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Plant Cell Physiol. 39(5): 526-532 (1998) JSPP © 1998 Polypeptide Composition of Envelopes of Spinach Chloroplasts: Two Major Proteins Occupy 90% of Outer Envelope Membranes Hiroyuki Koike, Maki Yoshio', Yasuhiro Kashino and Kazuhiko Satoh Department of Life Science, Faculty of Science, Himeji Institute of Technology, Harima Science Garden City, Hyogo, 678-1297 Japan Outer and inner envelope membranes of spinach chloroplasts were isolated using floatation centrifugation followed by sedimentation sucrose density gradient centrifugation after disruption of intact chloroplasts by freezing and thawing. Two major fractions with buoyant densities of 1.11 and 1.08 g cm"3 and a minor fraction with a density of 1.15 g cm"3 were obtained. They were identified as inner and outer envelope and thylakoid fractions, respectively, by analyzing their polypeptide composition by high-resolution SDS-PAGE and the N-terminal sequences of their protein components. Due to the refinement of the isolation procedure, most of the ribulose-l,5-ftwphosphate carboxylase/oxygenase (RuBisCO), which had always been observed as a contaminant, was eliminated from the outer envelope fraction. Application of high-resolution SDS-PAGE revealed that this fraction was rich in the low-molecular-mass outer envelope protein, E6.7 [Salomon et at. (1990) Proc. Natl. Acad. Sci. USA 87: 5778] and a protein with a molecular mass of 15 kDa which is homologous to the 16 kDa outer envelope protein of pea [Pohlmeyer et al. (1997) Proc. Natl. Acad. Sci. USA 94: 9504]. The two proteins account for 90% of the total proteins present in outer envelope membranes. Proteins which are suggested to function in translocation of nuclear-encoded polypeptides were not identified in the envelopes from spinach in the present study. Differences in the protein composition of outer envelope membranes are discussed based on the developemental stages of chloroplasts. Key words: Chloroplast envelope — SDS-PAGE — Spinach — Sucrose density gradient centrifugation. Chloroplast envelopes consist of two different membranes, outer and inner (Douce and Joyard 1990). The outer envelope membranes are thought to contain pores Abbreviations: hsp, heat shock protein; IEP, inner envelope protein; OEP, outer envelope protein; PVDF, polyvinylidene difluoride; RuBisCO, ribulose-l,5-6isphosphatecarboxylase/oxygenase. 1 Present address: Biological Function Section, Kansai Advanced Research Center, Communications Research Laboratory, Ministry of Posts and Telecommunications, Kobe, Hyogo, 651-24 Japan. which freely pass molecules of less than 10 kDa (Fliigge and Benz 1984). The inner envelope membranes act as a substantial barrier for both low and high molecular mass compounds. The membranes are sites of ion and metabolite translocation and synthesis of galactolipids (Douce and Joyard 1990, Flugge and Heldt 1991). The envelope membranes were first prepared by Mackender and Leech (1970) from Vicia faba. However, these isolated membranes were a mixture of the two envelope membranes. Methods to separate the two membranes were developed in the early 1980s (Cline et al. 1981, Block et al. 1983). The outer envelopes have been prepared in a highly purified state from pea chloroplasts, but the inner membranes are still contaminated with outer envelopes (Block et al. 1983, Cline et al. 1981). The SDS-PAGE profile of the inner and outer envelopes revealed that both membranes were contaminated with RuBisCO as well (Keegstra and Yousif 1986). This sometimes has disturbed the analysis of polypeptide composition of the envelopes by SDS-PAGE. Furthermore, due to the low resolution of the conventional SDS-PAGE system, estimation of the amounts of polypeptides in the envelope has not been performed, especially for low molecular mass components. It should be also emphasized that isolation and characterization of outer and inner envelopes from pea have been extensively studied, while those of spinach have not been much investigated. Many proteins are embedded in both membranes, but their functions are still largely unknown. Among them, the phosphate translocator in the inner envelope has been studied most extensively (Douce and Joyard 1990, Flugge and Heldt 1991). Recently, several proteins have been suggested to participate in the translocation of nuclear-encoded polypeptides into the chloroplast (Cline and Henry 1996); two GTP-binding proteins with molecular masses of 34 (OEP34) and 86 kDa (OEP86), two hsp70 homologues, and the 75 kDa protein (OEP75) in outer envelopes, and the 97 (IEP97), 44, and 36 kDa proteins in the inner envelopes (Schnell et al. 1994, Kessler et al. 1994, Hirsch et al. 1994, Wu et al. 1994, Seedorf et al. 1995, Tranel et al. 1995). Although their function is not known, 6.7 (E6.7), 16 (OEP 16), and 37 (E37) kDa proteins are known to be components of envelopes and are cloned (Salomon et al. 1990, Dress-Werringloer et al. 1991, Pohlmeyer et al. 1997). In the present study, a procedure to separate the inner and outer envelopes from spinach chloroplasts has been 526 Polypeptide composition of chloroplast envelopes 527 refined with special attention to getting rid of contamination of RuBisCO. Highly purified outer envelopes were obtained and a new component with a molecular mass of 15 kDa, with minimal disturbance by RuBisCO small subunit, was identified by high-resolution SDS-PAGE and amino acid sequencing of its N-terminus. It was revealed that the new component is a second major constituent of outer envelopes and that the protein is homologous to a recently reported outer envelope protein of 16 kDa (OEP16) from pea (Pohlmeyer et al. 1997). By analysis of distribution pattern of polypeptides among the outer and inner envelope fractions after fractionation of sedimentation sucrose density gradient centrifugation, other polypeptides were allocated to either outer or inner envelope membranes. Determination of constituent proteins—Protein compositions were analyzed by SDS-PAGE according to the method of Laemmli (1970) as modified by Ikeuchi and Inoue (1988). Amounts of proteins separated by the SDS-PAGE were determined by an image analyzer (Biolmage, U.S.A.) after staining the gel with Coomassie brilliant blue (CBB) R-250. N-terminal amino acid sequences of proteins blotted to PVDF membranes were determined by an ABI 473A protein sequencer. When N-termini of the proteins were blocked, the internal sequences were determined by the following procedures; the protein band was cut out from the gel and subjected to lysyl-endopeptidase treatment. The digested fragments were separated by SDS-PAGE and transferred to PVDF membranes; then their sequences were determined. The sequences were analyzed either by the BLAST (Altschul et al. 1990) or TFASTA (Lipman and Pearson 1985) programs in GCG software package installed on DEC3000. Materials and Methods Results and Discussion Preparation of intact chloroplasts—Spinach was purchased from a local farmer. Intact chloroplasts were prepared according to Siegenthaler and Dumont (1990) with some modifications. Leaves were homogenized for 10 s in the grinding medium containing 350 mM sorbitol, 25 mM 3-(/V-morpholino)propanesufonic acid (MOPS)-KOH (pH 7.6), 2 mM EDTA, and 2 mM Na-isoascorbate. The homogenate was filtered through two sheets of double-layered gauze. The crude intact chloroplasts were precipitated (2,000 x g, 4 min) and suspended in the grinding medium. The suspension was layered on grinding medium containing 40% Percoll, and intact chloroplasts were recovered as sediments. The intact chloroplasts thus obtained were washed by grinding medium from which Na-isoascorbate was omitted, resuspended in hypertonic SET buffer (1.3 M sucrose, 2 mM EDTA and 10 mM TricineNaOH (pH7.5)), at a Chi concentration of ca. 3mgml~', in order to tear the inner envelopes from the outer envelopes, and then rapidly frozen in liquid nitrogen. Preparation of envelope membranes—The envelope membranes were prepared by the method of Cline et al. (1981) with some modifications (Yoshio et al. 1995). The intact chloroplasts (25-30 mg Chi) were ruptured by two cycles of freezing and thawing and the volume was adjusted to 21 ml with the hypertonic SET buffer. The broken-chloroplast suspension was then placed on the bottom of a 35-ml centrifugation tube. Eight ml of 1.2 M, 3 ml of 1.1M, and 4 ml of 0.2 M sucrose, each containing 10 mM TricineNaOH (pH7.6), 2 mM EDTA, and 5 mM MgCl2 (TEM buffer) were layered successively onto the suspension. The crude envelope membranes were isolated by floatation centrifugation at 122,000 x g (max) for 15 h at 4°C. After this centrifugation, envelope membranes with yellow color were found in the interface between 0.2 M and 1.1 M sucrose. The envelope fraction was concentrated by Amicon Diaflow membrane PM 10, and the sucrose concentration was adjusted to 0.2 M. The crude envelope membranes were layered on a linear sucrose gradient from 0.3 to 1.1 M sucrose containing TEM buffer and were subjected to centrifugation at 122,000 xg (max) for 15 h at 4°C to separate the outer and inner envelopes. The separated bands were collected by a fraction collector taking 1 ml/fraction from the bottom. Absorbances at 275 and 678 nm of each fraction were monitored as indicators of proteins and Chi a, respectively. Buoyant densities of the fractions were determined by hand refractometers type No. 1 and No. 2 (ATAGO, Japan). Separation of outer and inner envelope membranes— For preparation of chloroplast envelopes, two procedures have been adopted to rupture intact chloroplasts; freezing and thawing (Cline et al. 1981) and mechanical homogenization (Block et al. 1983). In the present study, we used the freezing and thawing method because better separation was obtained (data not shown). When intact chloroplasts were broken by rapid freezing in liquid nitrogen, the yield of envelope membranes after floating centrifugation was better compared with slow freezing at — 20° C, probably because the separation of the outer envelope from inner envelope membrane due to differences in the permeability to the solute is transient, so that the chloroplast volume returns to its original value during the slow freezing (Robinson 1985). As is shown below, the floatation centrifugation is critical for reducing contamination by stromal soluble proteins, such as RuBisCO, and thylakoids in envelope preparations. Fig. 1A shows a separation pattern of membrane fragments after the sucrose linear gradient centrifugation. When monitored at 275 nm, two peaks and a shoulder were separated, centering around fractions 6, 16 and 21, respectively (Fig. 1A, closed circles). The small peak with yellowgreen color around fraction 6 corresponded to the main peak when monitored at 678 nm, which is the absorption maximum of Chi a in thylakoid membranes. The absorption spectrum of this fraction coincided with that of thylakoid membranes (data not shown). The density of this fraction was estimated to be 1.15 g cm" 3 , which agrees well with that of thylakoid membranes (Douce and Joyard 1982). The densities of the yellow fractions that show a peak and a shoulder in the middle of the tube were 1.11 (No. 16) and 1.08 (No. 22) gcm~ 3 , respectively. These values agree with those of inner and outer envelopes prepared from pea, respectively (Cline et al. 1981), but the value 1.11 g cm" 3 is slightly lower than that for inner envelope membranes of 528 Polypeptide composition of chloroplast envelopes spinach reported by Block et al. (1983). The two yellow fractions thus separated were, therefore, tentatively assigned to inner and outer envelope fractions, respectively. In order to confirm the above assignment, polypeptide compositions of these fractions were analyzed by high-resolution SDS-PAGE. In this analysis, a fixed volume of each fraction was subjected to SDS-PAGE in order to directly compare the amount of polypeptides in each fraction (Fig. IB). Fraction 22, tentatively assigned as an outer envelope fraction, was mainly composed of 15, 9.1, 7.8 and 7.6 kDa proteins. The 65 kDa and 16 kDa proteins indicated by arrowheads in Fig. IB were identified as large and small subunits of RuBisCO from their SDS-PAGE profile and N-terminal amino acid sequence, respectively (data not shown). Although RuBisCO was found in fraction 22, it was focuss- ed on fraction 25. It has been reported that significant amounts of RuBisCO were found in outer envelope fractions (Keegstra and Yousif 1986). However, the present study revealed that most of the RuBisCO can be separated from outer envelope fractions by a combination of floatation and sedimentation sucrose gradient centrifugation. The insertion of the 1.2 M sucrose layer between the homogenate and the 1.1 M sucrose solution was found to be critical in preventing diffusion of stromal soluble proteins at the stage of floatation centrifugation (see Materials and Methods). Refinement of the separation procedure resulted in the great reduction of contamination by RuBisCO in the crude envelope membrane fraction and clear separation of the outer envelopes from the enzyme in the following sedimentation sucrose gradient centrifugation. Application of a high-resolution SDS-PAGE system further enabled us to separate the 15 kDa protein from the small subunit of RuBisCO which has an apparent molecular mass of 16 kDa in the present SDS-PAGE system. 