The Plant Journal (2014) 80, 862–869
doi: 10.1111/tpj.12686
A protein with an inactive pterin-4a-carbinolamine dehydratase domain is required for Rubisco biogenesis in plants Leila Feiz1, Rosalind Williams-Carrier2, Susan Belcher2, Monica Montano1,†, Alice Barkan2 and David B. Stern1,* Boyce Thompson Institute for Plant Research, Cornell University, Tower Road, Ithaca, NY 14853, USA, and 2 Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA
1
Received 29 July 2014; revised 15 September 2014; accepted 17 September 2014; published online 3 October 2014. *For correspondence (e-mail [email protected]). † Current address: University of Wisconsin-Madison, Department of Surgery, 600 Highland Ave., Madison, WI 53792
SUMMARY Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a critical role in sustaining life by catalysis of carbon fixation in the Calvin–Benson pathway. Incomplete knowledge of the assembly pathway of chloroplast Rubisco has hampered efforts to fully delineate the enzyme’s properties, or seek improved catalytic characteristics via directed evolution. Here we report that a Mu transposon insertion in the Zea mays (maize) gene encoding a chloroplast dimerization co-factor of hepatocyte nuclear factor 1 (DCoH)/pterin4a-carbinolamine dehydratases (PCD)-like protein is the causative mutation in a seedling-lethal, Rubiscodeficient mutant named Rubisco accumulation factor 2 (raf2-1). In raf2 mutants newly synthesized Rubisco large subunit accumulates in a high-molecular weight complex, the formation of which requires a specific chaperonin 60-kDa isoform. Analogous observations had been made previously with maize mutants lacking the Rubisco biogenesis proteins RAF1 and BSD2. Chemical cross-linking of maize leaves followed by immunoprecipitation with antibodies to RAF2, RAF1 or BSD2 demonstrated co-immunoprecipitation of each with Rubisco small subunit, and to a lesser extent, co-immunoprecipitation with Rubisco large subunit. We propose that RAF2, RAF1 and BSD2 form transient complexes with the Rubisco small subunit, which in turn assembles with the large subunit as it is released from chaperonins. Keywords: carbon fixation, C4, Calvin cycle, Zea mays, photosynthesis, chaperone, protein folding.
INTRODUCTION All life depends on ribulose-1,5-bisphosphate carboxylase (Rubisco), which incorporates atmospheric CO2 through the carboxylation of the substrate ribulose-1,5-bisphosphate, the rate-limiting step of the Calvin–Benson pathway. In parallel with this main reaction, Rubisco catalyzes a competitive oxygenation side reaction, which is wasteful in terms of the net fixation of carbon dioxide. The steady increase in the atmospheric proportion of oxygen over millenia has increased the proportion of oxygenation to carboxylation reactions, leading to Rubisco being quite inefficient and slow at fixing carbon in the current environment (Whitney et al., 2011). To obtain the carbon required for growth and development, C3 plants allocate almost 30% of their nitrogen and more than 50% of their leaf proteins to Rubisco, making this enzyme a major sink for nitrogen and other resources required for optimal plant productivity. These effects are mitigated in C4 plants, where Rubisco is localized to bundle sheath chloroplasts in a concentrated CO2 environment. These factors have long made Rubisco a target for 862
engineering, either to improve its catalytic properties or to create a ‘C4-like environment’ for Rubisco in C3 plants (Evans, 2013). Although the screening of Rubisco from different species led to the identification of enzymes with higher specificity for CO2 and higher catalytic rates, incompatibility of host chaperones with heterologous Rubisco subunits prevented the assembly of novel Rubisco forms in plants or in Escherichia coli, hindering efforts to achieve a better enzyme via genetic selection or mutagenesis approaches (Parry et al., 2013). Form-I Rubisco, which exists in cyanobacteria, algae and plants, is a hexadecamer composed of eight large (50 kDa) and eight small (13–15 kDa) subunits, denoted here as LS and SS, respectively. The assembly pathway of cyanobacterial Rubisco has been delineated using bacterial and cellfree systems, assisted by the E. coli chaperonins GroEL/ GroES and the Rubisco-specific chaperone RBCX (Saschenbrecker et al., 2007; Liu et al., 2010; Bracher et al., 2011). In these systems, the chaperonin cage composed of GroEL and GroES binds unfolded LS in a complex of © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd
A new Rubisco biogenesis protein 863 RESULTS Identification of the causal mutation in raf2-1 The raf2 mutant was initially identified during large-scale phenotyping of approximately 2000 mutants in the maize Photosynthetic Mutant Library (PML; Stern et al., 2004) as a pale-green mutant (Figure 1a), with a reduced content of Rubisco and normal levels of representative subunits of PSI (PsaD), the cytochrome b6f complex (PetA), PSII (PsbA) and the ATP synthase (AtpB) (Figure 1b). This protein profile is similar to that of the bsd2, raf1 and cps2 mutants, which are deficient for established Rubisco biogenesis factors (Barkan, 1993; Feiz et al., 2012). raf2 seedlings survive on seed reserves for several weeks (Figure 1a), but the mutation is ultimately lethal. Their pale-green phenotype is more subtle than that of bsd2 and raf1 mutants, and they survive longer on soil, correlating with the fact that they accumulate approximately 10% of the wild-type (WT) level of LS, as compared with approximately 1% in the other mutants. RAF1 accumulates to normal levels in raf2
(a)
WT (%)
(b)
100 10
1
raf2
approximately 800 kDa, which then releases LS for further folding and assembly mediated by RBCX. In this step, RBCX dimers stabilize an octameric core of LS, LS8-(RBCX2)8, prior to the sequential replacement of RBCX with SS monomers, creating the holoenzyme. In algae and plants, nucleus-encoded SS and plastidencoded LS assemble to form the holoenzyme, following the import of cytosolically synthesized SS into the plastid. How this assembly occurs is largely unknown, however, mainly because LS is insoluble in both bacterial hosts and in cell-free systems. The role of the chloroplast Cpn60Cpn21/10 chaperonin complex, a protein cage homologous to E. coli GroEL/GroES, in Rubisco biogenesis was shown by the co-purification of Cpn60 with Rubisco (Barraclough and Ellis, 1980; Feiz et al., 2012), and by the characterization of Rubisco-deficient mutants of Oryza sativa (rice; Kim et al., 2013) and Zea mays (maize; Barkan, 1993; Feiz et al., 2012) with lesions in genes encoding an alpha isoform of Cpn60 (denoted CPS2 in maize). Whereas direct evidence for a role for RBCX in plant Rubisco assembly is lacking, maize mutants lacking either Bundle Sheath Defective 2 (BSD2), which harbors a zincfinger domain related to that in the chaperone DnaJ (Brutnell et al., 1999), or Rubisco Accumulation Factor 1 (RAF1), which lacks domains of known function, cannot assemble LS into holoenzyme. Instead, nascent LS accumulates in a CPS2-containing complex of approximately 800 kDa. This observation led to speculation that BSD2 and RAF1 in chloroplasts serve the role of cyanobacterial RBCX, by releasing LS from the chaperonin complex and ultimately mediating its assembly into holoenzyme (Feiz et al., 2012). To investigate the post-chaperonin stage(s) at which BSD2 and RAF1 play roles, we co-expressed these proteins together with chloroplast chaperonin and maize Rubisco in E. coli, or added them to an in vitro system containing recombinant LS and chaperonins. Our lack of success in solubilizing or assembling Rubisco in these systems led us to believe that additional and perhaps unknown factors might be required. Here we describe the genetic identification of the maize gene Rubisco accumulation factor 2 (raf2) that is required for Rubisco biogenesis. RAF2 is conserved among photosynthetic eukaryotes and is related to a mitochondrial protein: dimerization co-factor of hepatocyte nuclear factor 1 (DCoH)/pterin-4a-carbinolamine dehydratases (PCD)-like protein. Orthologs of raf2 are also found in photosynthetic bacteria harboring a-carboxysomes, a carbon-concentrating structure that is functionally and structurally associated with Rubisco (Rae et al., 2013). RAF2 was shown previously to lack PCD activity and to have acquired a chloroplast-specific domain of unknown function. It has therefore been speculated that RAF2 has evolved a new function (Naponelli et al., 2008), which our study now reveals to be a Rubisco assembly chaperone.
RbcL PsaD AtpB PetA PsbA RAF1 RbcL
WT Zm-raf2-1
(c)
CBB
Zm-raf2-1(+217)
Figure 1. Overview of the raf2 mutant phenotype. (a) Wild-type (WT) and homozygous raf2-1 plants at the seedling stage. (b) Immunoblot analyses of Rubisco large subunit and representative subunits of other photosynthetic complexes. Total proteins from equal surface area of the seedling leaf tip or dilutions (as indicated) were analyzed by probing with antibodies raised against the proteins indicated on the left; CBB, Coomassie blue staining. (c) The raf2 locus corresponds to GRMZM2G139123, an intron-less locus on chromosome 1. The location of the Mu insertion relative to the translation initiation codon is shown. The 9-bp target duplication is underlined.
