (A), SQDG (O),DOPE (m), DOPC (A),and DOPG (0)at different initial surface pressures.
12
31
1 E
v
E
2
v)
27-
trFd
g 0)
0
m
5 25u)
apoFd holoFd
23 -
2-
I I
0
5
10
time (min.)
FIG.1. Surface pressure increasesafterthe injection of prefd, trfd, apofd, and holofd underneath a lipid monolayer of the total lipid extract of the chloroplast outer envelope membrane. The intial surface pressure was 23 mN/m.
18
22
26
30
34
initial surface pressure (mNlm) FIG.3. Surface pressure increases after the injection of trfd underneath monolayers of MGDG (O), DGDG (A), SQDG (0), DOPE (m),DOPC (A),and DOPG (0)at different initial surface pressures.
Precursor Ferredoxin-Chloroplast Membrane Interactions Lipid
4040
The inefficient insertion of prefd and trfd in DGalDG and DOPC monolayers suggests that large lipid headgroups (47) prevent the penetration of prefd and trfd between lipids. This suggestion was verified by studying the interaction of prefd with 1,2-dioleoyl-sn-glycerol monolayers which lipid does not contain a headgroup (datanotshown).Injection of prefd underneath themonolayer at an initial surface pressure of 20 mN/m caused a rapid increase of the surface pressure to a value of 31 mN/m which corresponds to the collapse pressure of the 1,2-dioleoyl-sn-glycerol monolayer (30). This large effect was not the result of anonspecific absorption of the protein to the monolayer because apofd only caused an increase in surface pressure of less than 1 mN/m. Trfd is positively charged ( + 5 ) , therefore it is likely that electrostaticinteractionsare involvedin the insertion between the negatively charged lipids. T o investigate this possibility, trfd and prefd were injected underneath SQDG and DOPG monolayers in subphases which contain low salt (50 0 206 0 40 80 100 mM NaCl) and high salt concentrations (400 mM NaC1) (Fig. 4).High salt concentrationswill mask the charge on both trfd mol % MGDG in DOPC and prefd and on thelipids by which electrostatic interactions FIG.5. Effect of MGalDG concentration in DOPC monoare reduced. The insertion of prefd is independent of the salt layers on the interaction with trfd. The initial surface pressure concentration while in contrast,theinsertion of trfd is was 20 mN/m. strongly reduced,indicating thatonly trfd-SQDG interactions are mediated largely by electrostaticinteractions.Similar results were obtained with DOPG (data not shown). The outerenvelope membrane contains only 5% MGalDG. In view of the ability of prefd and trfd to interactefficiently with MGalDG, but not with the much more abundant PC,we determined the dependence of trfd insertion on theMGalDG content in DOPC monolayers (Fig.5 ) . Trfd insertion appeared to be independent of the MGalDG concentration above 25%. Even at 5% MGalDG in DOPC, the interaction is comparable to the 100% value. At 10% a maximum in the interaction was observed for which we do not yet have a good explanation. The strong interaction of prefd and trfd with MGalDG was not specific for the galactose headgroup because MGluDG, which contains a glucose headgroup, caused comparable interactions (data not shown). T o determine whether lipid-protein interactions could be involved in the targeting process of precursor proteins between chloroplasts and mitochondria which both depend on 18 22 2364 30 post-translational protein import, we compared the interacinitial surface pressure (mNlm) tion of prefd and trfd with lipid extracts of the chloroplast FIG.6. Surface pressure increases after the injection of prefd (squares) and trfd (triangles) underneath monolayers of a total lipid extract of the chloroplast outer envelope membrane (open symbols)and the total lipid extract of the mitochondrial outer membrane (closed symbols).
a , 0
I
3
6
time (min.)
FIG.4. Salt dependence of the interaction of prefd (- - -) and trfd (-) with SQDG. 1 and 3, 50 mM NaC1; 2 and 4 , 400 mM NaCl concentrations. The initialsurface pressure was 25 mN/m.
and mitochondrial outer membrane (Fig. 6). Both prefd and trfd efficiently insert in the chloroplast lipid monolayer, even above an initial pressure of 30 mN/m. The insertion in the mitochondrial lipid extract is significantly lower. The specificity of targeting sequences for the chloroplast lipid extract was analyzed by injection of P2,L18W underneath monolayers of membrane lipid extracts of chloroplasts andmitochondria (Fig. 7). The presequence has a strong interaction with the mitochondrial lipid extract comparable toliterature values (43, 44), buttheinteraction with the chloroplast lipid extract is significantly lower. Injection of Pz,L18W and trfd underneath monolayers of purifiedlipids ataninitial surface pressure of 30 mN/m (Table I) shows that both have their strongest interaction with the negatively charged SQDG and DOPG. The more stronger interactionof P2,L18W with these lipids is probably caused by its higher chargedensity and its amphiphilic surface
Precursor Ferredoxin-Chloroplast Membrane Interactions Lipid
0 26
22
18
34
30
initial surface pressure (mNlm)
FIG. 7. Surface pressure increasesafter the injection of PzeL18Wunderneath monolayers of total lipid extracts of the chloroplast outer envelope membrane (squares)and the total lipid extract of the mitochondrial outer membrane (circles).
TABLE I Surface pressure increasei n m N j mcaused by the presequence and trfd when injected underneath monolayers consistingof individual lipids at a n initial surface pressureof 30 mN/m. T h e results are average of three independent measurements with a standard deviation ranging between 0.25 and 0.35 mN/m Lipid Peptide
trfd P,,LlSW
DOPG
SQDG
DOPE
MGalDG
DOPC
DGalDG
6.0 10.5
4.4 9.0
1.0
3.4 2.6
0. 4.0
0. 2.1
4.5
seeking nature (45). Similar tendencies can be obtained for DOPE, DOPC, and DGalDG butinterestingly,trfdhas a stronger interaction with the plant lipid MGalDG. DISCUSSION
In this study itis demonstrated that the import-competent precursor of ferredoxinefficiently inserts into monolayers consisting of a lipid extract of its biological target, the outer membrane of the chloroplastenvelope. Moreover, it is shown that insertion is mediated by the transit sequence and can also be observed forthe chemically prepared full length transit peptide. The results confirm and extend the original observations on lipid-peptide interactions observed for fragments of thetransitpeptide of pSSU (21) which suggests that penetration into the target lipid-water interface is amore general property of transit sequences. The major difference being the muchlarger effects observed forthe intact precursor and the transit peptide of ferredoxin as compared to the fragments of the transit peptideof pSSU. The lipid specificity of the insertion of prefd was found to be rather broad and remarkable. Efficient penetration was observed forthe individual chloroplast membranelipid classes MGalDG (a galactolipid), SQDG (a negatively charged glycolipid), and PG (a negatively charged phospholipid) aswell as for zwitterionic PE which does not occur in chloroplasts. In contrast, insertion into the abundant envelope lipids PC (a zwitterionic lipid) and DGalDG (a galactolipid) is much less efficient. This specificity which was also reported for the fragments of the transit peptide of pSSU (21) suggests that MGalDG, SQDG, and PG contribute most to the efficient
4041
insertion of the precursor in monolayers of the total outer envelope membrane lipids. How can we understand this rather unusual lipid specificity? The most plausible explanation is that the transit sequence likes to reside intheinterface between the alkyl methyl groups and the more polar residues of the glycerol backbone and thelipid headgroups. This area hasbeen called the hydrogen belt (46) because it is believed that here extensive hydrogen networksexist between the lipid molecules. The transit sequence which is richinhydroxylated amino acidscouldpossibly participateinthis hydrogenbonding network. In order to reach this area the transit sequence will have to penetrate the headgroup layer. PC and DGalDG are two lipids with relativelylargeheadgroups(47) which are expected to seal this layer more tightly, thereby inhibiting penetration. The other headgroups are expected to be more loosely packed in the monolayer situation because of their small headgroup size and/or electrostatic repulsions. In this case the hydrogen belt area can be reached more efficiently. This model explains the very efficient insertion of prefd in monolayers of the headgroupless diacylglycerol (this study) and offers an explanation for the increased binding of pSSU to thermolysin-pretreated chloroplasts incubated with phospholipase C (48). The diacylglycerols expected to result from this treatment could facilitate insertion of the precursor in the outer envelope membrane. Our results further demonstrate that already low concentrations of MGalDGconfer monoefficient penetration of the transit peptide into the PC layer, suggesting that this galactolipid acts as an insertion site for the transit sequence. Penetration of the ferredoxin precursor in the monolayer of the target lipids appears to involve not only the transit sequence but possibly,also parts of themature sequence because the maximal pressureincreases observed for the 40% smaller (see Fig. 1). Wecannot transitpeptideare exclude the possibility that there are subtle differences in lipid-polypeptide interaction between thetransit sequence and the transit peptide which contribute to thisdifference. In fact, the interaction of the transit peptide with the anionic lipids depends more on electrostatic interactions as can be inferred from the differentresponse