Proc. Natl. Acad. Sci. USA Vol. 86, pp. 1198-1202, February 1989 Biochemistry Identification of an assembly in the small subunit of ribulose-1,5-bisphosphate carboxylase ( import/chimeric / structure/cyanobacteria) CATHERINE C. WASMANN, ROBERT T. RAMAGE*, HANS J. BOHNERT*t, AND JAMES A. OSTREM*t *Department of Biochemistry, and tDepartments of Molecular and Cellular Biology and Sciences, University of Arizona, Tucson, AZ 85721 Communicated by Diter von Wettstein, November 21, 1988

ABSTRACT The mature small subunit (SSU) of ribulose- holoenzyme. In the of higher newly synthe- 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39) in sized LSU (5, 6) and recently imported SSU (7, 8) associate with higher plants contains a highly conserved sequence of 16 amino a subunit binding protein complex. It has been proposed that the acids that is absent in the SSUs of cyanobacteria. To determine chloroplast subunit binding proteins belong to a class of poly- whether this region of the SSU of higher plants has a specific peptides that facilitate the posttranslational assembly of oligo- function, portions of the SSU genes (rbcS) of pea (Pisum meric protein complexes (9). In contrast, there is evidence that sativum) and the cyanobacterium Anacystis nidulans were fused cyanobacterial holoenzyme assembles without the aid of aux- to create chimeric genes that either lacked or contained the iliary proteins. Expression of cyanobacterial Rubisco coding sequence for the 16 conserved amino acids. Precursor in Escherichia coli produces LSU and SSU that assemble to proteins synthesized in vitro from the chimeric genes were form active holoenzyme (10). Both cyanobacterial and higher incubated with isolated pea chloroplasts to assay import and plant SSUs isolated from purified holoenzyme reassemble assembly into the holoenzyme. Fusion proteins lacking the spontaneously in vitro with cyanobacterial LSU octameric 16- sequence were imported and processed but failed cores to form active holoenzyme (11, 12). to assemble with endogenous large subunit. Addition of the Differences in the assembly pathway in cyanobacteria and region from a pea rbcS containing the 16 amino acids to the rbcS higher plants may be reflected in those regions of LSU or of Anacystis enabled the imported SSU fusion protein to SSU that have diverged in sequence composition. Of the two assemble with pea large subunit. This 16-amino acid sequence subunits LSU has retained the greatest degree of similarity is encoded by a separate exon in certain rbcS genes of higher among a wide range of photosynthetic organisms. The amino plants. We propose that the conserved 16-amino acid sequence acid sequence of Anacystis LSU has 81% identity with constitutes a domain acquired to facilitate assembly of the spinach LSU in comparison with -40% identity between eukaryotic holoenzyme. their respective SSU sequences. One obvious difference between cyanobacterial and cyanelle SSUs and the mature Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubis- SSU in higher plants is the addition of a highly conserved co; EC 4.1.1.39) catalyzes carbon dioxide fixation in all sequence of 16 amino acids that starts -50 residues from the The most common form the amino terminus of higher plant SSUs (13, 14). We hypothe- photosynthetic organisms. of sized that this sequence might be required for processes, such enzyme is composed of eight large subunits (LSUs), which as import or assembly, that are specific to the higher plant contain the activation and catalytic sites, and eight small holoenzyme. When we replaced portions of the mature pea subunits (SSUs) whose functions are unknown. In higher SSU with analogous regions from two cyanobacterial plants the holoenzyme consists of an octomeric core of four SSU genes we found that pea/cyanobacterial fusions that pairs of LSUs capped by four SSUs at either end of the lacked the conserved 16-amino acid sequence from pea SSU molecule (1). were imported and processed by isolated pea chloroplasts but Two types of subcellular organization, which may have a