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 862–869
864 Leila Feiz et al.
WT (%) 100 10 1 LS
RAF2
(b)
cps2
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The effect of RAF2 on the accumulation of rbcL and rbcs transcripts was investigated by gel blot analysis of total seedling leaf RNA from WT and raf2 mutants (Figure 3a). Known Rubisco-biogenesis mutants cps2, raf1 and bsd2 were analyzed in parallel. Neither the primary nor the 50 -processed rbcL transcripts were affected qualitatively or quantitatively in raf2 mutants, although rbcL mRNA exhibited increased abundance in raf1 mutants, as noted previously (Feiz et al., 2012). rbcs transcripts also appeared to be unaltered in raf2 mutants, indicating that RAF2 promotes Rubisco biogenesis at a post-transcriptional level.
bsd2
The raf2 locus, GRMZM2G139123, encodes a member of the PCD family. Some PCD family members are involved in the recycling of the multi-function co-factor tetrahydrobiopterin (Thony et al., 2000), and others are involved in transcription factor dimerization (Rose et al., 2004). Plants typically encode two PCD proteins. One localizes to mitochondria, is enzymatically active and has been proposed to be involved in molybdenum co-factor metabolism (Naponelli et al., 2008). The other, represented by RAF2 and its Arabidopsis ortholog (At5g51110), localizes to chloroplasts, as shown by a proteomic analysis (Friso et al., 2010) and GFP fusion data (Naponelli et al., 2008). At-RAF2 and maize RAF2 lacked catalytic activity when expressed in E. coli (Naponelli et al., 2008), but their functions were not previously tested through mutant analysis. Both proteome (Friso et al., 2010) and transcriptome (Li et al., 2010) data suggest approximately 2.5-fold higher accumulation of RAF2 protein and RNA in maize bundle sheath versus mesophyll chloroplasts; this is consistent with a role in Rubisco assembly, as Rubisco is restricted to bundle sheath chloroplast in maize, a C4 plant. RAF2 orthologus lack catalytic signatures, but also harbor a motif that distinguishes them from their mitochondrial and bacterial relatives (Figure S2). It is possible that this motif is relevant to the Rubisco-related function of RAF2. An antibody raised against recombinant RAF2 detected a protein of approximately 18 kDa in total leaf extracts from
Rubisco subunit genes are normally transcribed and translated in raf2, but a majority of LS is trapped in a chaperonin-containing complex
raf1
The raf2 gene product
plants carrying the WT Raf2 allele, consistent with the size of RAF2 lacking the predicted chloroplast transit peptide (18.5 kDa). This protein was absent in raf2-1 mutants, thereby confirming it to be RAF2 (Figure 2a). Like RAF1 (Figure 1b), BSD2 accumulated to the WT level in raf2-1 mutants, indicating that there were no pleiotropic effects on the abundances of known Rubisco chaperones. When recombinant RAF2 was analyzed by blue native gel (BNG) electrophoresis, it appeared to migrate as dimers and tetramers (Figure 2b). Animal PCD proteins are known to dimerize (Hevel et al., 2008), a bacterial homolog of RAF2 crystallizes as a dimer (Wheatley et al., 2014) and a brief report stated that Arabidopsis RAF2 forms dimers (Valkai, 2004), consistent with our observation for maize RAF2.
raf2
mutants (Figure 1b), indicating that raf2 does not mediate its effects by affecting the expression of raf1. Genetic crosses between raf2-1 and the WT had verified that the pale-green phenotype and low Rubisco levels cosegregated as a single, recessive Mendelian trait. The raf21 mutant arose in a maize line harboring active Mu transposons, and a deep sequencing strategy described previously by Williams-Carrier et al. (2010) was used to identify Mu insertions that co-segregated with the raf2 locus. Among the few candidate loci identified in this manner, GRMZM2G139123 was the only one predicted to encode a chloroplast-localized protein. The Mu insertion in this locus was located in the middle of its single exon, and is predicted to be a severe, likely null, allele (Figure 1c). Gene-specific PCR of additional raf2-1 mutant individuals and several phenotypically WT cousins confirmed that this insertion was tightly linked to raf2 (Figure S1). In addition, the bacterial homolog of raf2 is encoded in the same operon as Rubisco subunits, and was recently shown to increase the accumulation of bacterial Rubisco when co-expressed with Rubisco subunits and GroELS (Wheatley et al., 2014). Taken together, these findings provide strong support for the conclusion that RAF2 is required for Rubisco accumulation in maize.
720 480 242
RAF2
146
RAF1 3
66
RAF2 4
BSD2 20
RAF2 2
Figure 2. Characterization of endogenous and recombinant RAF2 protein. (a) Immunoblot analysis of soluble proteins extracted from equal surface areas of wild-type (WT), raf2, raf1, bsd2 and cps2 leaves. Proteins detected are shown to the left of each panel. (b) Soluble recombinant RAF1 and RAF2 proteins were analyzed in a 4–16% Blue Native Gel, followed by Coomassie staining. RAF13 marks the migration of RAF1 trimers, and RAF22 and RAF24 mark the positions of presumed RAF2 dimers and tetramers.
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 862–869
866 Leila Feiz et al. control. Immunopurifications with RAF2 antibody employed either cross-linked or non-cross-linked material, whereas the other three antibodies were used in immunopurification reactions only from cross-linked material. Eluted proteins were analyzed by immunoblotting after reversing the cross links (Figure 5). Column 1 shows the levels of LS, SS, RAF2, BSD2, RAF1 and an unrelated protein, chloroplast malic enzyme (ME), in the input material. Non-specific binding to beads was minimal, as gauged by immunopurification with anti-RNC2 (column 3). The a-RAF2 beads co-purified substantial quantities of both LS and SS from uncross-linked lysates (column 2), and these signals were augmented with prior cross-linking (column 4), suggesting that the interactions were labile. Furthermore, BSD2 co-purified with RAF2 only from cross-linked lysates (column 4), suggestive of a transient or weak interaction. The reciprocal interaction was not reproducibly observed, however, making the conclusion ambiguous (column 6). Evidence for a RAF2–RAF1 interaction was similarly inconclusive. The a-RAF1 (column 5) and a-BSD2 (column 6) beads likewise bound both LS and SS proteins above the background level; we had previously reported the RAF1–LS interaction (Feiz et al., 2012). BSD2 and RAF1 also showed a clear interaction in these reciprocal experiments.
1 α-RAF2
2 +
3
4 +
5
+
α-RNC2
+
α-RAF1
+
α-BSD2
+
WT lysate WT X-linked lysate
6
+
+
+
+
+
LS SS RAF2 RAF1
BSD2 ME Figure 5. Interaction between Rubisco subunits and chaperones. The indicated antibodies were bound to protein A beads and used for immunoprecipitation from total soluble proteins extracted from cross-linked (X-linked) or non-cross-linked wild-type (WT) leaves. The eluates were separated in a 13% SDS-polyacrylamide gel and analyzed by immunoblotting for the proteins indicated on the left. RNC2 is a nucleolar protein used as a negative control. Lane 1 was loaded with 0.4% of the input, and eluates were diluted 25-fold for self-detection (e.g. detection of RAF2 protein with immunoprecipitation by a-RAF2).
