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The Plant Cell, Vol. 3, 709-717, July 1991 O 1991 American Society of Plant Physiologists Targeting of Proteins to the Outer Envelope Membrane Uses a Different Pathway than Transport into Chloroplasts Hsou-min Li," Thomas Moore,b and Kenneth Keegstra".' a Department of Botany, University of Wisconsin, Madison, Wisconsin 53706 Department of Vegetable Crops, University of California, Davis, California 95616 The chloroplasticenvelope is composed of two membranes, inner and outer, each with a distinct set of polypeptides. Like proteins in other chloroplastic compartments, most envelope proteins are synthesized in the cytosol and posttranslationally imported into chloroplasts. Considerable knowledge has been obtained concerning protein import into most chloroplastic compartments. However, very little is known about the biogenesis of envelope membrane proteins. We isolated a cDNA clone from pea that encodes a 14-kilodalton outer envelope membrane protein. The precursor form of this protein does not possess a cleavable transit peptide and its import into isolated chloroplasts does not require either ATP or a thermolysin-sensitive component on the chloroplastic surface. These findings, together with similar observations made with a spinach chloroplastic outer membrane protein, led us to propose that proteins destined for the outer membrane of the chloroplastic envelope follow an import pathway distinct from that followed by proteins destined for other chloroplastic compartments. INTRODUCTION Most proteins present in chloroplasts are encoded by the nuclear genome and synthesized on cytosolic ribosomes (Chua and Schmidt, 1979). The transport of these cytosolically synthesized proteins to chloroplasts has been studied extensively. The outlines of this transport process are well described although the mechanistic details are still poorly understood. The structural complexity of chloroplasts adds to the challenge of understanding targeting to this organelle. Chloropiasts consist of three distinct membranes: the inner and outer membranes of the envelope and the thylakoid membrane. These three membranes in turn enclose three aqueous spaces: the intermembrane space of the envelope, the stroma, and the thylakoid lumen. Thus, cytosolically synthesized proteins not only must be targeted to chloroplasts but also must be directed to the proper compartment within chloroplasts. Most chloroplastic proteins are synthesized as precursor proteins with N-terminal extensions called transit peptides. Transit peptides are necessary and sufficient to direct the import of proteins into chloroplasts. Protein import to the inside of chloroplasts is initiated by an energy-dependent binding of precursors to the envelope, followed by an energy-dependent translocation across the envelope. Once in the stroma, precursor proteins are either processed to their mature size or further sorted to the thylakoid membrane or the thylakoid lumen (for a review, see ' To whom correspondence should be addressed. Keegstra, 1989). Targeting of envelope proteins to their proper membrane locations is not yet well understood. The chloroplastic envelope is the site where the organelle interacts with other cellular compartments. The envelope not only regulates the transport of metabolites between the stroma and the cytosol but also mediates the import of nuclear-encodedchloroplastic proteins into chloroplasts, as described above. The envelope plays a major role in the biosynthesis of various lipids including galactolipids, the predominant lipids in chloroplastic membranes (Douce and Joyard, 1990). Therefore, knowledge about the chloroplastic envelope, especially its protein constituents, is fundamental for understanding the function and biogenesis of chloroplasts. Despite the importance of chloroplastic envelope proteins, little is known about the biogenesisof these proteins, especially at the molecular level. One reason for this is that most envelope proteins are present in very low quantities compared to other chloroplastic proteins. Only a few genes encoding envelope proteins have been isolated. These include the genes encoding two inner membrane proteins from spinach chloroplasts: a 37-kD protein (Dreses-Werringloer et al., 1991) and the phosphate translocator, which is the most abundant protein in the envelope (Flügge et al., 1989). The import of these two proteins shows characteristics similar to the import of the stromal and the thylakoid