failed to assemble with endogenous pea LSU. Addition of bearing on the assembly of the Rubisco holoenzyme, have this sequence to the Anacystis SSU enabled the imported been described for the LSU and SSU genes (rbcL and rbcS, cyanobacterial SSU to assemble into the holoenzyme. These respectively). In the cyanobacterium Anacystis nidulans and results suggest that this sequence is directly involved in the in the cyanobacteria-like organelles (cyanelles) of Cyano- assembly of LSU and SSU in higher plants and provide phora paradoxa, the genes are closely linked and cotran- additional evidence that fundamental differences exist in the scribed (2, 3). In the second type of organization, found in pathway of holoenzyme assembly in higher plants and higher plants and green algae, rbcL and rbcS are located in photosynthetic prokaryotes. different subcellular compartments. rbcL is transcribed and translated to yield a 53-kDa polypeptide in the chloroplast. The nuclear-encoded SSU is synthesized in the cytoplasm as MATERIALS AND METHODS a precursor polypeptide (pSSU) of =20 kDa, which is Construction of Chimeric SSU Genes. Standard techniques imported into the chloroplast. Binding and import of pSSU is for site-directed mutagenesis (15, 16) were used to create mediated by the transit peptide, a sequence that constitutes restriction sites for EcoRI at the conserved Glu-Phe residues the amino-terminal end of the precursor (4). Cyanobacterial- (amino acids 43 and 44) in the rbcS genes of Anacystis type SSUs do not cross an intracellular membrane and lack (pANP1155, ref. 17) and pea (pSS15, ref. 18) and for HindIII the amino-terminal transit peptide. at the conserved Lys-Leu residues (amino acids 59 and 60) in Segregation of rbcS and rbcL genes into different subcel- Anacystis. The deoxyoligonucleotides used (5'-dTTGATC- lular compartments in higher plants may have necessitated GAATTCAACGA and 5'-dGTGGAAGCTTCCCCTGTTT new mechanisms for assembly of LSU and SSU into the Abbreviations: Rubisco, ribulose-1,5-bisphosphate carboxylase/ The publication costs of this article were defrayed in part by page charge oxygenase; SSU, small subunit ofRubisco; pSSU, precursor to SSU; payment. This article must therefore be hereby marked "advertisement" LSU, large subunit of Rubisco. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed.

1198 Downloaded by guest on September 29, 2021 Biochemistry: Wasmann et al. Proc. Natl. Acad. Sci. USA 86 (1989) 1199 for Anacystis and 5'-dTTGGAATTCGAGTTGGA for pea) activity 1100 Ci/mmol, 1 Ci = 37 GBq). Proteins were were prepared by Nancy Istock (Oligonucleotide Synthesis separated on NaDodSO4/polyacrylamide (15%) gels accord- Facility, University of Arizona). Chimeric SSU genes con- ing to Laemmli (24). Gels were prepared for fluorography taining the amino-terminal portion of the pea rbcS were using EN3HANCE (DuPont). Portions of the dried gels prepared as follows. DNA from a pea rbcS modified to containing the labeled SSU precursors were excised and contain a restriction site for EcoRI was digested with EcoRI prepared for liquid scintillation counting as described by and ligated with an EcoRI fragment containing the coding Wasmann et al. (25). The amount of precursor used in each sequence for the carboxyl-terminal portions of a SSU from import reaction was determined from the number of methio- either another higher plant (Mesembryanthemum crystallin- nine residues in each precursor and the specific activity ofthe um pMS5, ref. 19) or from a cyanobacterial-type SSU from radioactively labeled precursor synthesized in individual Anacystis or Cyanophora (pCP029, ref. 14). A synthetic reactions. (Precursor forms pSPPR and pSPPM linker was used to introduce a recognition site for the have six methionines, pSPPC and pSPPA have five methio- restriction endonuclease Sph I at the 5' end of the coding nines, pTPAN has seven methionines, and pSPCYP has eight sequence of the Anacystis rbcS. methionines; mature forms SPPR and SPPM have three A gene for a chimeric cyanobacterial SSU containing the methionines, SPPC and SPPA have two methionines, TPAN conserved 16-amino acid sequence from pea was prepared by has four methionines, and SPCYP has five methionines.) inserting the EcoRI-HindIII fragment from the modified pea In Vitro Transport Experiments. Transport experiments rbcS into the EcoRI and HindIII sites of the modified with and without protease treatment of isolated pea chloro- Anacystis rbcS (Fig. 1). Genes encoding precursor forms of plasts were performed essentially as described by Bartlett et the wild-type (pea-EcoRI, pSPPR) and chimeric SSUs al. (26) and Wasmann et al. (20). Individual import reactions (pealMesembryanthemum, pSPPM; pealAnacystis, pSPPA; contained approximately equal molar quantities of precursor pealCyanophora, pSPPC; transit peptide/Anacystis, pT- and chloroplasts equivalent to 200 ,ug of total chlorophyll in PAN; and cyanobacterial plus (assembly domain), pSPCYP) a total volume of 600 ,ul. The import reactions were stopped were prepared by ligation of a Sph I-BamHI DNA fragment after 30 min by adding 2 ml ofsorbitol/Hepes (4°C) containing containing the coding sequence for a transit peptide of a pea 400 nM nigericin to inhibit any further transport (27). The pSSU from pSP64/SSU (20) with Sph I-BamHI DNA frag- diluted chloroplast suspensions were underlayered with 2 ml ments containing the chimeric SSUs. of 40% (vol/vol) Percoll and pelleted at 3000 x g for 3 min in Manipulations of DNA and gel electrophoresis were per- a Beckman model J-6B centrifuge. Chloroplasts recovered in formed essentially as described by Maniatis et al. (21). The the pellet were suspended in 50 ,l of sorbitol/Hepes/ nucleotide sequences of the fusion junctions located at the nigericin. The chloroplasts were pelleted by brief centrifu- restriction sites for Sph I (not shown), EcoRI, and HindIII gation in an Eppendorf microcentrifuge, and the supernatant (Fig. 1) in the chimeric genes encoding pSPPA, pTPAN, and was removed. pSPCYP were confirmed by dideoxynucleotide sequencing Chloroplasts were lysed by adding 60 ,l of 20 mM (22). Tris-HCl, pH 8.3 at 4°C/50 mM dithiothreitol/100 ,uM ami- Synthesis and Quantitation of Radioactively Labeled SSU. nohexanoic acid/100 ,M benzamidine/100 ,M phenylmeth- Plasmid DNA was purified, linearized, and transcribed with ylsulfonyl fluoride/200 ,M leupeptin. To correct for vari- SP6 polymerase as described by Reiss et al. (23). Uncapped ability due to loss of chloroplasts during the pelleting and were translated in rabbit reticulocyte lysates accord- washing steps two aliquots of the lysed chloroplast suspen- ing to the manufacturer's instructions (Bethesda Research sion were used to determine chlorophyll content according to Laboratories) in the presence of [35S]methionine (specific Pick (28). The chloroplast lysates were fractionated into Assembly CS1 Domain CS2 CS3 *.*. * 0 * * 0* * * *-*V SPPR N0VPPIGK KKFETLSYLP PLTDLLEYLLRtKGVPCEFELEK GFVYRENNSRYTDG YWTVIdKNF GTTDASSVLKELDEWAATPQAFV IGF C ISf IANTPESY *+ -0 00 00- *- -o - - -+ O+- +o +00- 00+ 00o0o *- - 0 - o 0o -00

SPPM PPPIGK KKIIFETLSYLP PLTRDQLLKEVEYLLRKQNPCLEFEPTN GFVYRENGUTPGYYDG YWTNWKLPNF rCTDPS@WAELEEAKAYC F IIit1 ICGFUVOV ISF II ZAYKPASD O +0+ -0 00 00+. @+- * --0 o 00- 00 0- 0 - +- ++ 0- I+ - + 01 00- SPPC N0VWPPIGK KKFETLSYLP PLTDLLKEYLLRKGVPCLEFSFTAED ------VWTLWLPLF GTSEEVLSEI"QCKQOFPNAYI RWAFDSIROTWNF LVYKPL + -0 00 + o0- +--o -0 ,-- 0 * 0 -- 0- * 0 + -0 + 0 I0 SPPA NOVIPPIGK KKFETLSYLP PLThLLKEVEYLLRKGUVPCLEFNENSNPEE ------YWTMPLPLF DCSPQOVLDEYCRSEYGMI ItVAGFDUIKOCVTVSF IYVNUPOY -o *+* oo @0- +- -o 0 - - o -- 0 0 + -+0 -- +- +0-o0- 0 * - + 0 0 + +0 TPAN NHEFPINSNTLPKE RRFETFSYLP PLSDROIAAOIEYNIEQGFNPLIEFNENSNPEE ------TWTPMKL IF DCKSP@VLDEVCRtSEYGCY I RVAGFM IKOWTSF IIVNRPY 0 +0 -0 00 +- *+ o-+ -o - - - 0 -- 0 0 * - +0 -- *- 0.0.- 0 1+ - * 00 * +0

SPCYP NHEFPINSNTLPKE RRFETFSYLP PLSDIAAOIEYNIEOGFNPLIEFELEK GFWYRENNKSPRYYDG R ITVMLKFDCKSPQOVLDEfCRSEYyDI RVAGFONIKaCTQVSF IV - o00 +- -0 00 O-+ -o - - + O+- +O +00- + oo * - + -- *- - o - +0-0 + 00 * +0