These results lead to two main conclusions. First, RAF2 engages in interactions with LS and SS that resemble those observed for the known Rubisco chaperones RAF1 and BSD2. These results, in conjunction with the sequestration of nascent LS in the ‘LSc’ complex in raf2 mutants (Figure 4), imply that RAF2 likewise functions as a chaperone during Rubisco assembly. The second and striking result is that in each case the SS signal was several times stronger than the LS signal, in comparison with the signal in the input (column 1). This suggests that the chaperones have a stronger affinity for SS than LS, and/or that they interact more frequently with SS than with LS. Various models that account for these results are discussed below. DISCUSSION In this study we have described RAF2, a member of the PCD family, as a protein required for wild-type levels of Rubisco accumulation in maize. RAF2 joins RAF1 and BSD2 as Rubisco chaperones that are broadly represented in and restricted to the green lineage, yet have been placed in the Rubisco assembly pathway through genetic analyses in maize. In a recent structural analysis of a bacterial RAF2 homolog encoded in the a-carboxysome operon, co-expression with Rubisco and GroEL/GroES in E. coli increased the concentration of soluble, assembled Rubisco (Wheatley et al., 2014), attesting to an evolutionarily conserved role for this protein. Our data show that neither transcription nor translation of LS is reduced in raf2 mutants, yet Rubisco abundance is decreased dramatically. To investigate the fate of LS in this mutant, we used native gel electrophoresis of newly synthesized proteins (Figure 4). The data suggest that similar to raf1 and bsd2 (Feiz et al., 2012), newly synthesized LS in raf2 is trapped in the approximately 800-kDa LSc complex, the formation of which is dependent on CPS2, a chaperonin a-isoform. This implies that similar to RAF1 and BSD2, RAF2 is required for LS to exit the chaperonin cage successfully, a process that is facilitated by RBCX in at least some cyanobacteria (Saschenbrecker et al., 2007). During the in vitro assembly of cyanobacterial Rubisco, transient intermediates anchored by RBCX dimmers, including RBCX2(2)-LS2, RBCX2(4)-LS4 and RBCX2(8)-LS8, undergo sequential replacement of RBCX2 by SS monomers (Liu et al., 2010; Bracher et al., 2011). We considered the possibility that RAF1, RAF2 and/or BSD2 might mimic RbcX function in chloroplasts, but we were unable to obtain direct evidence of the composition and order of formation of these putative intermediates. As a first step, we attempted to stabilize transient interactions by in planta cross-linking prior to immunoprecipitation of each chaperone and any associated partners (Figure 5). Although these experiments validated the interaction of RAF1 and BSD2, as well as binding to LS by each chaperone, a surprising result was that not only RAF2, but also RAF1 and BSD2
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 862–869
866 Leila Feiz et al. control. Immunopurifications with RAF2 antibody employed either cross-linked or non-cross-linked material, whereas the other three antibodies were used in immunopurification reactions only from cross-linked material. Eluted proteins were analyzed by immunoblotting after reversing the cross links (Figure 5). Column 1 shows the levels of LS, SS, RAF2, BSD2, RAF1 and an unrelated protein, chloroplast malic enzyme (ME), in the input material. Non-specific binding to beads was minimal, as gauged by immunopurification with anti-RNC2 (column 3). The a-RAF2 beads co-purified substantial quantities of both LS and SS from uncross-linked lysates (column 2), and these signals were augmented with prior cross-linking (column 4), suggesting that the interactions were labile. Furthermore, BSD2 co-purified with RAF2 only from cross-linked lysates (column 4), suggestive of a transient or weak interaction. The reciprocal interaction was not reproducibly observed, however, making the conclusion ambiguous (column 6). Evidence for a RAF2–RAF1 interaction was similarly inconclusive. The a-RAF1 (column 5) and a-BSD2 (column 6) beads likewise bound both LS and SS proteins above the background level; we had previously reported the RAF1–LS interaction (Feiz et al., 2012). BSD2 and RAF1 also showed a clear interaction in these reciprocal experiments.
1 α-RAF2
2 +
3
4 +
5
+
α-RNC2
+
α-RAF1
+
α-BSD2
+
WT lysate WT X-linked lysate
6
+
+
+
+
+
LS SS RAF2 RAF1
BSD2 ME Figure 5. Interaction between Rubisco subunits and chaperones. The indicated antibodies were bound to protein A beads and used for immunoprecipitation from total soluble proteins extracted from cross-linked (X-linked) or non-cross-linked wild-type (WT) leaves. The eluates were separated in a 13% SDS-polyacrylamide gel and analyzed by immunoblotting for the proteins indicated on the left. RNC2 is a nucleolar protein used as a negative control. Lane 1 was loaded with 0.4% of the input, and eluates were diluted 25-fold for self-detection (e.g. detection of RAF2 protein with immunoprecipitation by a-RAF2).
These results lead to two main conclusions. First, RAF2 engages in interactions with LS and SS that resemble those observed for the known Rubisco chaperones RAF1 and BSD2. These results, in conjunction with the sequestration of nascent LS in the ‘LSc’ complex in raf2 mutants (Figure 4), imply that RAF2 likewise functions as a chaperone during Rubisco assembly. The second and striking result is that in each case the SS signal was several times stronger than the LS signal, in comparison with the signal in the input (column 1). This suggests that the chaperones have a stronger affinity for SS than LS, and/or that they interact more frequently with SS than with LS. Various models that account for these results are discussed below. DISCUSSION In this study we have described RAF2, a member of the PCD family, as a protein required for wild-type levels of Rubisco accumulation in maize. RAF2 joins RAF1 and BSD2 as Rubisco chaperones that are broadly represented in and restricted to the green lineage, yet have been placed in the Rubisco assembly pathway through genetic analyses in maize. In a recent structural analysis of a bacterial RAF2 homolog encoded in the a-carboxysome operon, co-expression with Rubisco and GroEL/GroES in E. coli increased the concentration of soluble, assembled Rubisco (Wheatley et al., 2014), attesting to an evolutionarily conserved role for this protein. Our data show that neither transcription nor translation of LS is reduced in raf2 mutants, yet Rubisco abundance is decreased dramatically. To investigate the fate of LS in this mutant, we used native gel electrophoresis of newly synthesized proteins (Figure 4). The data suggest that similar to raf1 and bsd2 (Feiz et al., 2012), newly synthesized LS in raf2 is trapped in the approximately 800-kDa LSc complex, the formation of which is dependent on CPS2, a chaperonin a-isoform. This implies that similar to RAF1 and BSD2, RAF2 is required for LS to exit the chaperonin cage successfully, a process that is facilitated by RBCX in at least some cyanobacteria (Saschenbrecker et al., 2007). During the in vitro assembly of cyanobacterial Rubisco, transient intermediates anchored by RBCX dimmers, including RBCX2(2)-LS2, RBCX2(4)-LS4 and RBCX2(8)-LS8, undergo sequential replacement of RBCX2 by SS monomers (Liu et al., 2010; Bracher et al., 2011). We considered the possibility that RAF1, RAF2 and/or BSD2 might mimic RbcX function in chloroplasts, but we were unable to obtain direct evidence of the composition and order of formation of these putative intermediates. As a first step, we attempted to stabilize transient interactions by in planta cross-linking prior to immunoprecipitation of each chaperone and any associated partners (Figure 5). Although these experiments validated the interaction of RAF1 and BSD2, as well as binding to LS by each chaperone, a surprising result was that not only RAF2, but also RAF1 and BSD2
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 862–869
A new Rubisco biogenesis protein 867 Cpn21/10
Chaperoninbound LS RAF13
LS
BSD2X
Native LS
RAF22
Folded SS
Chloroplast
tp-SS
Cytosol
L8S8 Rubisco
Figure 6. Model for roles of RAF2, RAF1 and BSD2 in Rubisco assembly. From top left: newly synthesized large subunit (LS) interacts with the chaperonin complex, which leads to correct folding (Native LS). RAF2, RAF1 and BSD2 would bind small subunit (SS) to form dynamic intermediates that then act to capture the folded LS from the chaperonin complex. In the absence of RAF2, RAF1 or BSD2, LS would be unable to escape from the chaperonin cycle, ultimately leading to aggregation and proteolysis. ‘Chaperonin-bound LS’ is equivalent to LSc (Figure 4).
co-immunopurified strongly with SS. This observation raises the possibility that unlike the cyanobacterial mechanism, in which SS folds independently and inserts into the octameric core of LS at the end of the pathway, the import, folding and assembly of SS may play central role(s) in the formation of chloroplast holoenzyme. Such a scenario would postulate a very different set of assembly intermediates. A model that is consistent with our co-immunopurification data is presented in Figure 6. The notion that the binding of chaperones to SS comprises an early step in Rubisco assembly has not received recent attention, but was raised when endogenous LS synthesized in a chloroplast extract could only
form Rubisco when purified SS was added to the system (Hubbs and Roy, 1992). Consequently, it was suggested that the assembly of Rubisco large and small subunits in plants may follow a different pathway than that in cyanobacteria: the two subunits may be intertwined from very early stages, and include intermediates such as (LS-SS)2, (LS-SS)2(LS-SS)2 or LS2SS2 (Roy and Andrews, 2000). We propose that unassembled SS monomers or oligomers form dynamic intermediates with RAF1, BSD2 and RAF2, which then act cumulatively or sequentially to capture folded LS from chaperonins (Figure 6). Our current model is that BSD2 and RAF1 are essential components of these hypothetical intermediates, because Rubisco assembly fails in their absence. Recent data from the cyanobacterium Thermosynechococcus elongatus reinforces the apparent differences between plant and prokaryotic pathways: in this organism the main role of RAF1 is to act at the end of the assembly pathway, to exchange its binding to LS for the incorporation of SS (Kolesinski et al., 2014). The raf2-1 null allele still accumulates 10% of the WT Rubisco level, suggesting that RAF2 accelerates Rubisco assembly but is not absolutely required. Among the many possibilities are that RAF2 acts as a dimerization factor for SS, facilitates an interaction between LS and SS without a direct physical contact with RAF1 and BSD2, and/or presents SS monomers or dimers to the other chaperones. Similarly, it could stabilize a post-chaperonin complex involving BSD2, RAF1 and LS. RAF2 forms multimers, and PCD proteins are known to bind clients such as HNF1a in vertebrates and PhhA in Pseudomonas (Song et al., 1999). In the case of the HNF1a homeodomain transcription factor, its regulatory function is activated through dimerization mediated by PCD (Rose et al., 2004). Animal PCD possesses a 17-amino acid helical stretch that is believed to mediate both homodimerization and heterologous interactions (Endrizzi et al., 1995; Rose
Figure 7. Prediction of RAF2 secondary structure. PHYRE 2 (Kelley and Sternberg, 2009) was used to model 97 residues (56%) of Zea mays (maize) chloroplast RAF2 (query sequence) using mouse PCD (fold library id d1ru0a; http://scop.berkeley.edu/sunid=0) as a template. Alignment confidence in this region was 100%; spirals, a-helix; horizontal filled gray arrows, b-sheet. Black and light-gray highlighting indicate insertions and deletions in RAF2 relative to mouse PCD, respectively, and residues outlined by boxes are required for catalysis based on the Catalytic Site Atlas (http://www.ebi.ac.uk/thornton-srv/databases/CSA). Thick black underlining denotes the recognition helix of mouse DCoH, encompassing amino acids 43–60, which is involved in dimerization of HNF1 and self-tetramerization. Additional designations, as assigned by DSSP (http://swift.cmbi.ru.nl/gv/dssp), are: T, hydrogen bond turn; B, residues in isolated b-bridges; S, bend.