of the transitpeptide and the precursor to an increased salt concentration. The interaction of the precursor with SQDG and PG was hardly affected by the presence of high salt, consistent with the proposed insertion model. Also the slightdifferencesinlipid specificity between the precursor and the transit peptide, in particular at lower surface pressures, point in that direction. However, it shouldbe realized that both prefd and the transit peptide are similarly effective in efficient inhibition of import of radiolabeled prefd into chloroplasts (29). Recently it was reported that the transit peptide of the y-subunit of the chloroplast ATP synthase from Chlamydomonas reinhardtii (49) was surface active, like the transit peptideof ferredoxin. The resultsof that study on lipid-transit peptide interactions in monolayers cannot be compared to our study because of the limited range of lipids tested and the choice of unusual low and biologically irrelevant initialsurface pressures in that study. Despitethefactsthatprefd efficiently insertsinto P E monolayers and that PE is an abundant component in the outer mitochondrial membrane, theferredoxin precursor specifically inserts into the lipid extract of the outer membrane of itschloroplast uersus that of themitochondrion.This suggests that transit sequence chloroplast-lipid interactions are involved in chloroplast targeting. One attraction of this hypothesis is that itprovides an evolutionary explanation on
4042
Precursor Ferredoxin-Chloroplast Membrane Interactions Lipid
3. Reiss, B., Wasman, C. C., and Bohnert, H. J. (1978) Mol. & Gen. Genet, the nature of the chloroplast targeting signal. Chloroplasts 2 0 9 , 116-121 are believed to be themost recently acquiredorganelle in the 4. Smeekens. J.. Geerts.. D... Bauerle.. C... and Weisbeek.. P. (1989) . , Mol. Gen. Genet. 2 6 1 , 178-182 plant cell and therefore a novel targeting sequence had to 5. Van den Broek, G., Tinko, M. P., Kausch, A. P., Cashmore, A. R., Van develop. The blue-green algae, believed t o be the ancestors of Montagne, M., and Herrera-Estrella, J. (1985) Nature 3 1 3 , 358-363 6. Von Heijne, G., Steppuhn, J., and Herrmann, R. G. (1989) Eur. J. Biochem. the chloroplast(50), already had thespecific lipids now exclu180.535-545 sively found in the chloroplast (51) and thus only a signal 7. Keegstra, K. (1989) Cell 56,247-253 8. Olsen, L. J., and Keegstra, K. (1992) J . Biol. Chem. 267,433-439 recognizing these specific lipids was required to direct the 9. Flugge, U.I. (1990) J. Bioenerg. Biomembr. 2 2 , 769-787 chloroplast precursor to the organelle. The observed prefer- 10. Olsen, L. J., Theg, S. M., Selman, 9. R., and Keegstra, K. (1989) J. Biol. Chem. 264,6724-6729 ence of themitochondrialprepeptide for insertinginthe 11. Cline, K. (1985) J. Biol. Chem. 260,3691-3696 mitochondrial outermembrane lipid extract and the high 12. T h S. M., Bauerle, C., Olsen, L. J.,Selman, B. R., andKeegstra, K. (E89) J. Biol. Chem. 264,6730-6736 efficiency of this process could together with mitochondrial 13. Paj"-p., and Blobel, G. (1987) Proc. Natl. Acad. Sci. U. S. A. 8 4 , 3288membrane receptors (52) and a membrane potential across 3ZYZ 14. Robinson, C., and Ellis, R. J. (1984) Eur. J. Biochem. 142,337-342 (53) contributetothe theinnermitochondrialmembrane 15. Cornwell, K. L., and Keegstra, K. (1987) Plant Physiol. 8 5 , 780-785 fidelity of correct targeting precursors to mitochondria and 16. Pain, D., Kanwar, Y. S., and Blobel, G. (1988) Nature 331,232-237 17. Schnell, D. J., Blobel, B., and Pain, D. (1990) J. Cell B i d . 1 1 1 , 1825-1838 chloroplasts. 18. Flugge, U. I., Weber, A,, Fischer, K., Lottspeich, F., Eckershorn, C. WaeThere are several arguments for an interaction of transit gemann, K., and Soll, J. (1991) Nature 3 5 3 , 364-367 19. Douce, R., Block, M. A,, Dorne, A. J., and Joyard, J. (19&1)Subcell. Biochem. sequences with thelipid part of the outer chloroplast envelope 1 fi * 1-QA ", membrane during import. First, the insertionwe observed in 20. Perry, S. E., Buvinger, W. E., Bennett, J., and Keegstra, K. (1991) J . Biol. Chem. 2 6 6 , 11882-11889 monolayers occurs at surface pressures,generally assumed to 21. van't Hof, R., Demel, R. A,, Keegstra, K., and De Kruijff, 9. (1991) FEBS Lett. 291,350-354 occur in biological membranes. Second, the outer envelope J. G., Moorman, A. F. M., and Verkley, F. N. (1978) Biochem. membrane has,like the mitochondrial outer membrane, a low 22. Huisman, Bio hys Res Commun. 