FIG. 1. Amino acid sequences ofwild-type and chimeric SSUs. The transit peptide (sequence not shown) is common to all precursors. SPPR, pea SSU encoded by a pea rbcS gene modified to contain an EcoRl site; SPPM, pea/Mesembryanthemum; SPPC, pea/Cyanophora; SPPA, pealAnacystis; TPAN, Anacystis SSU encoded by an Anacystis rbcS modified to contain an Sph I site; SPCYP, Anacystis SSU modified to contain the conserved 16-amino acid sequence found in higher plant SSUs. Construction ofthe precursors is described in text. The first six amino acids of TPAN and SPCYP are encoded by the Sph I linker fused to the amino terminus of the coding sequence of the Anacystis rbcS. Regions of the polypeptides with similar sequence and charge are boxed. The conserved sequences, CS1, CS2, and CS3, in the amino-terminal, central, and carboxyl-terminal regions of the mature SSU polypeptide, respectively, and the conserved 16-amino acid sequence, Assembly Domain, in higher plant SSUs as well as the chimeric SSU, SPCYP, are indicated. Amino acids that are completely conserved in our list of 11 higher plant, 2 green algae, 2 cyanobacterial, and Cyanophora SSU sequences are marked by asterisks (*). Amino acids with positively charged (+), negatively charged (-), and hydroxyl side chains (o) are indicated. Dashes have been inserted in the amino acid sequences of SPPA, SPPC, and TPAN to align conserved domains in cyanobacterial and higher plant SSUs. Locations of the recognition sites of the restriction endonucleases [EcoRI (v) and HindIII (v)] used to construct the fused genes are indicated. Downloaded by guest on September 29, 2021 1200 Biochemistry: Wasmann et al. Proc. Natl. Acad. Sci. USA 86 (1989) soluble and membrane fractions by centrifugation (20,000 x a. a. a. g, 60 min). The supernatants were removed and used to a. a. cL a- prepare samples equivalent to 4 ,ug of chlorophyll from the cn 0c CO, lysed chloroplast suspension for NaDodSO4/PAGE or non- denaturing PAGE. RESULTS Conserved Regions in the Wild-Type and Chimeric SSU Precursors. Three regions of conserved sequences are pres- -mbp ent in SSUs of both higher plants and cyanobacteria, which holo. - we have designated CS1, CS2, and CS3 (Fig. 1). Within CS1 and CS2 7 of the 10 amino acids in each region are identical in all SSU sequences published to date. Although the con- servation of sequence is less striking for CS3, 6 of the amino acids are identical, whereas charge (Arg' -- Lys') and side-chain replacements (Ile -* Val or Leu, Gly -* Ala, ref. 29) bring the number of conserved amino acids to 11 (Fig. 1). FIG. 3. Assembly of pea and chimeric pea/cyanobacterial SSU By fusing a part of the pea rbcS encoding the transit peptide into the holoenzyme. Nomenclature of the pSSUs is given in the and CS1 of pea to DNA sequences encoding the carboxyl- legend for Fig. 1. Labeled polypeptides recovered from protease- terminal portion ofCyanophora orAnacystis SSU we created treated chloroplasts after the 30-min import reaction shown in Fig. 2 genes for chimeric precursor were resolved by nondenaturing PAGE (6% gel, 4 ,.g of chlorophyll proteins (pSPPC and pSPPA, equivalent per lane). A photograph of the fluorogram is shown. The respectively) that encode these three conserved regions but position of Rubisco holoenzyme (holo) is shown at left, and that of lack the for a conserved 16-amino acid se- the subunit binding protein complex (bp) is shown at right. quence present only in higher plant SSUs. A higher plant- type chimera containing CS1 from pea together with CS2 and contrast, SPPC and SPPA, in which the carboxyl-terminal CS3 from M. crystallinum was created to determine whether regions ofcyanobacterial-type SSUs replaced that ofpea, did a fusion that did not fundamentally alter the primary structure not migrate with the pea holoenzyme and were not resolved of higher plant-type SSU was imported and assembled as as distinct polypeptide species under nondenaturing condi- efficiently as wild-type pea pSSU. tions (Fig. 3). These unassembled SSUs were, however, Import and Assembly of Wild-Type Pea pSSU and Chimeric present in the soluble protein