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 862–869
868 Leila Feiz et al. et al., 2004), and it has been suggested that sequence variation within this region could serve as a basis for client protein specificity (see Figure S2). Indeed, structural modeling of RAF2 using mouse PCD as a template shows conservation of this a-helical stretch (Figure 7). Whether this domain of maize RAF2 mediates multimerization and/or the formation of multiprotein complexes involved in Rubisco assembly, awaits further experimentation. EXPERIMENTAL PROCEDURES Gene cloning, gene expression and protein analysis The raf2 mutant was identified as a mutant specifically lacking Rubisco during large-scale immunoblot analyses of mutants in the PML collection (http://pml.uoregon.edu/photosyntheticml.html). Co-segregating Mu insertions were identified in an approach involving the deep-sequencing of Mu-gene junctions, as described previously (Williams-Carrier et al., 2010). PCR verification of the genotypes of raf1 mutant plants and their WT cousins used the gene-specific primer pair ZmRAF2-For (50 -GCCGACCTGCTGGGC GACTTG-30 ) and RAF2-Rev1 (50 -GGACGAGCCTCCACCCGACG-30 ) to amplify the WT allele, or one of them with a mixture of Muspecific primers, EoMu1 and EoMu2, to identify the mutant allele. A list of all primers is presented in Table S1. Gene expression and protein analysis, including Blue Native Gel electrophoresis, was performed according to Feiz et al. (2012). Antisera against RbcL, PsaD, AtpB, PetA and PsbA were purchased from Agrisera Antibodies (http://www.agrisera.com). The production of antiserum against RAF1 was described previously (Feiz et al., 2012).
Production of recombinant RAF2 and of anti-RAF2 and BSD2 antisera The raf2 and bsd2 coding regions preceded by glutathioneS-transferase were expressed in E. coli from pGEX 4T-1 (GE Healthcare, http://www.gehealthcare.com). The clarified cell lysates were applied to a Glutathione Sepharose 4 Fast Flow column (GE Healthcare), followed by on-column digestion by thrombin. Recombinant RAF2 and BSD2 were further purified by Superdex200 size-exclusion chromatography. Antisera were generated by Lampire Biological Laboratories (http://www.lampire.com).
Co-immunoprecipitation To prepare anti-RAF2, anti-RAF1, anti-BSD2 and anti-RNC2-bound protein A affinity beads, Pierce Protein A Plus Agarose beads (500 ll, 50% suspension; Thermo Scientific, http://www. thermoscientific.com) were added to disposable 5-ml polypropylene columns and washed three times with PBS, prior to overnight incubation with 4 ml of PBS and 700 ll of each antibody at 4°C. The beads were washed three times with PBS and three times with 0.2 M sodium borate, pH 9.0, and cross-linked to antibodies by suspending the beads in 5 ml of 0.2 M sodium borate containing 10 mg mL1 dimethyl pimelimidate and rocking for 1 h at 25°C. To stop the reaction, the beads were washed three times in ethanolamine, pH 8, and then incubated in the same buffer for 1 h. Affinity beads were used after three washes with PBS, an incubation with 0.2 M glycine, pH 2.5, for 10 min, and five more washes in PBS. The introduction of formaldehyde for in vivo cross-linking was performed as described previously (Feiz et al., 2012). Cross-linked leaf tissue (10 g) was collected from the top 4 cm of the second
and third leaves, cut into 1-mm pieces, and added to 20 ml cold extraction buffer (20 mM Tris-HCl, pH 9.0, 250 mM NaCl, 50 mM NaHCO3, 4 mM MgCl2, 0.5% Nonidet P-40 and an EDTA-free protease inhibitor cocktail; Roche, http://www.roche.com). The lysate was filtered through an Express PLUS Membrane (0.22 lm; EMD Millipore, http://www.emdmillipore.com) to remove particulates, and then pre-cleared by incubation with 500 ll of protein A on ice for 30 min. Equal quantities of the cross-linked lysate were rotated with each affinity bead at 4°C for 90 min. The beads were washed with 20–30 volumes of extraction buffer containing 0.1% Nonidet P-40, and proteins were eluted from the beads by two sequential incubations in 500 ll of elution buffer (5 mM sodium phosphate, pH 6.8, and 3% SDS). The eluates were pooled, concentrated by methanol/chloroform precipitation (Wessel and Flugge, 1984), air dried, dissolved in 100 ll of SDS loading buffer and heated to 100°C for 15 min to reverse the formaldehyde cross-linking. Co-immunoprecipitated proteins were analyzed by SDS-PAGE using 13.5% acrylamide gels. To prevent IgG heavychain interference with protein detection, clean-blot IP detection reagent (HRP; Thermo Scientific) was used at a dilution of 1:400.
ACKNOWLEDGEMENTS This research was supported by research grant award no. US4443-11 from BARD, The United States–Israel Binational Agricultural Research and Development Fund, to D.B.S., and by National Science Foundation grant IOS–0922560 to A.B. M.M. was supported by the NSF Research Experience for Undergraduates, Plant Genome Research Program (REU, PGRP), award no. DBI-0756560 to Goerg Jander and Mike Scanlon.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article. Figure S1. Validation of raf2 gene identification. Figure S2. Conservation of the RAF2 sequence. Table S1. Accession numbers of RAF2 sequences from green species.
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A new Rubisco biogenesis protein 869 large-scale quantitative proteomics using the first maize genome assembly. Plant Physiol. 152, 1219–1250. Hevel, J.M., Pande, P., Viera-Oveson, S., Sudweeks, T.J., Jaffree, L.S., Hansen, C.M. and Ayling, J.E. (2008) Determinants of oligomerization of the bifunctional protein DCoHa and the effect on its enzymatic and transcriptional coactivator activities. Arch. Biochem. Biophys. 477, 356– 362. Hubbs, A. and Roy, H. (1992) Synthesis and assembly of large subunits into ribulose bisphosphate carboxylase/oxygenase in chloroplast extracts. Plant Physiol. 100, 272–281. Kelley, L.A. and Sternberg, M.J. (2009) Protein structure prediction on the Web: a case study using the Phyre server. Nat. Protoc. 4, 363–371. Kim, S.-R., Yang, J.-I. and An, G. (2013) OsCpn60 alpha 1, encoding the plastid chaperonin 60 alpha subunit, is essential for folding of rbcL. Mol. Cells, 35, 402–409. Klockenbusch, C. and Kast, J. (2010) Optimization of formaldehyde cross-linking for protein interaction analysis of non-tagged integrin b1. J. Biomed. Biotechnol. 2010, 927585. Kolesinski, P., Belusiak, I., Czarnocki-Cieciura, M. and Szczepaniak, A. (2014) Rubisco Accumulation Factor 1 from Thermosynechococcus elongatus participates in the final stages of ribulose-1,5-bisphosphate carboxylase/oxygenase assembly in Escherichia coli cells and in vitro. FEBS J. 281, 3920–3932. Li, P.H., Ponnala, L., Gandotra, N. et al. (2010) The developmental dynamics of the maize leaf transcriptome. Nat. Genet. 42, 1060–U1051. Liu, C., Young, A.L., Starling-Windhof, A. et al. (2010) Coupled chaperone action in folding and assembly of hexadecameric Rubisco. Nature, 463, 197–202. Naponelli, V., Noiriel, A., Ziemak, M.J. et al. (2008) Phylogenomic and functional analysis of pterin-4a-carbinolamine dehydratase family (COG2154) proteins in plants and microorganisms. Plant Physiol. 146, 1515–1527. Parry, M.A.J., Andralojc, P.J., Scales, J.C., Salvucci, M.E., Carmo-Silva, A.E., Alonso, H. and Whitney, S.M. (2013) Rubisco activity and regulation as targets for crop improvement. J. Exp. Bot. 64, 717–730. Rae, B.D., Long, B.M., Badger, M.R. and Price, G.D. (2013) Functions, compositions, and evolution of the two types of carboxysomes: polyhedral
microcompartments that facilitate CO2 fixation in cyanobacteria and some proteobacteria. Microbiol. Mol. Biol. Rev. 77, 357–379. Rose, R.B., Pullen, K.E., Bayle, J.H., Crabtree, G.R. and Alber, T. (2004) Biochemical and structural basis for partially redundant enzymatic and ranscriptional functions of DCoH and DCoH2. Biochemistry, 43, 7345– 7355. Roy, H. and Andrews, T.J. (2000) Rubisco: assembly and mechanism. In Photosynthesis (Leegood, R., Sharkey, T. and Caemmerer, S. eds). Netherlands: Springer, pp. 53–83. Saschenbrecker, S., Bracher, A., Rao, K.V., Rao, B.V., Hartl, F.U. and Hayer-Hartl, M. (2007) Structure and function of RbcX, an assembly chaperone for hexadecameric Rubisco. Cell, 129, 1189–1200. Song, J., Xia, T.H. and Jensen, R.A. (1999) PhhB, a Pseudomonas aeruginosa homolog of mammalian pterin 4a-carbinolamine dehydratase/DCoH, does not regulate expression of phenylalanine hydroxylase at the transcriptional level. J. Bacteriol. 181, 2789–2796. Stern, D.B., Hanson, M.R. and Barkan, A. (2004) Genetics and genomics of chloroplast biogenesis: maize as a model system. Trends Plant Sci. 9, 293–301. Thony, B., Auerbach, G. and Blau, N. (2000) Tetrahydrobiopterin biosynthesis, regeneration and functions. Biochem. J. 347, 1–16. Valkai, I. (2004) 28D, a new component of the phytochrome B signal transduction, in Arabidopsis thaliana. Acta Biol. Szeged, 48, 87. Wessel, D. and Flugge, U.I. (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents and lipids. Anal. Biochem. 138, 141–143. Wheatley, N.M., Sundberg, C.D., Gidaniyan, S.D., Cascio, D. and Yeates, T.O. (2014) Structure and Identification of a pterin dehydratase-like protein as a Ribulose-bisphosphate Carboxylase/Oxygenase (RuBisCO) assembly factor in the alpha-carboxysome. J. Biol. Chem. 289, 7973–7981. Whitney, S.M., Houtz, R.L. and Alonso, H. (2011) Advancing Our Understanding and Capacity to Engineer Nature’s CO2-Sequestering Enzyme, Rubisco. Plant Physiol. 155, 27–35. Williams-Carrier, R., Stiffler, N., Belcher, S., Kroeger, T., Stern, D.B., Monde, R.A., Coalter, R. and Barkan, A. (2010) Use of Illumina sequencing to identify transposon insertions underlying mutant phenotypes in high-copy Mutator lines of maize. Plant J. 63, 167–177.
