A conserved basic residue cluster is essential for the protein quality control function of the Arabidopsis calreticulin 3

Abstract

Calreticulin (CRT) is a highly conserved chaperone-like lectin that regulates Ca(2+) homeostasis and participates in protein quality control in the endoplasmic reticulum (ER). Most of our CRT knowledge came from mammalian studies, but our understanding of plant CRTs is limited. Many plants contain more than two CRTs that form two distinct groups: CRT1/CRT2 and CRT3. Previous studies on plant CRTs were focused on their Ca(2+)-binding function, but recent studies revealed a crucial role for the Arabidopsis CRT3 in ER retention of a mutant brassinosteroid receptor, brassinosteroid-insensitive 1-9 (bri1-9) and in complete folding of a plant immunity receptor EF-Tu Receptor (EFR). However, little is known about the molecular basis of the functional specification of the CRTs. We have recently shown that the C-terminal domain of CRT3, which is rich in basic residues, is essential for retaining bri1-9 in the ER; however, its role in assisting EFR folding has not been studied. Here, we used an insertional mutant of CRT3, ebs2-8 (EMS mutagenized bri1 suppressor 2-8), in the bri1-9 background as a genetic system to investigate the functional importance of two basic residue clusters in the CRT3's C-terminal domain. Complementation experiments of ebs2-8 bri1-9 with mutant CRT3(M) transgenes showed that a highly conserved basic tetrapeptide Arg(392)Arg (393)Arg(394)Lys(395) is essential but a less conserved basic tetrapeptide Arg(401)Arg(402)Arg(403)Arg(404) is dispensable for the quality control function of CRT3 that retains bri1-9 in the ER and facilitates the complete folding of EFR.

Keywords: brassinosteroid; chaperone-like lectin; plant innate immunity; protein folding; protein quality control; receptor kinase.

Figure 1. Sequence alignment of the Arabidopsis CRT3 and its plant homologs. Amino acid sequence alignment of Arabidopsis CRT3 and its plant homologs was performed using the ClustalW program at Mobyle web tool (http://mobyle.pasteur.fr/cgi-bin/portal.py). Names and accession numbers of the CRT3 homologs used for analyses are as follows: AtCRT3 (NP_563816; At: Arabidopsis thaliana), BrCRT3 (translated from nucleotide sequences EX116073.1, EX026998.1 and EX117522.1, Br: Brassica rapa), VvCRT3 (translated from nucleotide sequence XM_002276397.1, Vv: Vitis vinifera), MtCRT3 (translated from nucleotide sequence BT052978.1, Mt: Medicago truncatula), OsCRT3 (BAC06263; Os: Oryza sativa), GhCRT3 (translated from nucleotide sequence DT563804.1; Gh: Gossypium hirsutum) MdCRT3 (translated from nucleotide sequences CN943087.1 and CV082227.1; Md: Malus domestica), SmCRT3 (translated from nucleotide sequence FE490612.1 and FE490611.1; Sm: Selaginella moellendorffii), AfCRT3 (translated from nucleotide sequences DR940519.1 and DT750168.1, Af: Aquilegia formosa), PtCRT3 (translated from nucleotide sequences BF777977.1, CO198952.1 and CO158387.1; Pt: Pinus taeda), ZmCRT3 (translated from nucleotide sequence AY105822; Zm: Zea mays), HvCRT3 (translated from nucleotide sequence AK248906.1; Hv: Hordeum vulgare) and TaCRT3 (EF452301.1; Ta: Triticum aestivum). Aligned amino acid sequences were shaded using the BoxShade 3.31 web server (http://mobyle.pasteur.fr/cgi-bin/portal.py). Residues identical in more than 8 sequences are shaded in red and similar ones are shaded in cyan. Two basic tetrapeptides in the C terminus of AtCRT3 are marked by blue bar, and the region that has > 50% basic residues is indicated by a green box.

Figure 2. Mutations of the basic Arg³⁹²Arg³⁹³Arg³⁹⁴Lys³⁹⁵ tetrapeptide destroy the activity of the gCRT3M transgene to rescue a T-DNA insertional ebs2 mutation. (A) Images of 5-week-old soil-grown plants of wild-type (WT) plant, bri1-9, ebs2-8 bri1-9 and 4 representative gCRT3^M ebs2-8 bri1-9 transgenic lines. (B) Immunoblot analysis of CRT3^M abundance in transgenic plants. Total protein extracts of 5-week-old soil-grown plants shown in (A) were separated by 10% SDS/PAGE and analyzed by immunoblotting with anti-CRT3 antibody. Coomassie blue staining of a small subunit of the Arabidopsis Rubisco gel was used as a loading control. The protein samples of 4 chosen gCRT3^M ebs2-8 bri1-9 transgenic plants were diluted 2 fold before sample loading.

Figure 3. The Endo-H sensitivity assay of BRI1 and bri1-9. Equal amounts of total proteins extracted from leaves of 5-week-old soil-grown plants shown in Figure 2A were treated with (+) or without (−) Endo Hf for 1.5 h at 37°C, separated by 7% SDS/PAGE and analyzed by immunoblotting with an anti-BRI1 antibody. Coomassie blue staining of RbcS serves as a loading control. Arrows indicated the positions of Endo-H-resistant (bri1-9^R) and Endo-H-sensitive bri1-9 (bri1-9^S) forms.

Figure 4. The basic tetrapeptide Arg³⁹²Arg³⁹³Arg³⁹⁴Lys³⁹⁵ of CRT3 is crucial for the complete folding of the plant immunity receptor EFR. (A) An immunoblot analysis of EFR. Total protein extracts from WT, bri1-9, ebs2-8bri1-9 and 4 selected gCRT3^M ebs2-8 bri1-9 transgenic lines were separated by 10% SDS/PAGE and analyzed by immunoblotting with an anti-EFR antibody. Coomassie blue staining of RbcS serves as a loading control. (B) The efl18 growth inhibition assay. Shown here are images of 7-d-old seedlings of WT, bri1-9, ebs2-8bri1-9 and 4 selected gCRT3^M ebs2-8 bri1-9 transgenic lines grown on half-strength MS agar medium (top panel) and 17-d-old seedlings of the same genotypes after 10-d treatment with 100 nM elf18 (bottom panel). (C) The Endo-H assay of EFR. Equal amounts of total proteins extracted from leaves of 5-week-old soil-grown plants were treated with (+) or without (−) Endo Hf for 1.5 h at 37°C, separated by 7% SDS/PAGE and analyzed by immunoblotting with an anti-EFR antibody. Coomassie blue staining of RbcS serves as a loading control.