membrane proteins. A homologous gene for the phosphate translocator was also identified in pea, but in 710 The Plant Cell this case it was reported to encode the import receptor for the small subunit of ribulose-1,5-bisphosphate carboxylase (SS), and was localized to the contact sites between the two membranes of the envelope (Schnell et al., 1990). Resolution of the controversy regarding the function and the chloroplastic location of the protein encoded by this gene will require further work. Another spinach gene that has been isolated encodes a 6.7-kD outer membrane protein (Salomon et al., 1990). The import of this protein is, however, very different from other chloroplastic proteins. The protein is synthesized without a cleavable transit peptide and its import into chloroplasts is not ATP dependent. At present it is unclear whether this distinct import mechanism is unique to this protein or is a more general feature shared by other chloroplastic outer membrane proteins. Also, because the specificity of the insertion process was not demonstrated, it is possible that the insertion into the chloroplastic outer membrane occurred because chloroplasts were the only organelle present in the experimental system. To understand better the biogenesis of chloroplastic envelope proteins, an effort was made to obtain cDNA clones encoding chloroplastic envelope proteins. We report here the isolation of a pea cDNA clone encoding a chloroplastic outer envelope membrane protein. The import of this protein into chloroplasts was investigated. The results indicate that the import of this protein possesses characteristics distinct from those of most chloroplastic proteins but are in good agreement with what has been observed with the 6.7-kD outer membrane protein. RESULTS Isolation and Characterization of a cDNA Clone Encoding a Chloroplastic Outer Envelope Membrane Protein Monoclonal antibodies were prepared against total envelope proteins. As shown in Figure 1A, lane 2, one of the antibodies recognized a 14-kD outer envelope membrane protein. This antibody was used to screen a Xgt11 cDNA expression library. A partial-length clone was isolated and the cDNA insert was used to synthesize nucleic acid probes to screen another Xgt11 cDNA library. Among the positive clones, A14kom was chosen for further study because it contained the largest insert. The cDNA insert of X14kom was subcloned into the expression vector pSP65 (Promega), resulting in the plasmid pSP14kom. When the cDNA insert in the plasmid was subjected to sequential in vitro transcription and translation, it directed the synthesis of a protein that migrated with an apparent molecular mass of 14 kD when analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) (Figure 1A, lane 3). This molecular mass is identical to that of the protein recognized by the monoclonal antibody. Because A 1 2 3 B CL HS .-18.4 9766-1 ,-14.3 - 6.2 49- 29-I 14Figure 1. A14kom Represents a Full-Length Clone with Respect to the Protein It Encodes. (A) Size comparison between the protein recognized by the monoclonal antibody and the protein encoded by A14kom. Pea chloroplastic outer membrane proteins (lane 1) were probed with a monoclonal antibody prepared against envelope proteins (lane 2). Lane 3, in vitro translation product from the cDNA insert of A14kom in plasmid pSP14kom. Lane 1 was stained with Amido Black. Lanes 2 and 3 were run on the same gel and blotted onto one piece of membrane. The two lanes were then cut apart and treated for antibody hybridization (lane 2) or fluorography (lane 3) by incubating the membrane with 20% 2,5-diphenyloxazole in toluene. Molecular mass markers in kilodaltons are indicated at left. (B) Hybrid selection of the mRNA corresponding to the cDNA insert of A14kom. Pea-leaf poly(A)* RNA was incubated with linearized plasmid pSP14kom. The hybrid-selected mRNA was used to program an in vitro wheat germ translation system. CL, translation product from the cDNA insert of pSP14kom; HS, translation product from the hybrid-selected mRNA. Samples were analyzed by SDS-PAGE. A fluorograph of the dried gel is shown. Molecular mass markers in kilodaltons are indicated at right. nuclear-encoded chloroplastic proteins are usually synthesized as higher molecular weight precursors in the cytosol, we were concerned that the cDNA insert of \14kom may not encode a full-length precursor protein. To investigate this possibility, the cDNA insert was used to hybrid-select its corresponding mRNA from pea-leaf