Arabidopsis Calmodulin-binding Protein IQ67-Domain 1 Localizes to Microtubules and Interacts with Kinesin Light Chain-related Protein-1

Katharina Bürstenbinder,Tatyana Savchenko,Jens Müller,Aaron W Adamson,Gina Stamm,Raymond Kwong,Brandon J Zipp,Dhurvas Chandrasekaran Dinesh,Steffen Abel

doi:10.1074/jbc.m112.396200

Abstract

Calcium (Ca(2+)) is a key second messenger in eukaryotes and regulates diverse cellular processes, most notably via calmodulin (CaM). In Arabidopsis thaliana, IQD1 (IQ67 domain 1) is the founding member of the IQD family of putative CaM targets. The 33 predicted IQD proteins share a conserved domain of 67 amino acids that is characterized by a unique arrangement of multiple CaM recruitment motifs, including so-called IQ motifs. Whereas IQD1 has been implicated in the regulation of defense metabolism, the biochemical functions of IQD proteins remain to be elucidated. In this study we show that IQD1 binds to multiple Arabidopsis CaM and CaM-like (CML) proteins in vitro and in yeast two-hybrid interaction assays. CaM overlay assays revealed moderate affinity of IQD1 to CaM2 (K(d) ∼ 0.6 μm). Deletion mapping of IQD1 demonstrated the importance of the IQ67 domain for CaM2 binding in vitro, which is corroborated by interaction of the shortest IQD member, IQD20, with Arabidopsis CaM/CMLs in yeast. A genetic screen of a cDNA library identified Arabidopsis kinesin light chain-related protein-1 (KLCR1) as an IQD1 interactor. The subcellular localization of GFP-tagged IQD1 proteins to microtubules and the cell nucleus in transiently and stably transformed plant tissues (tobacco leaves and Arabidopsis seedlings) suggests direct interaction of IQD1 and KLCR1 in planta that is supported by GFP∼IQD1-dependent recruitment of RFP∼KLCR1 and RFP∼CaM2 to microtubules. Collectively, the prospect arises that IQD1 and related proteins provide Ca(2+)/CaM-regulated scaffolds for facilitating cellular transport of specific cargo along microtubular tracks via kinesin motor proteins.

Highlights

Plant-specific IQD genes encode putative CaM targets of unknown functions
Deletion mapping of IQD1 demonstrated the importance of the IQ67 domain for CaM2 binding in vitro, which is corroborated by interaction of the shortest IQD member, IQD20, with Arabidopsis CaM/CaM-like proteins (CMLs) in yeast
Using a genetic yeast two-hybrid interaction screen, we identified kinesin light chain-related-1 (KLCR1) as an IQD1 binding protein in Arabidopsis, which prompted us to reinvestigate the subcellular localization of IQD1 at high resolution

Summary

Background

Plant-specific IQD genes encode putative CaM targets of unknown functions. Results: IQD1 interacts with KLCR1, binds to Arabidopsis CaM/CMLs, and localizes to microtubules. The Ca2ϩ sensor relays undergo conformational changes upon Ca2ϩ binding and transduces the signal via interaction with terminal Ca2ϩ responders, thereby modulating the biochemical activities of these target proteins and establishing a large number of cellular processes and subsequent plant responses [1,2,3]. CaMs and CMLs, encoded by 7 CaM and 50 CML genes in Arabidopsis, regulate a broad range of structurally diverse Ca2ϩ responders that are predicted to exceed 700 target proteins [9, 10] These include, among other functional categories, proteins implicated in generating Ca2ϩ signatures, enzymes in signaling and metabolic pathways, and transcriptional regulators [11, 12]. The first described IQD gene was functionally identified in a screen for Arabidopsis mutants with altered production of glucosinolates [16], a class of secondary metabolites in crucifers whose degradation products possess profound biological activ-

The abbreviations used are

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION