Calmodulin is involved in the dual subcellular location of two chloroplast proteins

Abstract

Cell compartmentalization is an essential process by which eukaryotic cells separate and control biological processes. Although calmodulins are well-known to regulate catalytic properties of their targets, we show here their involvement in the subcellular location of two plant proteins. Both proteins exhibit a dual location, namely in the cytosol in addition to their association to plastids (where they are known to fulfil their role). One of these proteins, ceQORH, a long-chain fatty acid reductase, was analyzed in more detail, and its calmodulin-binding site was identified by specific mutations. Such a mutated form is predominantly targeted to plastids at the expense of its cytosolic location. The second protein, TIC32, was also shown to be dependent on its calmodulin-binding site for retention in the cytosol. Complementary approaches (bimolecular fluorescence complementation and reverse genetics) demonstrated that the calmodulin isoform CAM5 is specifically involved in the retention of ceQORH in the cytosol. This study identifies a new role for calmodulin and sheds new light on the intriguing CaM-binding properties of hundreds of plastid proteins, despite the fact that no CaM or CaM-like proteins were identified in plastids.

Keywords: Chloroplast envelope; Subcellular location; calmodulin (CaM); chloroplast; plant biochemistry; plant molecular biology; subcellular fractionation; subcellular organelle.

Figure 1.

Interaction of the natural plant ceQORH protein with calmodulin. A, affinity purification of Arabidopsis ceQORH from crude plant cell extracts. Purification was performed on a CaM affinity resin (Stratagene). Lane M, prestained protein molecular weight markers; lane CE, crude solubilized plant proteins diluted in CaM-binding buffer containing 0.1% Nonidet P-40; lane P; unbound proteins; lane W, wash; lane E, four successive elution fractions are presented. B, production of recombinant Arabidopsis ceQORH and E. coli K12 QOR (ecQOR) proteins in E. coli SDS-PAGE analysis of crude bacterial extracts containing Arabidopsis ceQORH or E. coli ecQOR proteins. S, soluble fraction of the crude bacterial proteins; I, insoluble fraction of the crude bacterial proteins (inclusion bodies). C, affinity purification of Arabidopsis ceQORH and E. coli ecQOR produced in bacteria (see B). Purification was performed on a CaM affinity resin (Stratagene). As a control, the bacterial ecQOR protein was also tested. Lane S, soluble bacterial proteins diluted in CaM-binding buffer; lane P, unbound proteins; lane W, wash; lane E, pooled elution fractions. Note that the recombinant Arabidopsis ceQORH protein interacts with the CaM affinity resin (and is thus eluted from the column), whereas this is not the case for the recombinant E. coli ecQOR protein.

Figure 2.

The CaM-binding domain is located in the C terminus of ceQORH. A, scheme of the successive deletions and constructs used to localize the CaM-binding domain in the ceQORH sequence. Sequences of the various deletions in ceQORH are indicated. Construct 1, ceQORH protein; construct 2, ceQORH-GFP (full-length ceQORH protein fused to GFP); construct 3, D(1–59)ceQORH-GFP (ceQORH lacking 60 residues in N terminus fused to GFP); construct 4, D(1–99)ceQORH-GFP (ceQORH lacking 100 residues in N terminus fused to GFP); construct 5, (6–100)ceQORH-GFP (ceQORH lacking both 6 residues in N terminus and 229 residues in C terminus fused to GFP); construct 6, (60–100)ceQORH-GFP (ceQORH lacking both 60 residues in N terminus and 229 residues in C terminus fused to GFP); construct 7, D(280–329)ceQORH-GFP (ceQORH lacking 49 residues in N terminus fused to GFP); construct 8, D(229–329)ceQORH-GFP (ceQORH lacking 100 residues in N terminus fused to GFP); and construct 9, D(175–329)ceQORH-GFP (ceQORH lacking 146 residues in N terminus fused to GFP). The CAM-binding domain in the ceQORH sequence is deduced from ability of the various constructs to interact with CaM (+ means CaM-binding, and − means lack of CaM binding) and supported by results of experiments shown in B and C. B, SDS-PAGE. Lanes 1–6 are crude bacterial extracts containing recombinant ceQORH fusions as described for A. Lanes M, prestained protein molecular weight markers. Western blotting was performed using a primary antibody raised against GFP (1/70,000), an anti-rabbit HRP-conjugated secondary antibody (1/10,000) and ECL. For CaM overlay, after transfer, washing and saturation, the membrane was incubated in the hybridization solution containing 0.1 μg/ml biotinylated CAM. Detection of the bound CaM protein was performed using a streptavidin–HRP conjugate and ECL. C, successive deletions in the C terminus of ceQORH to localize its CaM-binding domain. For SDS-PAGE, lanes 7–9 are crude bacterial extracts containing recombinant ceQORH fusions as described in A. M, prestained protein molecular weight markers. With CaM overlay, after transfer, washing, and saturation, the membrane was incubated in the hybridization solution containing 0.3 μg/ml of CaM-HRP conjugate. Detection of the bound CaM protein was performed using ECL.

