Arabidopsis thaliana plastoglobule-associated fibrillin 1a interacts with fibrillin 1b in vivo

Arabidopsis thaliana plastoglobule-associated fibrillin 1a interacts with fibrillin 1b in vivo Francisco Manuel Gámez-Arjona, Juan Carlos de la Concepción, Sandy Raynaud, and Ángel Mérida Instituto de Bioquímica Vegetal y Fotosíntesis. CSIC-US. Avda Américo Vespucio, 49. 41092-Sevilla. Spain Corresponding author: Ángel Mérida Instituto de Bioquímica Vegetal y Fotosíntesis. CSIC-USE Avda Américo Vespucio, 49 41092-Sevilla Spain Phone: +34-954489507 Mail: [email protected] Title Page and Abstract Abstract Plant fibrillins are a well-conserved protein family found in the plastids of all photosynthetic organisms, where they perform a wide range of functions. A number of these proteins have been suggested to be involved in the maintenance of thylakoids and the formation of plastoglobules, preventing their coalescence and favouring their clustering via an as-yet unidentified crosslinking mechanism. In this work we show that two members of this group, namely fibrillin 1a and 1b, interact with each other via a head-to-tail mechanism, thus raising the possibility that they form homo- or hetero-oligomers and providing a mechanism to understand the function of these proteins. Keywords Fibrillin, plastoglobules, Arabidopsis, protein-protein interaction Abbreviations FBN, fibrillin; Y2H, yeast Two-Hybrid; BiFC, bimolecular fluorescence complementation; CTP, chloroplast transient peptide Highlights  An interaction between plastidial proteins fibrillin 1a and 1b is shown  Fibrillin 1a-Fibrillin 1a and Fibrillin 1b-Fibrillin 1b interactions are also shown  Interaction takes place via a head-to-tail mechanism  These interactions may explain the functions of these proteins in the plastids Highlights  An interaction between plastidial proteins fibrillin 1a and 1b is shown  Fibrillin 1a-Fibrillin 1a and Fibrillin 1b-Fibrillin 1b interactions are also shown  Interaction takes place via a head-to-tail mechanism  These interactions may explain the functions of these proteins in the plastids Highlights 1 Introduction The term fibrillins (FBNs) in plants designates a large protein family that is present in all types of plastids, such as chloroplasts, chromoplasts, and leucoplasts (Eugeni Piller et al., 2012). These proteins were named fibrillins as they were first identified in fibrils, the thread- or tube-like structures found in bell pepper (Capsicum annuum) fruit chromoplasts (Deruere et al., 1994), which are the main sites of chromoplasts for pigment accumulation (Winkenbach et al., 1976). Fibrillins are found in all photosynthetic organisms, ranging from cyanobacteria to plants (Laizet et al., 2004). Most of them are located in plastoglobules (Ytterberg et al., 2006), although the presence of FBNs in the stroma and stromal lamellae thylakoids has also been shown (Eymery and Rey, 1999). FBNs can be divided into 12 phylogenetic groups and appear to be involved in a wide range of functions, such as abiotic stress tolerance, growth and development, and hormone signalling, or lipid transport between thylakoid membranes and plastoglobules (Simkin et al., 2007; Singh and McNellis, 2011). Members of group 1, which includes Arabidopsis FBN1a and FBN1b, have been reported to be involved in plastoglobule formation and thylakoid maintenance (Rey et al., 2000; Simkin et al., 2007). Indeed, a recent proteomics study has indicated that FBNs are the most abundant proteins in the plastoglobules of Arabidopsis leaf rosettes, and six FBNs, including the four major ones (FBN1a, 1b, 2 and 4), account for 53% of the plastoglobule protein mass in Arabidopsis (Lundquist et al., 2012). The addition of a bell pepper FBN orthologous to Arabidopsis FBN1a to carotenoids and polar lipids in the same stoichiometric ratio found in vivo reconstituted the fibrils observed in bell pepper fruit chromoplasts (Deruere et al., 1994), and over-expression of this FBN led to *Manuscript text Click here to view linked References 2 an increase in the number of plastoglobules organized into clusters in tobacco chloroplasts (Rey et al., 2000). In light of these results, it has been hypothesized that FBN may prevent plastoglobule coalescence and favour their clustering by acting as an interface between the aqueous environment and lipids as well as by mediating cross-linking via an unknown mechanism (Rey et al., 2000). In this work we demonstrate an interaction between Arabidopsis FBN1a and FBN1b proteins, as well as FBN1a-FBN1a and FBN1b-FBN1b interactions. Yeast Two-Hybrid analyses indicate that these interactions involve the N-terminal part of one protein and the C-terminal part of the other in a head- to-tail mechanism, thereby suggesting the possibility that these proteins form filaments in vivo. These interactions provide a mechanism