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 Ca²⁺ 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 Ca²⁺-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 Arg³⁹²Arg³⁹³Arg³⁹⁴Lys³⁹⁵ is essential but a less conserved basic tetrapeptide Arg⁴⁰¹Arg⁴⁰²Arg⁴⁰³Arg⁴⁰⁴ is dispensable for the quality control function of CRT3 that retains bri1-9 in the ER and facilitates the complete folding of EFR.

Keywords: :

• receptor kinase
• chaperone-like lectin
• brassinosteroid
• protein quality control
• protein folding
• plant innate immunity

Disclosure of Potential Conflicts of Interest

There is no potential conflict of interest for this study.

Acknowledgment

We would like to thank the Arabidopsis Biological Resource Center at the Ohio State University for supplying the ebs2-8 mutant (SALK_051336) and members of the Li lab for stimulating discussion throughout this study. Research on the CRT3 project was supported by grants from National Institutes of Health (grant number GM060519) and the National Science Foundation (grant number IOS1121496) to J.L.