fraction and were clearly pSSUs. Isolated chloroplasts were incubated with equal resolved by NaDodSO4/PAGE (SPPC, SPPA, Fig. 2). Thus, amounts of wild-type pea or chimeric pSSUs for 30 min. failure to assemble was not due to rapid degradation of Labeled protein recovered from chloroplasts incubated with unassembled subunits. In some experiments a small amount pSPPC or pSPPA corresponded in size to that predicted for ofthe pea/cyanobacterial SSUs appeared to migrate with the the pea/cyanobacterial fusion proteins minus the pea transit holoenzyme and subunit binding protein complex (Fig. 3). peptide (Fig. 2). Treatment with protease removed only the To test the hypothesis that the additional 16 amino acids in small amount of precursor bound to the outer envelope and the mature portion of higher plant SSUs constitute a domain demonstrated that the labeled polypeptides were inside the required for assembly ofthe holoenzyme these residues were chloroplast envelope. The labeled protein recovered from inserted into a cyanobacterial SSU. The chimeric pSSUs chloroplasts incubated with pSPPR or pSPPM corresponded consisted of the pea transit peptide sequence fused to either in size (14 kDa) to the mature pea SSU. an Anacystis SSU (TPAN, Fig. 1) or an Anacystis SSU Soluble protein recovered after import was analyzed by containing the putative assembly domain from pea SSU nondenaturing PAGE to assay the assembly ofimported SSU (SPCYP, Fig. 1). The efficiency of import of these proteins into the holoenzyme. Approximately 60o of the labeled was determined by incubating equal amounts of the pSSUs protein recovered after import of the higher plant precursors with isolated chloroplasts. pTPAN was imported -90% as (pSPPR, pSPPM) migrated with the holoenzyme (Fig. 3). In efficiently as wild-type pea pSSU (Fig. 4). However, only 7% of the imported protein appeared in the holoenzyme (TPAN, :r 2 c) Fig. 4). In contrast to the pea/cyanobacterial SSUs, unas- a_ a_ a_ CL sembled Anacystis SSU was resolved by nondenaturing n PAGE (TPAN, Fig. 5A). pSPCYP was imported only 30% as

_ + efficiently as the wild-type pea precursor (Fig. 4). However, =40% of imported cyanobacterial SSU containing the puta- tive assembly domain was present in the holoenzyme (SP- CYP, Fig. 4 and Fig. 5A). In the same experiment -67% of the imported wild-type pea SSU (SPPR) assembled into the holoenzyme. In these two cases (SPPR and SPCYP) unas- sembled SSUs were either associated with the binding protein complex (bp, Fig. 5A) or were not well resolved by nondenaturing PAGE. To determine whether differences in assembly of TPAN and SPCYP SSUs were due to differences in the amount of FIG. 2. Import and processing of pSSU by isolated chloroplasts. protein imported into the chloroplasts the import reactions Reticulocyte lysates containing equal amounts oflabeled pSSUs of pea were adjusted to recover approximately equal amounts of (SPPR), pea/Mesembryanthemum (SPPM), pealCyanophora (SPPC), labeled SSU in the soluble protein fraction after the 30-min or pealAnacystis (SPPA) were incubated with chloroplasts in uptake incubation. We found that the quantity of precursor incu- medium (26) for 30 min. Import was stopped by mixing the chloroplast suspensions with cold buffer containing 400 nM nigericin (27). Labeled bated with the isolated chloroplasts had no effect on either polypeptides were resolved by NaDodSO4/PAGE (15% gel, 4 Ag of the percentage of pSSU imported or on the percentage chlorophyll equivalent per lane). A photograph of the fluorogram is assembled into the holoenzyme (data not shown). Approxi- shown. -, Not treated with protease; +, protease-treated samples. The mately 40% of the imported Anacystis SSU containing the position of the mature pea SSU is indicated at left. conserved 16-amino acid sequence migrated with the pea Downloaded by guest on September 29, 2021 Biochemistry: Wasmann et al. Proc. Natl. Acad. Sci. USA 86 (1989) 1201 A B

Relative Import % Assembled

v v SPPR L j 100 67

v SPPA [ 37 16

TPAN 87 7