to explain the function of these FBNs in fibril formation or the maintenance of plastoglobule clusters. Materials and Methods Plasmid construction. Full-length ORFs from FBN1a and FBN1b were cloned (without the stop codon) into the pDONR221 entry vector (Invitrogen) by a BP reaction (Invitrogen). After sequence verification, the inserts were transferred into the binary vectors pXNGW (-nYFP) and pXCGW (-cCFP) (Yuan et al., 2013), respectively, for the BiFC assay using LR Clonase II (Invitrogen). This resulted in translational fusions between the ORFs and the YFP/CFP moieties driven by the CaMV 35S promoter. For Y2H screening, a cDNA fragment coding for amino acids 71 to 144 of FBN1b was amplified from Arabidopsis cDNA using the primers FBN1B_Y2H_F 3 and FBN1b_Y2H_R (see list of primers in Table S1). These primers introduced NdeI and EcoRI restriction sites at the 5’- and 3’-ends of the fragment, respectively. The fragment amplified was cloned into the yeast vector pGBKT7 (Clontech) by restriction with NdeI and EcoRI and subsequent ligation. For the BiFC assays of the N- and C-terminal part of FBN1b, the cDNA fragment coding for the FBN1b chloroplast transit peptide (CTP) was fused to the fragment coding for amino acids 71 to 144 by two sequential steps of PCR amplifications using cDNA as probe and primers FBN1b_CTP-bait_F and FBN1b_CTP-bait_R in the first step, and the products of the first PCR amplification and primers FBN1b_F and FBN1b_CTP-bait_GW_R in the second step (see primers list in Table S1). The C-terminal part of FBN1b (amino acid residues 220 to the end) was fused to the CTP region using the same strategy with the primers FBN1b_CTP-Ct_F and FBN1b_CTP-Ct_R in the first PCR amplification and FBN1b_F and FBN1b_R in the second step. Once the N- and the C-terminal regions of FBN1b had been fused to the CTP, both constructs were cloned into the binary vectors pXNGW (-nYFP) and pXCGW (-cCFP), respectively, using LR Clonase II (Invitrogen). The full-length ORF of VTE1 (At4g32770) gene was amplified from leaves mRNA by RT-PCR using the primers described in Table S1, and cloned into the pDONR221 entry vector (Invitrogen) by a BP reaction (Invitrogen). After sequence verification, the insert was transferred into the binary vectors pXNGW (-nYFP) and pEarlyGate103 (Earley, et al., 2006), for BiFC assays and fusion to GFP marker respectively. 4 Transient expression in Nicotiana benthamiana. The corresponding vectors were electroporated into Agrobacterium tumefaciens strain C58 (Wood et al., 2001). The saturated overnight bacterial cultures carrying the GFP or the YFP/CFP construct moieties were each adjusted to a final O.D.600nm of 0.2 and then co-infiltrated with equal amounts of an Agrobacterium suspension carrying a p19 suppressor of post-transcriptional gene silencing, following the method of Silhavy et al. (Silhavy et al., 2002). The Agrobacterium suspensions were then infiltrated into the leaves of three- to four-week-old N. benthamiana plants as described previously (Marillonnet et al., 2005). The infiltrated plants were kept in a controlled growth chamber under the above conditions for two days until analysis by confocal microscopy. Yeast Two-Hybrid screening. Screening of a commercial, normalized, Arabidopsis cDNA library in the yeast expression vector pGADT7-RecAB (Clontech) was performed using the Matchmaker Gold Yeast Two-Hybrid system (Clontech), following the manufacturer’s instructions. The bait used in the screening was a fragment corresponding to the N-terminal part of FBN1b (amino acid residues 71 to 144). The cDNA encoding for the bait was cloned into the yeast cloning vector pGBKT7 as described above. Confocal microscopy. A DM6000 confocal laser scanning microscope (Leica Microsystems) equipped with a 63x water-immersion objective was used to examine protein-protein interactions in BiFC assays involving N. benthamiana mesophyll cells. YFP/CFP expression and chlorophyll autofluorescence imaging 5 was performed by excitation with a 488 nm argon laser; fluorescence was detected at 500-525 and 630-690 nm, respectively. Results and Discussion Searching for proteins that interact with FBN1b using the yeast two-hybrid (Y2H) system. Previous studies from our group have identified the interaction of FBN1b with a protein involved in starch metabolism (unpublished results). In order to continue with this analysis, we searched for other proteins that could interact with FBN1b by screening an Arabidopsis cDNA library using the Y2H system and a fragment of the N-terminal part of FN1b (residues 71 to 144) as bait. We selected this fragment because it contains a hydrophilic domain (corresponding to Block 1) defined by Laizet et al. (2004) and may therefore be involved in functions distinct from the interaction with the lipids of plastoglobules, such as protein-protein