poly (A)+ RNA. When the selected mRNA was translated in vitro, it directed the synthesis of a protein with a size identical to Chloroplastic Envelope Protein Targeting that of the protein translated from the cDNA insert (Figure 1B). This result indicates that the cDNA insert in A14kom contains the entire protein coding region of the gene. Figure 2 shows the DNA sequence of the cDNA insert in X14kom. It contains an open reading frame of 82 amino acids with a calculated molecular mass of 9.0 kD, even though the translation products, both from the hybridselected mRNA and from the cDNA insert, migrated with an apparent molecular mass of 14 kD on an SDS gel (Figure 1B). No related sequences were found in the GenBank and EMBL sequence data bases. Localization of the Protein Encoded by \14kom To examine whether the protein encoded by X14kom could be imported into chloroplasts, the in vitro translation product from pSP14kom was incubated with isolated intact chloroplasts under conditions that normally lead to the import of Chloroplastic precursor proteins (see Methods). As shown in Figure 3A, the in vitro translation product associated with chloroplasts; however, no molecular 711 B TR C OM14- r S E T TR C S E T -SS Figure 3. Localization of the Protein Encoded by X14kom (OM14). (A) Fractionation of chloroplasts after import of the protein encoded by A14kom. Chloroplasts were incubated with the in vitro translation product from pSP14kom under standard import conditions. Intact chloroplasts were reisolated and subjected to further fractionation, as described in Methods. TR, in vitro translation product; C, total chloroplasts after import; S, stroma; E, envelope; T, thylakoid. (B) Fractional of chloroplasts after import of prSS. The import and fractionation conditions were the same as in (A) except the import reaction was performed on ice instead of room temperature to obtain enough envelope-bound precursor protein molecules. SS, small subunit of ribulose-1,5-bisphosphate carboxylase. GAATTCCAA 1 1 2 ATG GGA AAG GCG AAA GAA GCG GTT GTG GTG M G K A K E A V V V GCG GGT GCC CTA GCA TTT GTC TGG CTC GCT 1 A G A L A F V W L A ATT GAA CTC GCT TTC AAA CCC TTC CTT TCT 1 I E L A F K P F L S CAG ACC CGT GAC TCC ATC GAC AAA TCC GAC 3 4 5 1 Q T R D S I D K S D CGA CCC GGG ATC CTG ACG ATG CTC CTC CTC 1 R P G I L T M L L L CTC CTC CTC CTG AAA CTG ACG CCG GAG ATG 1 L L L L K L T P E M CCG ACA AAG ACG ACT GAT GAT TTG AGC GCG 6 1 P T K T T D D L S A GTT GTT ACT ATT CCT TTC TTG CAT TCT GTA 7 1 V V T I P F L H S V TTT CGT TAA ATTGTTAAGTGAAATAACATTAACTAT 81 F R * GTTCTTTGTTGTGTTTTCTGTTCTGATCTGATTTCTGGAA TACTGTAATTTTAGGATTAGGATTGAATGCCAGGTTGTTC ACTTCATACGTTGGACAATTGGTTTGTTAATTTGGTGTTT GTCTCTTTAGCTATTGCAAATGCAATCGTGTTGTGAAAAT AACAAAGCTTCTGAATCAAAAAAAAAAAAAGGAATTC Figure 2. Nucleotide Sequence of the cDNA Insert in X14kom and the Deduced Amino Acid Sequence. Amino acid numbers are indicated at left. The standard one-letter code for amino acids is used. weight shift was observed after import. This result suggested that the protein encoded by X14kom was synthesized as a precursor protein without a cleavable transit peptide. Fractionation studies of the chloroplasts after import of the translation product from pSP14kom were performed to obtain information on the suborganellar localization of the imported protein. A parallel experiment was performed with the precursor to SS (prSS) because the imported, mature-size SS can serve as a marker for the stromal fraction and the prSS bound on the surface of chloroplasts can serve as a marker for the envelope fraction. As shown in Figure 3B, prSS associated mostly with the envelope fraction, with a small portion associated with the thylakoid fraction. Some association with thylakoids was expected because the thylakoid fraction is always contaminated with envelope membranes (Cline and Keegstra, 1983). The same fractionation pattern as prSS was observed with the protein encoded by X14kom, indicating that this protein was directed to the Chloroplastic envelope during import into chloroplasts. Furthermore, the result shown in Figure 4, lane 2, demonstrated that the protein was thermolysin sensitive after import, indicating that it was located in the outer membrane. Thermolysin at this concentration has been shown to be unable to penetrate the outer membrane (Cline et al., 1984). This protein was thus named OM14 (for outer membrane 14-kD protein). 