8 2 , 1121-1131 protein/lipid (19) ratio and thus is likely to expose extended 23. Smeefens; S., Bauerle, C., Hageman, J., Keegstra, K., and Weisbeek, P. J. (1986) Cell 46,356-375 lipid domains to thecytosol. This is supportedby the finding 24. Smeekens, S., Van Steeg, H., Bauerle, C., Bettenbroek, H., Keegstra, K., and Weisbeek, P. J. (1987) Plant Mol. Biol. 9 , 377-388 that antibodies canrecognize the galactosyldiacylglycerolsat 25. Pilon, M., de Boer, A. D., Knols, S. L., Koppelman, M. H. G. M., van der the surface of the chloroplast envelope (54). Third, phosphoGraaf. R. M.. de Kruiiff. B.. and Weisbeek. P. J. (1990) J. Biol Chem. . ,~ lipase C treatment of chloroplasts blocks import and affects 2 6 5 , 3358-3361.. 26. Pilon, M., de Krugff, B., and Weisbeek, P. J. (1992) J. Biol. Chem. 2 6 7 , precursor binding (48). Fourth, significant bindingof precur2548-2556 sor to chloroplastsoccurs even in theabsence of ATP (9) and 27. Von Heijne, G., Steppuhn, J., and Herrmann,R. G. (1989) Eur. J. Biochem. 180.535-545 after protease treatment(10, 11). 28. Pagani; S., Bonomi, F., and Cerletti, P. (1984) Eur. J. Biachem. 142,361Our study does not give insight into the role of putative 366 M., Rietveld, A. G., Weisbeek, P. J., and De Kruijff, 9. (1992) J. proteinacious chloroplast receptors in chloroplast protein im-29. Pilon, Bid. Chem. 267,19907-19913 port. In our opinion theobserved precursor-lipid interaction 30. Demel, R. A., van Doorn, J. M., and van der Voorst, D. J. (1992) Biochem. Biophys. Acta 1 1 2 4 , 151-158 could play, besides a targeting function, other roles in the 31. Bradford, M. M. (1976) Anal. Biochem. 72,248-254 chloroplast protein importprocess. Penetration into the outer32. Van Deenen, L. L. M., and De Haas, G. H. (1964) Adu. Lipid Res. 2 , 168229 leaflet of the outer membrane could by a two-dimensional 33. Comfurius, P., andZwaal, R. F. A. (1977) Biochim. Biophys. Acta 488,3642 diffusion mechanism facilitate productive collisions with the 34. Wieslander, A,,Ulmius, J., Lindblom, G., and Fontell, K. (1978) Biochim. import apparatus. Moreover, the precursor-lipid interactions Biophys. Acta 5 1 2 , 241-253 35. Bligh, E. G., and Dyer, W. J. (1959) Can. J. Biochem. Physiol. 37,911-917 could give rise to specific structural motives in the transit Keegstra, K., and Jousif, A. E. (1986) Methods Enzymol. 118,316-325 sequences which are decoded in the import apparatus. In this36. 37. Cline, K., Werner-Washburne, M., Lubben, T. H., and Keegstra, K. (1985) J. Biol. Chem. 260,3691-3696 light it is of interest to note that transit peptides display a R.. Lambrechts. H.. Nicolav. " , K... and de Kruiiff. " . B.(1990) Biochim. considerable structural flexibility (29, 43). Furthermore, the 38. Hovius. Biophys.'Acta 1021,217-226 39. Morse, P. D.,and Dreamer,P. W. (1973) Biochim. Biophys. Acta298,769specific interactions in particular with MGalDG (a typical 7QO . non-bilayer lipid) could result in changes in local membrane 40. Cline, K., Andrews, J.,Mersey,B., Newcomh, E. H., andKeegstra, K. (1981) Proc. Natl. Acad. Sci. U. S. A . 78,3595-3599 organization which could influence the translocationprocess. 41. Block, M. A,, Dorne, A.-J., Joyard, J., and Douce, R. (1983) J. Biol. Chem. Finally lipid-transit sequence interactions could be of impor2 5 8 , 13281-13286 tance for the action of the matrix proteasewhich will have to 42. Demel, R. A,, Geurts van Kessel, W. S. M., Zwaal, R. F. A., Roelofsen, B., and Van Deenen, L. L. M. (1975) Biochim. Biophys. Acta 406,97-107 recognize the transitsequence at the exitof the translocation 43. Endo, T.,Kawamura, K., and Nakai, M.(1992) Eur. J . Biochem. 270,671675 machinery in the inner envelope membrane. "
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Acknowledgments-W. Heerma and C. Versluis (Department of Analytical Molecule Spectroscopy, University of Utrecht, The Netherlands) are gratefully acknowledged for performing the mass spectroscopic analysis of PZ5L18W.We thank K. Brouwer for preparing the manuscript. REFERENCES 1. de Boer, A. D., and Weisbeek, P. J. (1991) Biochim. Biophys. Acta 1 0 7 1 , 221-253 2. Chua, N.-H., and Schmidt, G.W. (1978) Proc. Natl. Acad. Sci. U. S. A . 7 5 , 6110-6114
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