v v SPCYP 30 39 FIG. 4. Efficiencies of import and assembly of the wild-type pea and chimeric pSSUs. (A) Schematic diagram of the pea and chimeric pea/cyanobacterial SSUs imported into isolated chloroplasts. Portions of the fusion proteins derived from the wild-type pea SSU are denoted by open bars; those parts derived from the Anacystis SSU are denoted by solid bars; the 16-amino acid assembly domain is represented by hatched bars. Locations of the restriction endonucleases [EcoRI (v) and HindII1 (v)] used in constructing the chimeric genes are indicated. (B) Portions of the gels containing radioactively labeled proteins resolved by NaDodSO4/PAGE or nondenaturing PAGE were excised and radioactivity was determined by liquid scintillation counting as described (25). The proportion of the chimeric SSUs imported relative to wild-type pea SSU is shown in the Relative Import column. The proportion of the imported protein associated with Rubisco holoenzyme is indicated in the % Assembled column. holoenzyme, whereas Anacystis SSU without this sequence anobacterial SSUs and pea/cyanobacterial chimeric SSUs did not associate to a significant extent with the holoenzyme that lack the 16-amino acid sequence failed to assemble with (Fig. SB). The results from this experiment clearly demon- pea LSU (Fig. 3, SPPC and SPPA; Fig. 5, TPAN). Addition strated that addition of the conserved domain present in of this sequence to a cyanobacterial SSU enabled imported higher plant SSUs enabled imported cyanobacterial SSU to cyanobacterial SSU to assemble with pea LSU (Fig. 5, participate in assembly of the holoenzyme. SPCYP). We propose that this region of the SSU polypeptide constitutes a domain necessary for assembly of the eukary- DISCUSSION otic holoenzyme. We have examined the function of a conserved 16-amino acid Information from studies of the genomic structure of rbcS sequence in higher plant SSUs by assaying the import and is consistent with this hypothesis. Genes for nuclear-encoded assembly of chimeric proteins in isolated chloroplasts. Cy- SSUs contain intervening sequences at one, two, or three A B highly conserved positions (refs. 30 and 31 and references cited a. a. in ref. 31). In SSU genes with three the amino acid c z c z sequences encoded by 1 and 3 have no homologues in a. U) prokaryotic SSUs or the cyanobacterial-like SSU of Cyano- cno Cl) o cn phora. Wolter et al. (31) proposed that exons 1 and 3 contain domains incorporated into the rbcS gene to fulfill new functions needed after transfer of rbcS from the of an endosym- bp biont to the nuclear genome of the host. Exon 1 codes for the holo transit sequence and the first two amino acids of the mature SSU; acquisition of exon 1 directed transport of the SSU through the chloroplast envelope. Exon 3 coincides precisely with the conserved 16-amino acid sequence in higher plant SSUs. The results presented here demonstrate that this domain facilitates assembly of the imported polypeptide. There is evidence that the primary structure of this domain affects holoenzyme assembly in . The SSU of the green alga Chlamydomonas reinhardtii also contains addi- tional amino acids relative to the cyanobacterial SSU at a FIG. 5. Assembly of pea SSU (SPPR), cyanobacterial SSU position analagous to the higher plant sequence (32). How- (TPAN), and cyanobacterial SSU modified to contain the conserved ever, the algal sequence is 7-10 amino acids longer than that 16-amino acid sequence from pea SSU (SPCYP). (A) Equal amounts of higher plants and has little similarity in sequence. When of precursor based on the number ofradioactive counts (cpm) and the radioactively labeled pSSU from Chlamydomonas is im- methionine content of each precursor were incubated with isolated ported into pea chloroplasts, a partially processed 18-kDa chloroplasts. (B) Amounts of precursor offered to isolated chloro- SSU polypeptide is recovered in the soluble fraction of the plasts were adjusted for differences in import efficiency so that chloroplasts (33). Neither the partially processed nor the roughly equivalent amounts of labeled SSU were