712 The Plant Cell Fluorography 1 2 Immunoblot 3 4 5 6 — 14.3 OM14 et al., 1985). Monospecific antibodies that reacted with OM14 were eluted with a low-pH solution. When analyzed on immunoblots, this eluted antibody recognized a single protein in Chloroplasts (Figure 4, lane 3). This protein was located in the outer membrane and had the same molecular weight and the same thermolysin sensitivity as OM14 (Figure 4, lanes 4 and 5). When this antibody preparation was used to probe cellular fractions enriched in mitochondria or microsomes, no cross-reactive protein was detected (data not shown). The same results were also obtained with the monoclonal antibody (data not shown). In addition, under the same condition that led to the import of OM14 into Chloroplasts, OM14 could not be imported into pea mitochondria (data not shown). From these studies, we concluded that OM14 is an authentic chloroplastic protein. Membrane Topology of OM14 protease — -f — + Figure 4. Identification of the Authentic OM14 in Chloroplasts. Chloroplasts after import of OM14 (lane 1) were treated with thermolysin (lane 2), revealing that the imported OM14 was thermolysin sensitive. A protein with this same property and the same molecular mass as OM14 was identified in Chloroplasts (lanes 3 to 5, see below). Lane 6 shows outer membrane proteins probed with a polyclonal antibody against total outer membrane proteins. This polyclonal antibody was incubated with the fusion protein containing OM14, expressed from M 4kom, to affinity purify monospecific antibody reactive with OM14. The purified monospecific antibody was then used to probe total chloroplastic protein (lane 3), Chloroplasts treated with thermolysin (lane 4), and outer membrane proteins (lane 5). Lanes 1 to 5 were run on the same gel and blotted onto one piece of membrane. The membrane was then cut into two parts and treated for fluorography or immunoblot analysis as indicated. Molecular mass markers in kilodaltons are indicated at right. Thermolysin treatment of samples are indicated at bottom. Identification of the Authentic OM14 in Chloroplasts It is not surprising that a chloroplastic outer membrane protein is susceptible to thermolysin digestion. However, a hydrophobic protein that nonspecifically associated with the surface of Chloroplasts might exhibit the same property. To verify that OM14 is a genuine chloroplastic protein, we tested the ability of a polyclonal antibody, raised against total outer membrane protein (Figure 4, lane 6), to react with OM14. If OM14 could be recognized by this antibody, it would provide independent evidence that OM14 is an authentic chloroplastic outer membrane protein. The polyclonal antibody preparation was incubated with a fusion protein containing OM14 that was immobilized on nitrocellulose membranes after expression of X14kom (Johnson Imported OM14 could not be removed from the outer membrane by alkaline or high salt extraction (data not shown), suggesting that OM14 was an integral membrane protein. Figure 5A shows the hydrophobicity analysis (Kyte and Doolittle, 1982) of the deduced OM14 polypeptide sequence. It predicts that the protein spans the outer membrane at least twice (using the criterion that the hydrophobicity value exceeds 1.58 for at least 1 residue; Jahnig, 1990) with a major hydrophilic domain connecting two membrane spanning domains. The conformation of the C-terminal end of the polypeptide is not clear; although it is hydrophobic, its hydrophobicity and its length are probably not sufficient for this region of the polypeptide to span the membrane another time. To investigate the topology of OM14 within the membrane, i.e., to determine whether the hydrophilic domain faces the cytosol or the intermembrane space, we employed the protease chymotrypsin. Chymotrypsin cleaves peptide bonds predominantly after tyrosine, tryptophan, or phenylalanine residues. In OM14 polypeptide, two phenylalanines are located in the hydrophilic domain with other target residues of Chymotrypsin all located in the hydrophobic regions, i.e., possibly buried in the membrane (Figure 5A). If the hydrophilic domain is located on the cytosolic site of the outer membrane so that the two target sites in the hydrophilic domain are exposed on the surface of Chloroplasts, the protein should be susceptible to Chymotrypsin digestion. On the other hand, if the hydrophilic domain is located in the intermembrane space, the protein should be relatively Chymotrypsin resistant. Digestion of translation products with Chymotrypsin revealed that the precursor form of OM14 was sensitive to