interactions. The cDNA fragment coding for the FBN1b fragment was fused to the DNA binding domain of the yeast GAL4 transcription factor by cloning into the expression vector pGBKT7 and subsequent transformation into the yeast strain Y2HGOLD (Materials and Methods). Expression of this construct was confirmed by immunoblotting yeast crude extracts employing polyclonal antibodies against the GAL4 DNA binding domain (data not shown). The fragment was used to screen a normalized, commercial Arabidopsis cDNA library in the pGADT7-Rec yeast expression vector (Clontech). Although 35 positive clones that grew on selective medium were obtained, 22 of these encoded for proteins with an 12 (2007) Fibrillin influence on plastid ultrastructure and pigment content in tomato fruit. Phytochem 68: 1545–1556 Singh DK, McNellis TW (2011) Fibrillin protein function: the tip of the iceberg? Trends Plant Sci 16: 432–441 Vidi PA, Kanwischer M, Baginsky S, Austin JR, Csucs G, Dörmann P, Kessler F, Bréhélin C (2006) Tocopherol cyclase (VTE1) localization and vitamin E accumulation in chloroplast plastoglobule lipoprotein particles. J Biol Chem 281: 11225–11234. Vidi PA, Kessler F, Bréhélin C (2007) Plastoglobules: a new address for targeting recombinant proteins in the chloroplast. BMC Biotechnol 7: 4 Winkenbach F, Falk H, Liedvogel B, Sitte P (1976) Chromoplasts of Tropaeolum majus L.: Isolation and characterization of lipoprotein elements. Planta 128: 23–28 Wood DW, Setubal JC, Kaul R, Monks DE, Kitajima JP, Okura VK, Zhou Y, Chen L, Wood GE, Almeida NF, et al (2001) The genome of the natural genetic engineer Agrobacterium tumefaciens C58. Science 294: 2317–2323 Ytterberg AJ, Peltier J-B, van Wijk KJ (2006) Protein profiling of plastoglobules in chloroplasts and chromoplasts. A surprising site for differential accumulation of metabolic enzymes. Plant Physiol 140: 984–997 Yuan L, Gu R, Xuan Y, Smith-Valle E, Loqué D, Frommer WB, WiréN von N (2013) Allosteric regulation of transport activity by heterotrimerization of Arabidopsis ammonium transporter complexes in vivo. Plant Cell 25: 974– 13 984 14 Figure Legends Figure 1. Localization of FBN1b fused to GFP in Nicotiana benthamiana chloroplasts. cDNA coding for the full-length FBN1b protein was fused to GFP protein and transiently expressed in N. benthamiana leaves. A: GFP fluorescence. B: chlorophyll autofluorescence. C: combination of images A and B. Figure 2. Interaction between FBN1b and FBN1a. cDNAs coding for the fulllength FBN1b (Panels A, B and C) and FBN1a proteins (Panels D, E, and F) were fused to the N-terminal half of YFP and co-transformed in N. benthamiana leaves together with the cDNA coding for FBN1b fused to the C-terminal moiety of CFP. A and D correspond to YFP/CFP fluorescence and B and E to chlorophyll autofluorescence. C and F are combinations of images A and B, and D and E, respectively. Figure 3. FBN1a and FBN1b clones found in Y2H screening. The amino acid sequences of the different clones of FBN1a and FBN1b identified in the Y2H screening of an Arabidopsis cDNA library are aligned with the FBN1a and FBN1b polypeptides. The initial amino acid of each clone is indicated by a number. Clones of the FBN1b gene are indicated by light grey boxes; dark grey boxes indicate FBN1a clones. Black boxes indicate the sequences encoding for the chloroplast transit peptide of both genes. The FBN1b fragment used as bait in the Y2H screening is indicated. The minimal fragment of FBN1a and FBN1b necessary to interact with the bait is delimited by two vertical parallel lines. Figure 4. Interaction between the N- and C-terminal parts of FBN1b. The cDNA fragment of FBN1b gene used in the Y2H screening (amino acids 71 to 144) was fused to the FBN1b CTP region and to the N-terminal half of YFP and co-transformed in N. benthamiana leaves together with the cDNA coding for the C-terminal part of FBN1b determined previously (amino acids 220 to the end) (see Figure 3) fused to the FBN1b CTP region and to the C-terminal moiety of CFP. A: YFP/CFP fluorescence. B: chlorophyll autofluorescence. C: combination of images A and B. A scheme of both constructs is indicated. N- 15 FBN1b: N terminal part of FBN1b. C-FBN1b: C-terminal part of FBN1b. Numbers indicate amino acid positions in the FBN1b polypeptide. 16 Table 1. Plastidial proteins found in Y2H screening of an Arabidopsis library using the N-terminal part of FBN1b as bait Number of independent clones AGI Code Protein 1 Atcg00810 Ribosomal protein L22 1 At4g04640 ATPase, F1 complex, gamma subunit protein 1 At3g04790 Ribose 5-P isomerase 2 At4g04020 Fibrillin1a 4 At4g22240 Fibrillin1b Table Figure Click here to download high resolution image Figure Click here to download high resolution image Figure Click here to download high resolution image Figure Click here to download high resolution image