1.20 Distribution and allocation of proteins—In order to allocate proteins to either inner or outer envelopes, their distribution patterns were determined by analyzing the densities of stained bands on the SDS-PAGE profile (Fig. 2). The distribution patterns of proteins were grouped 1.00 10 15 20 Bottom B 25 30 35 Top Fraction No. m 32 kDa -100 kDa - 45 kDa - 32 kDa -24 kDa -15 kDa - 9.1 kDa - 7.8 kDa ' 7.6 kDa Fig. 1 Fractionation of chloroplast envelopes by sedimentation sucrose density gradient centrifugation (A) and polypeptide patterns of fractions analyzed by SDS-PAGE (B). Crude envelope membranes obtained by freezing-thawing followed by floatation centrifugation were separated by 0.3-1.1 M sucrose gradient centrifugation. Separated membranes were collected from the bottom by 1 ml, and absorbances at 275 and 678 nm were monitored. For analysis of proteins, an equal volume of each fraction was concentrated and subjected to SDS-PAGE. Numbers in circles above the SDS-PAGE profile represent fraction numbers. The upper and bottom arrowheads show the proteins of large and small subunits of RuBisCO, respectively. 10 15 20 Fraction 25 30 35 No. Fig. 2 Distribution pattern of proteins typical of each type. Amounts of protein in each fraction after SDS-PAGE were analyzed by an image analyzer by calculating the integrated densities of the bands. • ; 32 kDa protein (type I), • ; 9.1 kDa protein (type II), A; 24 kDa protein (type III), • ; 15 kDa protein (type IV). The bottom profile represents a fractionation pattern of envelopes monitored at 275 nm, which was redrawn from Fig. 1A. IM and OM represent inner and outer envelope membranes, respectively. Polypeptide composition of chloroplast envelopes into four categories. The amount of the 32 kDa protein (type I) was the highest in fraction 16, decreased steeply in the lower density fractions (higher fraction number), and was almost negligible in fraction 22, which was tentatively assigned as an outer envelope fraction. The N-terminus of the protein was blocked, while its internal sequence (FISDLF) matched that of phosphate translocator (Fliigge et al. 1989), which is a marker protein for inner envelopes (Douce and Joyard 1990, Flugge and Heldt 1991). Therefore, fraction 16 was identified as an inner envelope fraction. The 45 kDa protein, which was also identified as inner envelope protein E37 (Dress-Werringloer et al. 1991) from its internal sequence (EXRIIEGD), showed the same distribution pattern as the 32 kDa protein. The proteins grouped into type I were the 100, 45, 39.5, and 32 kDa proteins. The 9.1 kDa protein, on the other hand, was focussed in fraction 22 (type II) and scarcely found in inner envelope fractions. The proteins grouped into type II were the 40, 26, 18, and 9.1 kDa proteins. The amount of the 24 kDa protein, whose N-terminus was blocked, was higher in inner envelope fraction, while a significant amount was also found in the outer envelope fraction (type III), although the total amount of the protein was not much compared with other types of proteins. The proteins belonging to type III were the 85, 57, 30, and 24 kDa proteins. Distribution of the 15 kDa protein and 7.8 and 7.6 kDa proteins showed two peaks; one at fraction 16 and another at fraction 22 with higher amounts; these proteins were categorized as type IV. Judging from the fact that inner membranes are normally contaminated with outer envelope membranes (Cline and Keegstra 1983, Cline et al. 1985), the peak which corresponds to inner envelope membrane fraction can be attributed to outer envelope mem- 529 branes which were not fully separated from inner envelope membranes. Thirty amino acid residues of the N-terminus of the 15 kDa protein were determined (Fig. 3A). Homology search of the protein against a DNA database revealed that it is homologous to the 16 kDa protein of outer envelopes (OEP16) from pea reported by Pohlmeyer et al. (1997). The outer envelope protein of pea has a molecular mass of 16 kDa and is assigned as an amino acid transporter protein. The protein had not been identified so far in spinach outer envelopes probably because it was hindered by a significant amount of the RuBisCO small subunit and by conventional SDS-PAGE systems. No other homologous protein was found in EMBL/GenBank/DDBJ database. However, cDNAs with homologous translated sequences were found by searching in expressed sequence tag (EST) database; one from maize, two from rice, and 13 from Arabidopsis thaliana (see Fig. 3A). Two sequences from rice and ten out of 13 sequences from A. thaliana are identical in the first 30 amino acid residues, so that only one from rice and three from A. thaliana are represented in Fig. 