Chymotrypsin before import (Figure 5B, lane 2). After being inserted into the outer membrane, OM14 was resistant to Chymotrypsin digestion (Figure 5B, lane 4). The protein Chloroplastic Envelope Protein Targeting the outer membrane at least twice with the connecting hydrophilic domain exposed to the intermembrane space. However, because Chymotrypsin does sometimes have a broader specificity, the possibility that OM14 has some other membrane topology cannot be excluded. A +3- 713 iW Hydrophobic 0 Hydrophilic Import Characteristics of OM14 -3| 20 I I I I I I I I I 40 I I 60 I I I I 80 Amino Acid Number B Chymotrypsin Sonication Thermolysin — + — — — — 1 2 — + — — -— 3 4 CH — + — - — + — — — 5 6 7 + — + — - + 8 9 OM Figures. Membrane Topology of OM14. (A) Hydrophobicity analysis of the deduced amino acid sequence of OM14. The analysis was carried out using the method of Kyte and Doolittle (1982) with a window of 19 residues as suggested by Jahnig (1990). Positions of Chymotrypsin target sites in the polypeptide are indicated. F, phenylalanine; W, tryptophan. (B) Chymotrypsin sensitivity of OM14 under various conditions. OM14 from the in vitro translation mixture (TR), chloroplasts after import of OM14 (CH), or isolated outer membrane vesicles from chloroplasts after the import reactions (OM) were treated with Chymotrypsin, thermolysin, and/or sonication, as indicated on the top of the figure. was Chymotrypsin resistant even in outer membrane vesicles isolated from chloroplasts of import reactions (Figure 5B, lane 6), indicating that most of the isolated outer membrane vesicles possessed a right-side-out orientation. The resistance of the protein to Chymotrypsin was not due to the inaccessibility of the protein in the isolated membrane vesicles because OM14 was still sensitive to thermolysin (Figure 5B, lane 9). Furthermore, the resistance to Chymotrypsin was lost when the protease had access to both sides of the outer membrane, i.e., after the membrane vesicles were sonicated in the presence of the protease (Figure 5B, lane 8). This result indicated that imported OM14 in intact chloroplasts was Chymotrypsin resistant because the protease target sites were located in the intermembrane space and thus protected from the protease. The same results were obtained with the authentic OM14 in chloroplasts by using immunoblot analysis (data not shown). Accordingly, we propose that OM14 spans Time course studies of the import of OM14 into chloroplasts revealed that the import process was time and temperature dependent. The data in Figure 6 demonstrated that, at 25°C, the initial rate of import was rapid and gradually reached a plateau. This process was influenced strongly by temperature because, at 4°C, not only did import proceed at a lower rate but much less protein was imported. The reason for this drastic change of import by low temperature is unknown. It could be an effect of the temperature on membrane fluidity, on the import competence of the protein, or on the association(s) of other required protein(s) with OM14. The energy requirement for the import of OM14 was also investigated. The in vitro translation product was gel 25° C 400- 0 0 5 10 15 20 I 25 I 30 Time (minutes) Figure 6. Import Time Course of OM14. Import reactions were conducted at either 25°C or 4°C. At each time point, 60 t^L of the reaction mixture was removed and the reaction terminated as described (Theg et al., 1989). Samples were analyzed by SDS-PAGE. Quantitation of samples in the gel is shown. 714 The Plant Cell filtered to remove most of the ATP in the translation mixture (Olsen et al., 1989).As a control, prSS synthesized in vitro and the translation mixture treated the same way were tested in parallel experiments. No translocation of prSS was observed when there was no ATP in the import reaction (data not shown; Olsen et al., 1989). However, as shown in Figure 7, the amount of OM14 imported was essentially the same regardless of the amount of ATP provided. Nigericin and valinomycin also had no effect on the import of OM14. We concluded, therefore, that neither ATP nor a proton-motive force was required for the import of OM14. It has been shown for several chloroplastic precursor proteins that protein import into chloroplasts requires some proteinaceous component(s) on the chloroplastic surface. When chloroplasts are pretreated with thermolysin, the amount of protein imported is reduced greatly (Cline et al., 1985). However, the same treatment had almost no effect on