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 862–869
doi: 10.1111/tpj.12686
A protein with an inactive pterin-4a-carbinolamine dehydratase domain is required for Rubisco biogenesis in plants Leila Feiz1, Rosalind Williams-Carrier2, Susan Belcher2, Monica Montano1,†, Alice Barkan2 and David B. Stern1,* Boyce Thompson Institute for Plant Research, Cornell University, Tower Road, Ithaca, NY 14853, USA, and 2 Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA
1
Received 29 July 2014; revised 15 September 2014; accepted 17 September 2014; published online 3 October 2014. *For correspondence (e-mail [email protected]). † Current address: University of Wisconsin-Madison, Department of Surgery, 600 Highland Ave., Madison, WI 53792
SUMMARY Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a critical role in sustaining life by catalysis of carbon fixation in the Calvin–Benson pathway. Incomplete knowledge of the assembly pathway of chloroplast Rubisco has hampered efforts to fully delineate the enzyme’s properties, or seek improved catalytic characteristics via directed evolution. Here we report that a Mu transposon insertion in the Zea mays (maize) gene encoding a chloroplast dimerization co-factor of hepatocyte nuclear factor 1 (DCoH)/pterin4a-carbinolamine dehydratases (PCD)-like protein is the causative mutation in a seedling-lethal, Rubiscodeficient mutant named Rubisco accumulation factor 2 (raf2-1). In raf2 mutants newly synthesized Rubisco large subunit accumulates in a high-molecular weight complex, the formation of which requires a specific chaperonin 60-kDa isoform. Analogous observations had been made previously with maize mutants lacking the Rubisco biogenesis proteins RAF1 and BSD2. Chemical cross-linking of maize leaves followed by immunoprecipitation with antibodies to RAF2, RAF1 or BSD2 demonstrated co-immunoprecipitation of each with Rubisco small subunit, and to a lesser extent, co-immunoprecipitation with Rubisco large subunit. We propose that RAF2, RAF1 and BSD2 form transient complexes with the Rubisco small subunit, which in turn assembles with the large subunit as it is released from chaperonins. Keywords: carbon fixation, C4, Calvin cycle, Zea mays, photosynthesis, chaperone, protein folding.
INTRODUCTION All life depends on ribulose-1,5-bisphosphate carboxylase (Rubisco), which incorporates atmospheric CO2 through the carboxylation of the substrate ribulose-1,5-bisphosphate, the rate-limiting step of the Calvin–Benson pathway. In parallel with this main reaction, Rubisco catalyzes a competitive oxygenation side reaction, which is wasteful in terms of the net fixation of carbon dioxide. The steady increase in the atmospheric proportion of oxygen over millenia has increased the proportion of oxygenation to carboxylation reactions, leading to Rubisco being quite inefficient and slow at fixing carbon in the current environment (Whitney et al., 2011). To obtain the carbon required for growth and development, C3 plants allocate almost 30% of their nitrogen and more than 50% of their leaf proteins to Rubisco, making this enzyme a major sink for nitrogen and other resources required for optimal plant productivity. These effects are mitigated in C4 plants, where Rubisco is localized to bundle sheath chloroplasts in a concentrated CO2 environment. These factors have long made Rubisco a target for 862
engineering, either to improve its catalytic properties or to create a ‘C4-like environment’ for Rubisco in C3 plants (Evans, 2013). Although the screening of Rubisco from different species led to the identification of enzymes with higher specificity for CO2 and higher catalytic rates, incompatibility of host chaperones with heterologous Rubisco subunits prevented the assembly of novel Rubisco forms in plants or in Escherichia coli, hindering efforts to achieve a better enzyme via genetic selection or mutagenesis approaches (Parry et al., 2013). Form-I Rubisco, which exists in cyanobacteria, algae and plants, is a hexadecamer composed of eight large (50 kDa) and eight small (13–15 kDa) subunits, denoted here as LS and SS, respectively. The assembly pathway of cyanobacterial Rubisco has been delineated using bacterial and cellfree systems, assisted by the E. coli chaperonins GroEL/ GroES and the Rubisco-specific chaperone RBCX (Saschenbrecker et al., 2007; Liu et al., 2010; Bracher et al., 2011). In these systems, the chaperonin cage composed of GroEL and GroES binds unfolded LS in a complex of © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd
A new Rubisco biogenesis protein 863 RESULTS Identification of the causal mutation in raf2-1 The raf2 mutant was initially identified during large-scale phenotyping of approximately 2000 mutants in the maize Photosynthetic Mutant Library (PML; Stern et al., 2004) as a pale-green mutant (Figure 1a), with a reduced content of Rubisco and normal levels of representative subunits of PSI (PsaD), the cytochrome b6f complex (PetA), PSII (PsbA) and the ATP synthase (AtpB) (Figure 1b). This protein profile is similar to that of the bsd2, raf1 and cps2 mutants, which are deficient for established Rubisco biogenesis factors (Barkan, 1993; Feiz et al., 2012). raf2 seedlings survive on seed reserves for several weeks (Figure 1a), but the mutation is ultimately lethal. Their pale-green phenotype is more subtle than that of bsd2 and raf1 mutants, and they survive longer on soil, correlating with the fact that they accumulate approximately 10% of the wild-type (WT) level of LS, as compared with approximately 1% in the other mutants. RAF1 accumulates to normal levels in raf2
(a)
WT (%)
(b)
100 10
1
raf2
approximately 800 kDa, which then releases LS for further folding and assembly mediated by RBCX. In this step, RBCX dimers stabilize an octameric core of LS, LS8-(RBCX2)8, prior to the sequential replacement of RBCX with SS monomers, creating the holoenzyme. In algae and plants, nucleus-encoded SS and plastidencoded LS assemble to form the holoenzyme, following the import of cytosolically synthesized SS into the plastid. How this assembly occurs is largely unknown, however, mainly because LS is insoluble in both bacterial hosts and in cell-free systems. The role of the chloroplast Cpn60Cpn21/10 chaperonin complex, a protein cage homologous to E. coli GroEL/GroES, in Rubisco biogenesis was shown by the co-purification of Cpn60 with Rubisco (Barraclough and Ellis, 1980; Feiz et al., 2012), and by the characterization of Rubisco-deficient mutants of Oryza sativa (rice; Kim et al., 2013) and Zea mays (maize; Barkan, 1993; Feiz et al., 2012) with lesions in genes encoding an alpha isoform of Cpn60 (denoted CPS2 in maize). Whereas direct evidence for a role for RBCX in plant Rubisco assembly is lacking, maize mutants lacking either Bundle Sheath Defective 2 (BSD2), which harbors a zincfinger domain related to that in the chaperone DnaJ (Brutnell et al., 1999), or Rubisco Accumulation Factor 1 (RAF1), which lacks domains of known function, cannot assemble LS into holoenzyme. Instead, nascent LS accumulates in a CPS2-containing complex of approximately 800 kDa. This observation led to speculation that BSD2 and RAF1 in chloroplasts serve the role of cyanobacterial RBCX, by releasing LS from the chaperonin complex and ultimately mediating its assembly into holoenzyme (Feiz et al., 2012). To investigate the post-chaperonin stage(s) at which BSD2 and RAF1 play roles, we co-expressed these proteins together with chloroplast chaperonin and maize Rubisco in E. coli, or added them to an in vitro system containing recombinant LS and chaperonins. Our lack of success in solubilizing or assembling Rubisco in these systems led us to believe that additional and perhaps unknown factors might be required. Here we describe the genetic identification of the maize gene Rubisco accumulation factor 2 (raf2) that is required for Rubisco biogenesis. RAF2 is conserved among photosynthetic eukaryotes and is related to a mitochondrial protein: dimerization co-factor of hepatocyte nuclear factor 1 (DCoH)/pterin-4a-carbinolamine dehydratases (PCD)-like protein. Orthologs of raf2 are also found in photosynthetic bacteria harboring a-carboxysomes, a carbon-concentrating structure that is functionally and structurally associated with Rubisco (Rae et al., 2013). RAF2 was shown previously to lack PCD activity and to have acquired a chloroplast-specific domain of unknown function. It has therefore been speculated that RAF2 has evolved a new function (Naponelli et al., 2008), which our study now reveals to be a Rubisco assembly chaperone.