3A. Comparing the determined sequence and those registered in the database, the protein seems to have no transit peptide. A massive amount of low-molecular-mass protein (less than 10 kDa) was found in the fraction centered at No. 22, tentatively assigned as an outer envelope fraction. Three proteins with molecular masses of 7.6, 7.8 and 9.1 kDa had identical N-terminal amino acid sequences (Fig. 3B) and were identified as an outer envelope membrane protein registered by Salomon et al. (1990). The protein is abbreviated as E6.7 because of its calculated molecular mass deduced from the cDNA sequence. Judging from the N-terminal sequence that starts from the first Met residue, it is (A) spinach pea maize rice A. thai. A. thai. A. thai. PRSSF AKVDVWDTGNPLLDRTVDGFLKIG M SGSLSSP.L. . .I.M. . .F.NL M. . . G. SGSFRSP. I. . .I.M. . .F.N M. .GG.SGSISSPRI. .AI.M. . .F.N .SGTVSTP.LS.A. .M INL. . .A M.S.T.SGTVSTP.LS.A..M...F.NL...A .T.SGTVSTP.LS.A..M...F.NL...A *1 (B) spinach spinach MESVAKPATTKEGXAKQAAIV S *1 *8 *2 *3 *4 *5 *6 *7 Fig. 3 N-terminal amino acid sequences and their alignments of 15 kDa proteins (A) and 7.6, 7.8 and 9.1 kDa (B) of outer envelopes. The top row represents the N-terminal amino acid sequence determined in the present study. Database accession numbers are *1; present study, *2; Z73553, *3; T25210, *4; D4O577, *5; T21031, *6; Z33853, *7; T45509, *8; M35665. .; identical amino acid to that of the present study. —; deletion. X; unidentified residue. A. thai.; Arabidopsis thaliana. Polypeptide composition of chloroplast'envelopes clear that the protein does not posses a transit sequence and is directly inserted into outer envelopes. The protein (E6.7) was also found in the inner envelope fraction, and the amount varied from season to season (data not shown). The reason why E6.7 with the identical N-terminal amino acid sequences migrated differently in the SDS-PAGE is not clear. Some of the protein molecules might be processed at the C-terminal side, giving rise to different molecular masses, because the C-terminus is thought to protruding to the cytosol (Salomon et al. 1990). Another possibility is that, due to its hydrophobic nature, some of the E6.7 molecules might not unfold completely even in the presence of a high concentration of SDS, providing different mobilities in the SDS-PAGE system. The protein was first identified and characterized as an outer envelope protein by a monospecific antibody (Joyard et al. 1983). However, due to the conventional SDS-PAGE systems which has a low resolution in the low molecular mass range, the presence of the proteins with different molecular masses in outer envelopes had not been elucidated. These proteins were clearly separated by the high-resolution SDS-PAGE system employed in the present study, and there are at least three different molecular mass forms in outer envelope membranes. Most of the proteins of the outer and inner envelopes are categorized either type I or IV. It should be noted, however, that there are also proteins of types II and III, although their amounts are small. In order to explain the distribution pattern of types II and III, we have made the following assumptions. (1) There are contact sites between the outer and inner envelopes (Cline et al. 1985, Pain et al. 1988, Schnell and Blobel 1993, Scott and Theg 1996). (2) Some of the proteins of outer envelopes are not evenly distributed, especially around the contact site. (3) On separation of outer and inner envelopes by freezing and thawing after hypertonic medium treatments, some outer envelope fragments around the contact sites remained attached to inner envelopes. If a portion of the outer envelopes other than contact site is broken easily by freezing and thawing, the outer envelope proteins localized out of the contact site can be found only in the outer envelope fraction, giving rise to type II. Judging from the facts that three bands on the SDS-PAGE have N-terminal amino acid sequences identical to that of E6.7 and that one of which (the 9.1 kDa protein) belongs to type II and the others (7.6 and 7.8 kDa proteins) to type IV (Fig. 2), some conformation or function of the protein could differ depending on the site of localization. In contrast, the proteins located on or close to the contact sites of the outer envelope membranes might show the type III feature. Alternatively, if we assume that the type III proteins are located in both outer and inner envelopes, the distribution pattern is accounted for simply by the total amount of