Figure 3.

Targeted mutagenesis identifies essential residues required for CaM-binding properties of ceQORH. A, scheme of the residues selected for targeted mutagenesis of the ceQORH sequence. The CAM-binding region in the ceQORH sequence is deduced from successive deletions of ceQORH domains (see Fig. 2). B, SDS-PAGE and CaM overlay. Lanes Ec, purified recombinant QOR from E. coli K12 used as a negative control; lanes M, prestained protein molecular weight markers; lanes 1, purified recombinant WT form of ceQORH from A. thaliana used as a positive control; lanes 10, purified mutagenized form of Arabidopsis ceQORH mutant 1; lanes 11, purified mutagenized form of Arabidopsis ceQORH mutant 2; lanes 12, purified mutagenized form of Arabidopsis ceQORH mutant 3. Note that, in Mut2, mutagenesis of only three residues is sufficient to abolish interaction of ceQORH with CaM.

Figure 4.

The ceQORH CaM-binding domain is neither essential for ceQORH targeting to parenchymal cell chloroplasts nor required for specific localization of ceQORH to the plastid envelope. A–C, lack of CaM-binding domain does not affect envelope localization of ceQORH. Cell fractionation was performed on plants stably expressing in A, ceQORH-GFP (ceQORH fused to GFP); in B, Mut2-ceQORH-GFP (ceQORH mutant affected in CaM-binding properties fused to GFP); and in C, 6–100-ceQORH-GFP (ceQORH lacking its 197 C-terminal residues, including the CaM-binding domain, fused to GFP). Lanes M, prestained protein molecular weight markers; lanes CE, crude cell extract; lanes Cp, chloroplast extract; lanes E, envelope; lanes S, stroma; lanes T, thylakoids. Note that to limit artifacts resulting from overexpression of the various ceQORH constructs, transgenic plants were selected for expression levels of recombinant proteins similar to endogenous ceQORH level. Each lane contains 20 μg of proteins. RBCL, large subunit of RuBisCO (stroma marker). LHCP, light-harvesting complex proteins (thylakoid membrane marker). TPT, Triose-P/phosphate translocator (envelope marker). Western blotting analyses were performed using the antibody raised against ceQORH. D, purified chloroplast envelope fractions do not contain detectable levels of CaM. CaM, 100 to 0.1 ng of purified recombinant CaM1 from Arabidopsis. Lane M, prestained protein molecular weight markers. CE, crude cell extract, envelope (0.6–60 μg of envelope proteins). Note that a second CaM detections experiment is shown, using longer exposure time to improve sensitivity of ECL detection.

Figure 5.

Membranes fractions of epidermal cells from Arabidopsis leaves are enriched in a high-molecular-weight CaM isoform when compared with crude leaf extract. A, detection of CaM isoforms in crude leaf extracts (CE) and epidermal tissue from Arabidopsis leaves. Western blotting was performed using antibodies raised against ceQORH, LHCP, and CaM-767. B, molecular mass of the CaM isoform enriched in epidermal cells is above (∼20 kDa) the one expected for classical (short) CaM isoforms (∼16 kDa). Epi, 5 μg of proteins from crude epidermal extract from Arabidopsis leaves. Fractionation of membrane and soluble fractions of epidermal tissue reveals that the high-molecular-weight (>20 kDa) CaM isoform is bound to membranes. M, prestained molecular mass markers; CE, 15 μg of proteins from crude leaf extracts from Arabidopsis; epidermal tissue (Epi) 15 μg of proteins from crude epidermal extract from Arabidopsis leaves; Sol, 15 μg of soluble proteins from epidermal tissue; Mb, 15 μg of membrane proteins from epidermal tissue. Note that a second CaM detection experiment is shown using longer exposure time to improve sensitivity of ECL detection. C, CaM is more abundant in epidermal cells (1/400 to 1/800 of all epidermal proteins) when compared with crude leaf extract (1/2000 to 1/20,000 of all cell proteins). CaM, 100 to 0.1 ng of purified recombinant CaM1 from Arabidopsis. M, prestained protein molecular weight markers; Epi, 4 μg of proteins from crude epidermal extract; CE, 20 μg of proteins from crude leaf extract. D, alignment of C termini from classical short CaM isoform (e.g. CaM1) with the CaM isoform identified in the PM from petunia (CaM53-Pet) and its closest homolog in Arabidopsis (CaM5-Ath). Note that CaM5 from Arabidopsis and CaM53 from petunia contain an additional C terminus sequence when compared with short CaM isoforms (CaM1). Conserved residues are bold (black for identity and gray for similarity). The underlined C residue is the CaM53 isoprelynation site.