the import of OM14 (data not shown). Combining this result with the observation of an ATPindependent import, we concluded that the “import” of OMI 4 to the chloroplastic outer membrane is very different from the “import” of proteins into the inside of chloroplasts. The former probably represents the integration of a protein into a membrane without any translocation event. It is thus more proper to refer the association of OM14 with chloroplasts as “insertion” instead of “import.” DISCUSSION Severa1 lines of evidence demonstrated that OM14 is a chloroplastic outer membrane protein. A monospecific antibody preparation, affinity purified by reaction with OM14 derived from the cloned gene, recognized a protein in the chloroplastic outer membrane that had the same molecular mass, same thermolysin sensitivity, and same membrane topology as OMI 4. In addition, proteins with cross-reactivity to this antibody could not be detected in other cell fractions. These observations demonstrated that OM14 is an authentic chloroplastic protein in vivo. Protein insertion into the chloroplastic outer membrane has now been studied with two proteins, OM14 of pea chloroplasts reported here and a 6.7-kD protein of spinach chloroplasts (Salomon et al., 1990). 60th proteins are synthesized without cleavable transit peptides and, in both cases, insertion into the outer membrane does not require ATP. For both proteins, thermolysin pretreatment of chloroplasts has no effect on their insertion. Because of these characteristics, it is important to demonstrate that the insertion is specific because it is possible that such small hydrophobic proteins might insert into any membrane. Consequently, we demonstrated that in vitro synthesized OM14 could be inserted into the outer envelope membrane O 100 200 300 1OOO Nig Val [ATP] (pM) & lonophores Figure 7. ATP and Proton-Motive Force Requirements for the lmport of OM14. lrnport reactions for the ATP requirementexperiment were carried out in the dark with the indicated amount of ATP added to each reaction mixture. The ionophore experiment was carried out in the light with each ionophore present at 2 wM. lmport reactions were carried out for 6 min. Reisolated chloroplasts after import were treated further with chymotrypsin in an attempt to remove noninserted protein molecules. Quantitation of samples analyzed by SDS-PAGE is shown. [ATP], ATP concentrations; Nig, nigericin; Val, valinomycin. of isolated chloroplasts but not mitochondria, indicating that the in vitro import of OM14 faithfully reflects the organelle specificity of protein targeting in vivo. The insertion of OM14 was not inhibited by synthetic peptide analogues of the transit peptide of prSS (data not shown). These peptides were shown to inhibit the binding and translocation of several precursors destined for other chloroplastic compartments (Perry et al., 1991). These observations provide additional evidence that OMI 4 used a different import receptor from that used by the majority of chloroplastic proteins. Alternatively, it is possible that OM14 interacted directly with the lipid components of the outer membrane so that it did not utilize a proteinaceous receptor. Two different mechanisms have been described for the insertion of proteins into mitochondrial outer membranes. Porin of Neurospora crassa and monoamine oxidase B of beef heart mitochondria require ATP and a trypsin-sensitive component on the mitochondrial surface for their insertion (Zhuang et al., 1988; Pfaller et al., 1990). On the other hand, insertion of porin into the Saccharomyces cerevisiae mitochondrial outer membrane does not require ATP, and a trypsin pretreatment of the mitochondria does not inhibit this insertion (Gasser and Schatz, 1983). In addition, cytochrome c of mitochondria, a protein present in the intermembrane space of the envelope, also does not require ATP or a protein on the mitochondrial surface for Chloroplastic Envelope Protein Targeting its translocation across the outer membrane (Nicholson et al., 1988). It is unclear whether these differences represent variations on a single pathway to the outer membrane or whether they reflect two different pathways. The specificity of OM14 insertion to the chloroplastic outer membrane was shown by its exclusive presence in the chloroplastic outer membrane in vivo and the failure to insert into mitochondrial outer membrane in vitro. Because OM14 does not possess a cleavable transit peptide, it is uncertain where the targeting information