RbcL PsaD AtpB PetA PsbA RAF1 RbcL
WT Zm-raf2-1
(c)
CBB
Zm-raf2-1(+217)
Figure 1. Overview of the raf2 mutant phenotype. (a) Wild-type (WT) and homozygous raf2-1 plants at the seedling stage. (b) Immunoblot analyses of Rubisco large subunit and representative subunits of other photosynthetic complexes. Total proteins from equal surface area of the seedling leaf tip or dilutions (as indicated) were analyzed by probing with antibodies raised against the proteins indicated on the left; CBB, Coomassie blue staining. (c) The raf2 locus corresponds to GRMZM2G139123, an intron-less locus on chromosome 1. The location of the Mu insertion relative to the translation initiation codon is shown. The 9-bp target duplication is underlined.
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 862–869
864 Leila Feiz et al.
WT (%) 100 10 1 LS
RAF2
(b)
cps2
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The effect of RAF2 on the accumulation of rbcL and rbcs transcripts was investigated by gel blot analysis of total seedling leaf RNA from WT and raf2 mutants (Figure 3a). Known Rubisco-biogenesis mutants cps2, raf1 and bsd2 were analyzed in parallel. Neither the primary nor the 50 -processed rbcL transcripts were affected qualitatively or quantitatively in raf2 mutants, although rbcL mRNA exhibited increased abundance in raf1 mutants, as noted previously (Feiz et al., 2012). rbcs transcripts also appeared to be unaltered in raf2 mutants, indicating that RAF2 promotes Rubisco biogenesis at a post-transcriptional level.
bsd2
The raf2 locus, GRMZM2G139123, encodes a member of the PCD family. Some PCD family members are involved in the recycling of the multi-function co-factor tetrahydrobiopterin (Thony et al., 2000), and others are involved in transcription factor dimerization (Rose et al., 2004). Plants typically encode two PCD proteins. One localizes to mitochondria, is enzymatically active and has been proposed to be involved in molybdenum co-factor metabolism (Naponelli et al., 2008). The other, represented by RAF2 and its Arabidopsis ortholog (At5g51110), localizes to chloroplasts, as shown by a proteomic analysis (Friso et al., 2010) and GFP fusion data (Naponelli et al., 2008). At-RAF2 and maize RAF2 lacked catalytic activity when expressed in E. coli (Naponelli et al., 2008), but their functions were not previously tested through mutant analysis. Both proteome (Friso et al., 2010) and transcriptome (Li et al., 2010) data suggest approximately 2.5-fold higher accumulation of RAF2 protein and RNA in maize bundle sheath versus mesophyll chloroplasts; this is consistent with a role in Rubisco assembly, as Rubisco is restricted to bundle sheath chloroplast in maize, a C4 plant. RAF2 orthologus lack catalytic signatures, but also harbor a motif that distinguishes them from their mitochondrial and bacterial relatives (Figure S2). It is possible that this motif is relevant to the Rubisco-related function of RAF2. An antibody raised against recombinant RAF2 detected a protein of approximately 18 kDa in total leaf extracts from
Rubisco subunit genes are normally transcribed and translated in raf2, but a majority of LS is trapped in a chaperonin-containing complex
raf1
The raf2 gene product
plants carrying the WT Raf2 allele, consistent with the size of RAF2 lacking the predicted chloroplast transit peptide (18.5 kDa). This protein was absent in raf2-1 mutants, thereby confirming it to be RAF2 (Figure 2a). Like RAF1 (Figure 1b), BSD2 accumulated to the WT level in raf2-1 mutants, indicating that there were no pleiotropic effects on the abundances of known Rubisco chaperones. When recombinant RAF2 was analyzed by blue native gel (BNG) electrophoresis, it appeared to migrate as dimers and tetramers (Figure 2b). Animal PCD proteins are known to dimerize (Hevel et al., 2008), a bacterial homolog of RAF2 crystallizes as a dimer (Wheatley et al., 2014) and a brief report stated that Arabidopsis RAF2 forms dimers (Valkai, 2004), consistent with our observation for maize RAF2.
raf2
mutants (Figure 1b), indicating that raf2 does not mediate its effects by affecting the expression of raf1. Genetic crosses between raf2-1 and the WT had verified that the pale-green phenotype and low Rubisco levels cosegregated as a single, recessive Mendelian trait. The raf21 mutant arose in a maize line harboring active Mu transposons, and a deep sequencing strategy described previously by Williams-Carrier et al. (2010) was used to identify Mu insertions that co-segregated with the raf2 locus. Among the few candidate loci identified in this manner, GRMZM2G139123 was the only one predicted to encode a chloroplast-localized protein. The Mu insertion in this locus was located in the middle of its single exon, and is predicted to be a severe, likely null, allele (Figure 1c). Gene-specific PCR of additional raf2-1 mutant individuals and several phenotypically WT cousins confirmed that this insertion was tightly linked to raf2 (Figure S1). In addition, the bacterial homolog of raf2 is encoded in the same operon as Rubisco subunits, and was recently shown to increase the accumulation of bacterial Rubisco when co-expressed with Rubisco subunits and GroELS (Wheatley et al., 2014). Taken together, these findings provide strong support for the conclusion that RAF2 is required for Rubisco accumulation in maize.
720 480 242
RAF2
146
RAF1 3
66
RAF2 4
BSD2 20
RAF2 2
Figure 2. Characterization of endogenous and recombinant RAF2 protein. (a) Immunoblot analysis of soluble proteins extracted from equal surface areas of wild-type (WT), raf2, raf1, bsd2 and cps2 leaves. Proteins detected are shown to the left of each panel. (b) Soluble recombinant RAF1 and RAF2 proteins were analyzed in a 4–16% Blue Native Gel, followed by Coomassie staining. RAF13 marks the migration of RAF1 trimers, and RAF22 and RAF24 mark the positions of presumed RAF2 dimers and tetramers.
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 862–869
866 Leila Feiz et al. control. Immunopurifications with RAF2 antibody employed either cross-linked or non-cross-linked material, whereas the other three antibodies were used in immunopurification reactions only from cross-linked material. Eluted proteins were analyzed by immunoblotting after reversing the cross links (Figure 5). Column 1 shows the levels of LS, SS, RAF2, BSD2, RAF1 and an unrelated protein, chloroplast malic enzyme (ME), in the input material. Non-specific binding to beads was minimal, as gauged by immunopurification with anti-RNC2 (column 3). The a-RAF2 beads co-purified substantial quantities of both LS and SS from uncross-linked lysates (column 2), and these signals were augmented with prior cross-linking (column 4), suggesting that the interactions were labile. Furthermore, BSD2 co-purified with RAF2 only from cross-linked lysates (column 4), suggestive of a transient or weak interaction. The reciprocal interaction was not reproducibly observed, however, making the conclusion ambiguous (column 6). Evidence for a RAF2–RAF1 interaction was similarly inconclusive. The a-RAF1 (column 5) and a-BSD2 (column 6) beads likewise bound both LS and SS proteins above the background level; we had previously reported the RAF1–LS interaction (Feiz et al., 2012). BSD2 and RAF1 also showed a clear interaction in these reciprocal experiments.