recovered in the soluble protein fraction. Labeled polypeptides recovered from intact completely processed Chlamydomonas SSU assembles with chloroplasts reisolated after 30-min import reactions were resolved pea LSU, although both forms assemble with Chlamydo- by nondenaturing PAGE (6% gel, 4 ,ug of chlorophyll equivalent per monas LSU in crude cell extracts. Mishkind et al. (33) lane). A photograph of the fluorograms is shown. The position of the suggest that the inability of the algal SSU to assemble with Rubisco holoenzyme (holo) is indicated at left and that of the subunit pea LSU may be due to evolutionary divergence in the binding protein complex (bp) is indicated at right. mechanisms of holoenzyme assembly. An equally attractive Downloaded by guest on September 29, 2021 1202 Biochemistry: Wasmann et al. Proc. Natl. Acad. Sci. USA 86 (1989) hypothesis is that both organisms share a step in assembly of 1. Chapman, M. S., Suh, S. W., Cascio, D., Smith, W. W. & the holoenzyme that is restricted, in this case, by differences Eisenberg, D. (1987) Nature (London) 329, 354-356. in the amino acid composition of the algal and higher plant 2. Shinozaki, K. & Sugiura, M. (1985) Mol. Gen. Genet. 200, 27- assembly domains. 32. Although higher plants and green algae may share similar 3. Starnes, S. M., Lambert, D. H., Maxwell, E. S., Stevens, import and assembly processes, evidence exists for funda- S. E., Porter, R. D. & Shively, J. M. (1985) FEMS Microbiol. mental differences in the assembly of higher plant Lett. 28, 165-169. and 4. Schmidt, G. W. & Mishkind, M. L. (1986) Annu. Rev. Bio- cyanobacterial holoenzymes. One proposed component of chem. 55, 879-912. the assembly pathway in higher plants, the subunit binding 5. Barraclough, R. & Ellis, R. J. (1980) Biochim. Biophys. Acta protein (9), has not been identified in extracts from cyano- 608, 19-31. (8). The apparent absence of binding protein in 6. Roy, H., Bloom, M., Milos, P. & Monroe, M. (1982) J. Cell cyanobacteria together with the specific ability of cyanobac- Biol. 94, 20-27. terial LSUs to combine in vitro with purified SSUs to form 7. Gatenby, A. A., Lubben, T. H., Alquist, P. & Keegstra, K. active holoenzyme (11) indicate that an assembly step me- (1988) EMBO J. 7, 1307-1314. diated by a subunit binding protein complex may not be 8. Ellis, R. J. & van der Vies, S. M. (1988) Photosyn. Res. 16, necessary in these organisms. The assembly process is 101-115. apparently 9. Hemmingsen, S. M., Woolford, C., van der Vies, S. M., Tilly, more complex in eukaryotes. Although higher K., Dennis, D. T., Georgopoulos, C. P., Hendrix, R. W. & plant SSUs assemble with cyanobacterial LSU (12, 34), Ellis, R. J. (1988) Nature (London) 333, 330-334. cyanobacterial SSU did not assemble to a significant extent 10. Gatenby, A. A., van der Vies, S. M. & Bradley, D. (1985) with higher plant LSU after import into chloroplasts (Figs. 4 Nature (London) 314, 617-620. and 5, TPAN). 11. Andrews, T. J. & Ballment, B. (1983) J. Biol. Chem. 258, 7514- We interpret our results to indicate that assembly in higher 7518. plants involves an obligatory step in which the cyanobacterial 12. Andrews, T. J. & Lorimer, G. H. (1985) J. Biol. Chem. 260, SSU is unable to participate. Addition of the region encom- 4632-4636. 13. Mazur, B. J. & Chui, C.-F. (1985) Nucleic Acids Res. 13, 2373- passing the conserved 16-amino acid sequence from pea to 2386. the cyanobacterial SSU enabled the imported SSU to assem- 14. Wasmann, C. C. (1985) Ph.D. thesis (Michigan State Univ., ble (Figs. 4 and 5, SPCYP), strongly implying that this East Lansing). sequence plays a specific and essential role in the assembly 15. Kunkel, T. A. (1985) Proc. Natl. Acad. Sci. USA 82, 488-492. process in higher plants. The possibility that the assembly 16. Nisbet, I. T. & Beilharz, M. W. (1985) Gene Anal. Tech. 2, 23- domain functions principally as a spacer for correct alignment 29. of other regions of the SSU polypeptide cannot be excluded. 