Figure 6.

CaM5 and ceQORH colocalize in epidermal cells. Confocal microscopy was performed on plants stably expressing ceQORH-GFP (ceQORH fused to GFP) and transiently expressing CaM5 fused to cyan fluorescent protein (CaM5-CFP). Chlorophyll, chlorophyll autofluorescence. Overlay, overlay of all three channels. Bar, 40 μm. Note that although CaM5 and ceQORH are colocated at the periphery of leaf cells, only CaM5 accumulates within the nucleus.

Figure 7.

Identification of the CaM isoform interacting with ceQORH: CaM5 and ceQORH interact in epidermal cells. A, confocal imaging (BiFC) performed on plants transiently expressing either ceQORH–YFPC (ceQORH fused to the C terminus of YFP) or Mut2–ceQORH–YFPC (Mut2 version of ceQORH fused to the C terminus of YFP) and CaM5-YFPN (CaM5 sequence fused to the N terminus of YFP). YFP, fluorescence of YFP resulting from BiFC. Chlorophyll, chlorophyll autofluorescence. Overlay, overlay of the two channels. Bar, 40 μm. B, Western blotting analyses performed to validate expression of YFP fusions. Crude cell extracts from WT plants were included as negative controls. Note that although both ceQORH–YFPC and Mut2–ceQORH–YFPC accumulate at a similar level in plant cells, BiFC is only detected using the WT ceQORH version (i.e. containing its CaM-binding domain). Note that BiFC is mostly detected at the periphery of leaf cells, i.e. where CaM5 and ceQORH colocalize within epidermal cells (see Fig. 6).

Figure 8.

ceQORH lacking its CaM-binding domain is targeted to chloroplasts in epidermal cells. Confocal microscopy was performed on plants stably expressing the following: A, GFP (GFP alone as a negative control of plastid localization; bar, 8 μm). B, TP-GFP (transit peptide of the small subunit of RuBisCO fused to GFP as a positive control of plastid localization; bar, 8 μm). C, ceQORH-GFP (plant ceQORH fused to GFP; bar, 10 μm). D, Mut2-ceQORH-GFP (ceQORH lacking its CaM-binding domain; bar, 5 μm). GFP, GFP fluorescence. Chlorophyll, chlorophyll autofluorescence. Overlay, overlay of all three channels.

Figure 9.

CaM5 is the CaM isoform that controls retention of ceQORH at the periphery of plant cells: ceQORH is targeted to the chloroplast envelope in epidermal cells of the cam5 mutant. Confocal microscopy was performed on both WT plants and cam5 mutant stably expressing ceQORH-GFP (plant ceQORH fused to GFP) or Mut2-ceQORH-GFP. ceQORH-GFP, GFP fluorescence. Chlorophyll, chlorophyll autofluorescence. Overlay, overlay of the two channels. The bar indicates 5 μm in the left column and 10 μm in the four right columns.

Figure 10.

CaM5 control of chloroplast targeting is not limited to ceQORH. A, like ceQORH, Tic32 accumulates at the periphery of epidermal cells when containing its CaM-binding domain. B, Tic32 lacking its CaM-binding domain is targeted to chloroplasts in epidermal cells. Confocal microscopy was performed on plants stably expressing Tic32-GFP (Tic32 fused to GFP; bar, 10 or 20 μm) and Del-Tic32-GFP (Tic32 protein lacking its C terminus; bar, 20 μm). GFP, GFP fluorescence. Chlorophyll, chlorophyll autofluorescence. Overlay, overlay of the two channels.

Figure 11.

Comparison of the CaM-binding domain of ceQORH from Arabidopsis and spinach to the major classes of CaM-binding domains. h indicates a hydrophobic residue, and B indicates a basic residue. In the classical nomenclature of these classes, each number is positioning a hydrophobic residue in their consensus sequences (see the work of Yap et al. (23) and Tidow and Nissen (30)). The ceQORH peptides show closest similarity to the 1-16 class, which contains two isoforms of a calcium/calmodulin dependent protein kinase kinase (CaMKKa and CaMKKb). Other classes are the 1-14 class (comprising the 1-14, 1-8-14, basic 1-8-14, and 1-5-8-14 subclasses), the 1-10 class (comprising the 1-10, 1-5-10, basic 1-5-10, and hydrophilic 1-4-10 subclasses), and the IQ class, to which the ceQORH CaM-binding domain appears more distally related. Residues that were mutagenized in ceQORH are indicated (dark blue in Mut1-ceQORH, light blue in Mut2-ceQORH, and both dark and light blue in Mut3-ceQORH).