1 α-RAF2
2 +
3
4 +
5
+
α-RNC2
+
α-RAF1
+
α-BSD2
+
WT lysate WT X-linked lysate
6
+
+
+
+
+
LS SS RAF2 RAF1
BSD2 ME Figure 5. Interaction between Rubisco subunits and chaperones. The indicated antibodies were bound to protein A beads and used for immunoprecipitation from total soluble proteins extracted from cross-linked (X-linked) or non-cross-linked wild-type (WT) leaves. The eluates were separated in a 13% SDS-polyacrylamide gel and analyzed by immunoblotting for the proteins indicated on the left. RNC2 is a nucleolar protein used as a negative control. Lane 1 was loaded with 0.4% of the input, and eluates were diluted 25-fold for self-detection (e.g. detection of RAF2 protein with immunoprecipitation by a-RAF2).
These results lead to two main conclusions. First, RAF2 engages in interactions with LS and SS that resemble those observed for the known Rubisco chaperones RAF1 and BSD2. These results, in conjunction with the sequestration of nascent LS in the ‘LSc’ complex in raf2 mutants (Figure 4), imply that RAF2 likewise functions as a chaperone during Rubisco assembly. The second and striking result is that in each case the SS signal was several times stronger than the LS signal, in comparison with the signal in the input (column 1). This suggests that the chaperones have a stronger affinity for SS than LS, and/or that they interact more frequently with SS than with LS. Various models that account for these results are discussed below. DISCUSSION In this study we have described RAF2, a member of the PCD family, as a protein required for wild-type levels of Rubisco accumulation in maize. RAF2 joins RAF1 and BSD2 as Rubisco chaperones that are broadly represented in and restricted to the green lineage, yet have been placed in the Rubisco assembly pathway through genetic analyses in maize. In a recent structural analysis of a bacterial RAF2 homolog encoded in the a-carboxysome operon, co-expression with Rubisco and GroEL/GroES in E. coli increased the concentration of soluble, assembled Rubisco (Wheatley et al., 2014), attesting to an evolutionarily conserved role for this protein. Our data show that neither transcription nor translation of LS is reduced in raf2 mutants, yet Rubisco abundance is decreased dramatically. To investigate the fate of LS in this mutant, we used native gel electrophoresis of newly synthesized proteins (Figure 4). The data suggest that similar to raf1 and bsd2 (Feiz et al., 2012), newly synthesized LS in raf2 is trapped in the approximately 800-kDa LSc complex, the formation of which is dependent on CPS2, a chaperonin a-isoform. This implies that similar to RAF1 and BSD2, RAF2 is required for LS to exit the chaperonin cage successfully, a process that is facilitated by RBCX in at least some cyanobacteria (Saschenbrecker et al., 2007). During the in vitro assembly of cyanobacterial Rubisco, transient intermediates anchored by RBCX dimmers, including RBCX2(2)-LS2, RBCX2(4)-LS4 and RBCX2(8)-LS8, undergo sequential replacement of RBCX2 by SS monomers (Liu et al., 2010; Bracher et al., 2011). We considered the possibility that RAF1, RAF2 and/or BSD2 might mimic RbcX function in chloroplasts, but we were unable to obtain direct evidence of the composition and order of formation of these putative intermediates. As a first step, we attempted to stabilize transient interactions by in planta cross-linking prior to immunoprecipitation of each chaperone and any associated partners (Figure 5). Although these experiments validated the interaction of RAF1 and BSD2, as well as binding to LS by each chaperone, a surprising result was that not only RAF2, but also RAF1 and BSD2
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 862–869
866 Leila Feiz et al. control. Immunopurifications with RAF2 antibody employed either cross-linked or non-cross-linked material, whereas the other three antibodies were used in immunopurification reactions only from cross-linked material. Eluted proteins were analyzed by immunoblotting after reversing the cross links (Figure 5). Column 1 shows the levels of LS, SS, RAF2, BSD2, RAF1 and an unrelated protein, chloroplast malic enzyme (ME), in the input material. Non-specific binding to beads was minimal, as gauged by immunopurification with anti-RNC2 (column 3). The a-RAF2 beads co-purified substantial quantities of both LS and SS from uncross-linked lysates (column 2), and these signals were augmented with prior cross-linking (column 4), suggesting that the interactions were labile. Furthermore, BSD2 co-purified with RAF2 only from cross-linked lysates (column 4), suggestive of a transient or weak interaction. The reciprocal interaction was not reproducibly observed, however, making the conclusion ambiguous (column 6). Evidence for a RAF2–RAF1 interaction was similarly inconclusive. The a-RAF1 (column 5) and a-BSD2 (column 6) beads likewise bound both LS and SS proteins above the background level; we had previously reported the RAF1–LS interaction (Feiz et al., 2012). BSD2 and RAF1 also showed a clear interaction in these reciprocal experiments.
1 α-RAF2
2 +
3
4 +
5
+
α-RNC2
+
α-RAF1
+
α-BSD2
+
WT lysate WT X-linked lysate
6
+
+
+
+
+
LS SS RAF2 RAF1
BSD2 ME Figure 5. Interaction between Rubisco subunits and chaperones. The indicated antibodies were bound to protein A beads and used for immunoprecipitation from total soluble proteins extracted from cross-linked (X-linked) or non-cross-linked wild-type (WT) leaves. The eluates were separated in a 13% SDS-polyacrylamide gel and analyzed by immunoblotting for the proteins indicated on the left. RNC2 is a nucleolar protein used as a negative control. Lane 1 was loaded with 0.4% of the input, and eluates were diluted 25-fold for self-detection (e.g. detection of RAF2 protein with immunoprecipitation by a-RAF2).
These results lead to two main conclusions. First, RAF2 engages in interactions with LS and SS that resemble those observed for the known Rubisco chaperones RAF1 and BSD2. These results, in conjunction with the sequestration of nascent LS in the ‘LSc’ complex in raf2 mutants (Figure 4), imply that RAF2 likewise functions as a chaperone during Rubisco assembly. The second and striking result is that in each case the SS signal was several times stronger than the LS signal, in comparison with the signal in the input (column 1). This suggests that the chaperones have a stronger affinity for SS than LS, and/or that they interact more frequently with SS than with LS. Various models that account for these results are discussed below. DISCUSSION In this study we have described RAF2, a member of the PCD family, as a protein required for wild-type levels of Rubisco accumulation in maize. RAF2 joins RAF1 and BSD2 as Rubisco chaperones that are broadly represented in and restricted to the green lineage, yet have been placed in the Rubisco assembly pathway through genetic analyses in maize. In a recent structural analysis of a bacterial RAF2 homolog encoded in the a-carboxysome operon, co-expression with Rubisco and GroEL/GroES in E. coli increased the concentration of soluble, assembled Rubisco (Wheatley et al., 2014), attesting to an evolutionarily conserved role for this protein. Our data show that neither transcription nor translation of LS is reduced in raf2 mutants, yet Rubisco abundance is decreased dramatically. To investigate the fate of LS in this mutant, we used native gel electrophoresis of newly synthesized proteins (Figure 4). The data suggest that similar to raf1 and bsd2 (Feiz et al., 2012), newly synthesized LS in raf2 is trapped in the approximately 800-kDa LSc complex, the formation of which is dependent on CPS2, a chaperonin a-isoform. This implies that similar to RAF1 and BSD2, RAF2 is required for LS to exit the chaperonin cage successfully, a process that is facilitated by RBCX in at least some cyanobacteria (Saschenbrecker et al., 2007). During the in vitro assembly of cyanobacterial Rubisco, transient intermediates anchored by RBCX dimmers, including RBCX2(2)-LS2, RBCX2(4)-LS4 and RBCX2(8)-LS8, undergo sequential replacement of RBCX2 by SS monomers (Liu et al., 2010; Bracher et al., 2011). We considered the possibility that RAF1, RAF2 and/or BSD2 might mimic RbcX function in chloroplasts, but we were unable to obtain direct evidence of the composition and order of formation of these putative intermediates. As a first step, we attempted to stabilize transient interactions by in planta cross-linking prior to immunoprecipitation of each chaperone and any associated partners (Figure 5). Although these experiments validated the interaction of RAF1 and BSD2, as well as binding to LS by each chaperone, a surprising result was that not only RAF2, but also RAF1 and BSD2
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 862–869
A new Rubisco biogenesis protein 867 Cpn21/10
Chaperoninbound LS RAF13
LS
BSD2X
Native LS
RAF22
Folded SS
Chloroplast
tp-SS
Cytosol
L8S8 Rubisco
Figure 6. Model for roles of RAF2, RAF1 and BSD2 in Rubisco assembly. From top left: newly synthesized large subunit (LS) interacts with the chaperonin complex, which leads to correct folding (Native LS). RAF2, RAF1 and BSD2 would bind small subunit (SS) to form dynamic intermediates that then act to capture the folded LS from the chaperonin complex. In the absence of RAF2, RAF1 or BSD2, LS would be unable to escape from the chaperonin cycle, ultimately leading to aggregation and proteolysis. ‘Chaperonin-bound LS’ is equivalent to LSc (Figure 4).