that mediates this specificity resides. A similar question arises with mitochondrial outer membrane proteins, which also lack cleavable presequences(Hartl et al., 1989). This question has been addressed with a 70-kD mitochondrial outer membrane protein, where the first 12 amino acid residues function as a matrix targeting sequence and the subsequent hydrophobic residues function as a “stop-transfer” signal that anchors the protein in the outer membrane (Nakai et ai., 1989). The situation with OM14 is more complex because it is predicted to span the membrane at least two times. Hypotheses for OM14 insertion can be derived by drawing analogies with integral membrane proteins of the endoplasmic reticulum (Blobel, 1980; Lipp et al., 1989). The first membrane-spanningdomain of OM14 may function as a signal-anchor sequence that initiates the insertion of the protein into the chloroplastic outer membrane. The second membrane-spanningdomain may then function as a stop-transfer sequence resulting in an Ni,-Ci, topology of the protein (“in” refers to the cytosol). This prediction is also in agreement with the “positive inside” hypothesis (von Heijne and Gavel, 1988) because the residues on the amino-terminal side of the first membranespanning domain has a net positive charge. Alternatively, the two membrane-spanning domains could pair together and spontaneously insert into the outer membrane, as described by the “helical hairpin hypothesis” (Engelman and Steitz, 1981). Regardless of the mechanistic details, the current data support the conclusion that chloroplastic outer membrane proteins utilize an import pathway that is distinct from that of protein transport to other chloroplastic compartments. Outer membrane protein targeting involves a direct insertion from the cytosol with no need of an extra peptide extension and its subsequent removal by way of processing. This feature is shared with proteins destined for the outer membrane of mitochondria and seems to be a general feature of protein targeting to the outer membrane of each organelle. The lack of an ATP requirement and the apparent independence of a proteinaceous receptor that have been observed with OM14 and E6.7 have also been observed with some, but not all, mitochondrial outer membrane proteins. It remains to be determined whether these features are general for all chloroplastic outer membrane proteins. 715 METHODS Antibody Preparations, cDNA Cloning, and Sequencing For monoclonalantibody preparation,total envelope proteinswere used to immunize 8-week-old Balb/c mice according to the procedure described (Stahliet al., 1983). The spleen cells from an immunized mouse were fused with NS-1 mouse myeloma cells, an 8-azaguanine resistant, nonsecreting cell line. Supernatants from cultured hybrid cells were assayed using ELISA, as described (Voller et al., 1980), with envelope proteins as immobilized antigens. Positive cell lines were expanded and rescreened by ELISA against both envelope and stromal proteins. Selected lines were cloned by limiting dilution, and frozen. Those lines that reacted only with envelope but not stromal proteins were analyzed further on immunoblots. The polyclonal antibody against purified outer membrane proteins was prepared as described in Marshall et al. (1990). Monospecific antibody affinity purified by OM14 was prepared as described (Johnson et al., 1985). cDNA libraries were made from pea-leaf poly(A)+RNA and were gifts of Dr. S. Gantt (Gantt and Key, 1986) and Dr. G. Coruzzi (Tingey et al., 1987).The screening process using antibodies was performed as described (Huynh et al., 1985). Synthesis of nucleic acid probes by in vitro transcription of a partia1 cDNA clone and the screening process using these probes were performed as described (Wahl et al., 1987). For DNA sequencing, the cDNA insert from X14kom was subcloned in both orientations into the Phagescript vector (Stratagene). A series of nested deletions was made using exonuclease 111 and S1 nuclease according to the procedure of Henikoff (1987) with modifications described by Greenler et al. (1989). Singlestranded DNA was isolated and sequenced using the dideoxy chain termination method (Sanger et al., 1980) with the Klenow fragment of DNA polymerase I and LP~*P-~ATP. Overlapping clones from the nested deletions were chosen by running T-tracking reactions before the full sequencing. Both strands of the cDNA were sequenced in their entirety. Sequence data analysis was carried out using the UWGCG software (Devereux et al., 1984). Hybrid selection of mRNA was performed as described (Maniatis et al., 1982). Pea-leaf poly(A)+ RNA was prepared as described (Cline et al., 1985). Precursor Protein Synthesis and lmport into lsolated Chloroplasts The cDNA insert from X14kom was subcloned into the pSP65 vector (Promega), resulting in the plasmid pSPl4kom. Tritiumlabeled precursor proteins were synthesized from pSP14kom by using in vitro transcription/translation systems as described (Smeekenset al., 1986).Where indicated, ATP was removed from the translation mixture by filtering the translation mixture through a Sephadex G-25 column after the translation reactions (Olsen et al., 1989). lntact chloroplasts were isolated from 10- to 15-day old pea (fisum sarivum cv Perfection) seedlings as described (Cline, 1986). lmport experiments were performed in import buffer (330 mM sorbitol/50 mM Hepes.KOH, pH 8.0) at room temperature 716 The Plant Cell as described (Li et al., 1990) except that the amount of precursor proteins was reduced to 5 x 105dpm for OM14 and 1 x 10' dpm for prSS. After import, intact chloroplasts were reisolated by centrifugation through a 40% Percoll cushion. Recovered chloroplasts were subjected to further fractionation or protease treatments. The experiment shown in Figure 6 was performed using the silicone oil/perchloric acid method as described (Theg et al., 1989) except that the oil volume was increased from 1O0 pL to 160 pL. Thermolysin pretreatment of chloroplasts was done as described by Cline et al. (1985). of monoclonal antibodies, Dr. Jerry Marshall for the polyclonal antibody against outer membrane proteins, and Jim Sloan for advice on DNA sequencing. This work was supported in part by a grant to K.K. from the Office of Basic Energy Sciences at the Department of Energy. The nucleotide sequence data reported in this paper will appear in the GenBank Nucleotide Sequence Database under the accession number M69105. Received April 16, 1991; accepted May 15, 1991. Post-lmport Treatments of Chloroplasts For fractionation of chloroplasts, reisolated intact chloroplasts from a 450-pL import reaction were hypotonically lysed in 450 pL of 10 mM Tris.HCI, pH 7.5/2 mM EDTA (TE) by incubating on ice for 1O min. Lysed chloroplasts were loaded onto a sucrose step gradient with 1.2 mL of 1.2 M sucrose, 1.5 mL of 1 M sucrose, and 1.5 mL of 0.46 M sucrose, and centrifuged in a Beckman SW 50.1 rotor at 47,000 rpm for 1 hr. Stromal, envelope(a mixture of inner and outer membranes), and thylakoid fractions were retrieved from the supernatant, the 0.46 M/1 M sucrose interface, and the pellet, respectively. Proteins in the stromal fraction were concentratedby acetone precipitation. The envelopefraction was diluted with TE buffer and pelleted by centrifugation in a Beckman JA-20 rotor at 20,000 rpm for 45 min. The thylakoid fraction was washed with TE buffer and pelleted by centrifugation in a microcentrifuge at 7,500 rpm for 5 min. A similar procedure was used to isolate outer membrane vesicles except that the chloroplasts were lysed hypertonically in 0.6 M sucrose by one cycle of freezing and thawing (Keegstra and Yousif, 1986) and the sucrose step gradient was made with 1.8 mL of 1 M sucrose, 1.6 mL of 0.8 M sucrose, and 1.3 mL of 0.46 M sucrose. The outer membrane fraction was retrieved from the 0.46 M/0.8 M sucrose interface. All sucrose solutions were made in TE buffer. Thermolysin treatment of chloroplasts or outer membrane vesicles was performed as described (Smeekens et al., 1986). Chymotrypsin treatment of chloroplasts or outer membrane vesicles was performed in import buffer containing 10 mM CaCI2and 30 pg/mL tosyl-L-lysine chloromethyl ketone-treated chymotrypsin. The reaction was incubated at room temperature for 30 min and the digestion was terminated by adding phenylmethanesulfonyl fluoride to 1 mM. 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H M Li, T Moore and K Keegstra Plant Cell 1991;3;709-717 DOI 10.1105/tpc.3.7.709 This information is current as of June 18, 2017 Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw153229 8X eTOCs Sign up for eTOCs at: http://www.plantcell.org/cgi/alerts/ctmain CiteTrack Alerts Sign up for CiteTrack Alerts at: http://www.plantcell.org/cgi/alerts/ctmain Subscription Information Subscription Information for The Plant Cell and Plant Physiology is available at: http://www.aspb.org/publications/subscriptions.cfm © American Society of Plant Biologists ADVANCING THE SCIENCE OF PLANT BIOLOGY