17. Shinozaki, K. & Sugiura, M. (1983) Nucleic Acids Res. 11, However, 6957-6964. the high degree of conservation of sequence in this 18. Coruzzi, G., Broglie, R., Cashmore, A. & Chua, N.-H. (1982) portion ofthe SSU polypeptide (94% identity between Lemna J. Biol. Chem. 258, 1399-1402. and tomato) suggests that this possibility is unlikely. 19. DeRocher, E. J., Ramage, R. T., Michalowski, C. B. & Results from the import experiments support previous Bohnert, H. J. (1987) Nucleic Acids Res. 15, 6301. work showing that the "passenger" protein fused to the SSU 20. Wasmann, C. C., Reiss, B., Bartlett, S. G. & Bohnert, H. J. transit peptide affects the efficiency of import into chloro- (1986) Mol. Gen. Genet. 205, 446-453. plasts (7, 20, 23). The results were unexpected in that import 21. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular was significantly reduced Cloning:A Laboratory Manual (Cold Spring Harbor Lab., Cold by heterologous fusions of the Spring Harbor, NY). mature portion of a higher plant SSU and a cyanobacterial 22. Sanger, F., Nicklen, S. & Coulsen, A. R. (1977) Proc. Natl. SSU (Fig. 4, SPPA and SPCYP). The import efficiency of the Acad. Sci. USA 74, 5463-5467. pea, pea/Mesembryanthemum, and the Anacystis SSU fused 23. Reiss, B., Wasmann, C. C. & Bohnert, H. J. (1987) Mol. Gen. to the transit peptide of pea were comparable (Fig. 4, SPPR Genet. 209, 116-121. and TPAN; SPPM, data not shown). Apparently alterations 24. Laemmli, U. K. (1970) Nature (London) 227, 680-685. that affect the structure or interaction of domains within the 25. Wasmann, C. C., Reiss, B. & Bohnert, H. J. (1988) J. Biol. mature portion of the SSU affect binding to or import across Chem. 263, 617-619. the chloroplast envelope. The near wild-type import of a 26. Bartlett, S. G., Grossman, A. R. & Chua, N.-H. (1982) in chimeric protein consisting ofAnacystis SSU fused to Methods in Chloroplast Molecular Biology, eds. Edelmann, the pea M., Hallick, R. B. & Chua, N.-H. (Elsevier, Amsterdam), pp. transit peptide confirmed that the SSU assembly domain is 1081-1091. not necessary for efficient import into the chloroplast. 27. Cline, K., Werner-Washburne, M., Lubben, T. H. & Keegstra, Recent crystallographic data on the structure of the higher K. (1985) J. Biol. Chem. 260, 3691-3696. plant form of the holoenzyme shows that, once assembled, 28. Pick, U. (1982) in Methods in Chloroplast Molecular Biology, residues in the assembly domain are in contact with barrel, eds. Edelmann, M., Hallick, R. B. & Chua, N.-H. (Elsevier, carboxyl-terminal, and amino-terminal domains of neighbor- Amsterdam), pp. 873-880. ing LSU polypeptides (35). It appears that contacts between 29. Dayhoff, M. 0. (1978) Atlas of Protein Sequence and Struc- the SSU and LSU or, alternatively, between SSU and other ture. (Natl. Biomed. Res. Found., Washington, DC), Vol. 5, proteins in the stroma (e.g., the subunit Suppl. 3. binding protein 30. Sugita, M., Manzara, T., Pichersky, E., Cashmore, A. & complex) are crucial for efficient assembly of the holoenzyme Gruissem, W. (1987) Mol. Gen. Genet. 209, 247-256. in higher plants. 31. Wolter, F. P., Fritz, C. C., Willmitzer, L. & Schreier, P. H. (1988) Proc. Natl. Acad. Sci. USA 85, 846-850. We thank Drs. Frank P. Wolter and Michael S. Chapman for 32. Goldschmidt-Clermont, M. & Rahire, M. (1986) J. Mol. Biol. making results available before their publication and Drs. Elizabeth 191, 421-432. Vierling, Richard Hallick, Karen Oishi, and C. William Birky for 33. Mishkind, M. L., Wessler, S. R. & Schmidt, G. W. (1985) J. critically reading the manuscript. This work was supported by Cell Biol. 100, 226-234. National Science Foundation Grant PCM 83-18166 to H.J.B. and in 34. van der Vies, S. M., Bradley, D. & Gatenby, A. A. (1986) part by U.S. Department of Agriculture Grant CGRP 87-2-2748 to EMBO J. 5, 2439-2444. H.J.B., Arizona Agriculture Experimental Station Grant ARZT 35. Chapman, M. S., Suh, S. W., Curmi, P. M. G., Cascio, D., 174452 to H.J.B., and a Sigma Xi Grant-in-Aid of Research to R.T.R. Smith, W. W. & Eisenberg, D. S. (1988) Science 241, 71-74. 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