co-immunopurified strongly with SS. This observation raises the possibility that unlike the cyanobacterial mechanism, in which SS folds independently and inserts into the octameric core of LS at the end of the pathway, the import, folding and assembly of SS may play central role(s) in the formation of chloroplast holoenzyme. Such a scenario would postulate a very different set of assembly intermediates. A model that is consistent with our co-immunopurification data is presented in Figure 6. The notion that the binding of chaperones to SS comprises an early step in Rubisco assembly has not received recent attention, but was raised when endogenous LS synthesized in a chloroplast extract could only
form Rubisco when purified SS was added to the system (Hubbs and Roy, 1992). Consequently, it was suggested that the assembly of Rubisco large and small subunits in plants may follow a different pathway than that in cyanobacteria: the two subunits may be intertwined from very early stages, and include intermediates such as (LS-SS)2, (LS-SS)2(LS-SS)2 or LS2SS2 (Roy and Andrews, 2000). We propose that unassembled SS monomers or oligomers form dynamic intermediates with RAF1, BSD2 and RAF2, which then act cumulatively or sequentially to capture folded LS from chaperonins (Figure 6). Our current model is that BSD2 and RAF1 are essential components of these hypothetical intermediates, because Rubisco assembly fails in their absence. Recent data from the cyanobacterium Thermosynechococcus elongatus reinforces the apparent differences between plant and prokaryotic pathways: in this organism the main role of RAF1 is to act at the end of the assembly pathway, to exchange its binding to LS for the incorporation of SS (Kolesinski et al., 2014). The raf2-1 null allele still accumulates 10% of the WT Rubisco level, suggesting that RAF2 accelerates Rubisco assembly but is not absolutely required. Among the many possibilities are that RAF2 acts as a dimerization factor for SS, facilitates an interaction between LS and SS without a direct physical contact with RAF1 and BSD2, and/or presents SS monomers or dimers to the other chaperones. Similarly, it could stabilize a post-chaperonin complex involving BSD2, RAF1 and LS. RAF2 forms multimers, and PCD proteins are known to bind clients such as HNF1a in vertebrates and PhhA in Pseudomonas (Song et al., 1999). In the case of the HNF1a homeodomain transcription factor, its regulatory function is activated through dimerization mediated by PCD (Rose et al., 2004). Animal PCD possesses a 17-amino acid helical stretch that is believed to mediate both homodimerization and heterologous interactions (Endrizzi et al., 1995; Rose
Figure 7. Prediction of RAF2 secondary structure. PHYRE 2 (Kelley and Sternberg, 2009) was used to model 97 residues (56%) of Zea mays (maize) chloroplast RAF2 (query sequence) using mouse PCD (fold library id d1ru0a; http://scop.berkeley.edu/sunid=0) as a template. Alignment confidence in this region was 100%; spirals, a-helix; horizontal filled gray arrows, b-sheet. Black and light-gray highlighting indicate insertions and deletions in RAF2 relative to mouse PCD, respectively, and residues outlined by boxes are required for catalysis based on the Catalytic Site Atlas (http://www.ebi.ac.uk/thornton-srv/databases/CSA). Thick black underlining denotes the recognition helix of mouse DCoH, encompassing amino acids 43–60, which is involved in dimerization of HNF1 and self-tetramerization. Additional designations, as assigned by DSSP (http://swift.cmbi.ru.nl/gv/dssp), are: T, hydrogen bond turn; B, residues in isolated b-bridges; S, bend.
© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd, The Plant Journal, (2014), 80, 862–869
868 Leila Feiz et al. et al., 2004), and it has been suggested that sequence variation within this region could serve as a basis for client protein specificity (see Figure S2). Indeed, structural modeling of RAF2 using mouse PCD as a template shows conservation of this a-helical stretch (Figure 7). Whether this domain of maize RAF2 mediates multimerization and/or the formation of multiprotein complexes involved in Rubisco assembly, awaits further experimentation. EXPERIMENTAL PROCEDURES Gene cloning, gene expression and protein analysis The raf2 mutant was identified as a mutant specifically lacking Rubisco during large-scale immunoblot analyses of mutants in the PML collection (http://pml.uoregon.edu/photosyntheticml.html). Co-segregating Mu insertions were identified in an approach involving the deep-sequencing of Mu-gene junctions, as described previously (Williams-Carrier et al., 2010). PCR verification of the genotypes of raf1 mutant plants and their WT cousins used the gene-specific primer pair ZmRAF2-For (50 -GCCGACCTGCTGGGC GACTTG-30 ) and RAF2-Rev1 (50 -GGACGAGCCTCCACCCGACG-30 ) to amplify the WT allele, or one of them with a mixture of Muspecific primers, EoMu1 and EoMu2, to identify the mutant allele. A list of all primers is presented in Table S1. Gene expression and protein analysis, including Blue Native Gel electrophoresis, was performed according to Feiz et al. (2012). Antisera against RbcL, PsaD, AtpB, PetA and PsbA were purchased from Agrisera Antibodies (http://www.agrisera.com). The production of antiserum against RAF1 was described previously (Feiz et al., 2012).
Production of recombinant RAF2 and of anti-RAF2 and BSD2 antisera The raf2 and bsd2 coding regions preceded by glutathioneS-transferase were expressed in E. coli from pGEX 4T-1 (GE Healthcare, http://www.gehealthcare.com). The clarified cell lysates were applied to a Glutathione Sepharose 4 Fast Flow column (GE Healthcare), followed by on-column digestion by thrombin. Recombinant RAF2 and BSD2 were further purified by Superdex200 size-exclusion chromatography. Antisera were generated by Lampire Biological Laboratories (http://www.lampire.com).
Co-immunoprecipitation To prepare anti-RAF2, anti-RAF1, anti-BSD2 and anti-RNC2-bound protein A affinity beads, Pierce Protein A Plus Agarose beads (500 ll, 50% suspension; Thermo Scientific, http://www. thermoscientific.com) were added to disposable 5-ml polypropylene columns and washed three times with PBS, prior to overnight incubation with 4 ml of PBS and 700 ll of each antibody at 4°C. The beads were washed three times with PBS and three times with 0.2 M sodium borate, pH 9.0, and cross-linked to antibodies by suspending the beads in 5 ml of 0.2 M sodium borate containing 10 mg mL1 dimethyl pimelimidate and rocking for 1 h at 25°C. To stop the reaction, the beads were washed three times in ethanolamine, pH 8, and then incubated in the same buffer for 1 h. Affinity beads were used after three washes with PBS, an incubation with 0.2 M glycine, pH 2.5, for 10 min, and five more washes in PBS. The introduction of formaldehyde for in vivo cross-linking was performed as described previously (Feiz et al., 2012). Cross-linked leaf tissue (10 g) was collected from the top 4 cm of the second
and third leaves, cut into 1-mm pieces, and added to 20 ml cold extraction buffer (20 mM Tris-HCl, pH 9.0, 250 mM NaCl, 50 mM NaHCO3, 4 mM MgCl2, 0.5% Nonidet P-40 and an EDTA-free protease inhibitor cocktail; Roche, http://www.roche.com). The lysate was filtered through an Express PLUS Membrane (0.22 lm; EMD Millipore, http://www.emdmillipore.com) to remove particulates, and then pre-cleared by incubation with 500 ll of protein A on ice for 30 min. Equal quantities of the cross-linked lysate were rotated with each affinity bead at 4°C for 90 min. The beads were washed with 20–30 volumes of extraction buffer containing 0.1% Nonidet P-40, and proteins were eluted from the beads by two sequential incubations in 500 ll of elution buffer (5 mM sodium phosphate, pH 6.8, and 3% SDS). The eluates were pooled, concentrated by methanol/chloroform precipitation (Wessel and Flugge, 1984), air dried, dissolved in 100 ll of SDS loading buffer and heated to 100°C for 15 min to reverse the formaldehyde cross-linking. Co-immunoprecipitated proteins were analyzed by SDS-PAGE using 13.5% acrylamide gels. To prevent IgG heavychain interference with protein detection, clean-blot IP detection reagent (HRP; Thermo Scientific) was used at a dilution of 1:400.
ACKNOWLEDGEMENTS This research was supported by research grant award no. US4443-11 from BARD, The United States–Israel Binational Agricultural Research and Development Fund, to D.B.S., and by National Science Foundation grant IOS–0922560 to A.B. M.M. was supported by the NSF Research Experience for Undergraduates, Plant Genome Research Program (REU, PGRP), award no. DBI-0756560 to Goerg Jander and Mike Scanlon.
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article. Figure S1. Validation of raf2 gene identification. Figure S2. Conservation of the RAF2 sequence. Table S1. Accession numbers of RAF2 sequences from green species.
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