outer and inner envelope membranes without any complicated assumptions, because the fractionation pattern for the 24 kDa (type III) protein was similar to the bottom trace of Fig. 2 monitored at 275 nm. However the presence of type II proteins can not be explained by this assumption. The fractionation pattern of outer and inner envelope membranes has been examined by monitoring the absorbance at 280 nm (Cline et al. 1981, 1985, Andrews et al. 1985). In some cases, distribution of enzyme activities such as Mg2+-ATPase (McCarty et al. 1984) or acyl-CoA synthetase and acyl-CoA thioesterase (Andrews and Keegstra 1983) were traced. However, monitoring of the absorbance at 280 nm determines only the total amount of protein in the fraction and that of enzyme activities traces one particular enzyme. Application of the high-resolution SDS-PAGE system made it possible to trace most of the particular proteins fractionated in each fraction. Analysis of the fractionation pattern also revealed that most of the RuBisCO normally contaminating the outer envelope fraction reported so far could be separated in principle, since the main peak of RuBisCO was focussed on fraction 25, while that of outer envelopes was on fraction 22. Components of the inner and outer envelope membranes—The outer and inner envelope proteins thus assigned are summarized in Fig. 4. The inner envelope membranes contained up to 50 bands, with 5 major components OM 100 kDa-85 kDa -57 kOa 45 kDa .40 kDa 39.5 kDa 32 kDa -30 kDa • 9.1 kDs II • - 7.8 kD£ • 7.6 kD£ Fig. 4 Polypeptide compositions of inner (IM, fraction 16 of Fig. IB) and outer (OM, fraction 22 of Fig. IB) envelope membranes. For details, see text. Polypeptide composition of chloroplast envelopes other than the large and small subunits of RuBisCO indicated by arrowheads. Molecular masses of the main polypeptides were 100, 45, 32, 24, 15, 7.8, and 7.6 kDa. The 7.8 and 7.6 kDa proteins could be separated if a smaller amount of the proteins was applied (for example, see fraction 13 of Fig. IB). Two of them (the 45 and 32 kDa proteins) were identified as E37 (Dreses-Werringloer et al. 1991) and phosphate translocator (Fliigge et al. 1989) from their internal amino acid sequences. Although the N-terminal or internal amino acid sequence of the highest molecular mass component (100 kDa) was not obtained due to blockage of its N-terminus and/or shortage in its amount, it may correspond to the 96 kDa protein of pea (IEP97) (Cline et al. 1981) or El 10 of spinach (Block et al. 1983) of inner envelopes, which was recently assigned as a participant in the polypeptide translocation machinery (Hirsch and Soil 1995). The outer envelopes contain at least 25 stained bands. The densitometric analysis of the gel revealed that the 15 kDa protein constituted 20% and the E6.7 (the 7.6, 7.8 and 9.1 kDa proteins) constituted 40% of the total outer envelope proteins, respectively. This corresponds to 15 and 75% for the 15 kDa protein and E6.7, respectively, on a molecular number basis. Thus, the two proteins were found to be major components of outer envelope membranes suggesting their important function(s) therein. This is compatible with the fact that the protein composition of outer envelopes is rather simple (Cline and Keegstra 1983, Block et al. 1983, Cline et al. 1985, Hirsch and Soil 1995). We could not identify proteins of the polypeptide translocation machinery (OEP86, 75 and 34), which are prominent components of outer envelope membranes of pea chloroplasts (Cline et al. 1985, Keegstra and Yousif 1986, Schnell and Blobel 1993, Hirsch and Soil 1995). In the isolation of pea chloroplast envelopes, leaves are harvested from young seedlings 2-3 weeks old (Cline et al. 1981, 1985, Schnell and Blobel 1993). In these seedlings, chloroplasts are under development, so many of the nuclear-encoded polypeptides still have to be incorporated into chloroplasts through the translocation machinery in order to construct them. Thus, chloroplasts have to prepare a large amount of translocation machinery in the envelope membranes. On the other hand, spinach is normally purchased from a market or local farmer (Block et al. 1983, Siegenthaler and Dumont 1990), as in the case of the present study. These plants are at least 2-3 months old. Chloroplasts isolated from them are fully developed and do not need to translocate much of nuclear-encoded polypeptides. Consequently, envelopes do not need to harbor much translocation machinery. 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