Functional analysis of the Hikeshi-like protein and its interaction with HSP70 in Arabidopsis

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Highlights

Abstract

Heat shock proteins (HSPs) refold damaged proteins and are an essential component of the heat shock response. Previously, the 70 kDa heat shock protein (HSP70) has been reported to translocate into the nucleus in a heat-dependent manner in many organisms. In humans, the heat-induced translocation of HSP70 requires the nuclear carrier protein Hikeshi. In the Arabidopsis genome, only one gene encodes a protein with high homology to Hikeshi, and we named this homolog Hikeshi-like (HKL) protein. In this study, we show that two Arabidopsis HSP70 isoforms accumulate in the nucleus in response to heat shock and that HKL interacts with these HSP70s. Our histochemical analysis revealed that HKL is predominantly expressed in meristematic tissues, suggesting the potential importance of HKL during cell division in Arabidopsis. In addition, we show that HKL regulates HSP70 localization, and HKL overexpression conferred thermotolerance to transgenic Arabidopsis plants. Our results suggest that HKL plays a positive role in the thermotolerance of Arabidopsis plants and cooperatively interacts with HSP70.

Introduction

As sessile organisms, plants are exposed to various environmental stresses, among which heat is one of the most critical stressors for the growth and productivity of plants. Heat stress damages plant cells by provoking protein aggregation, reactive oxygen species (ROS) production, and the hyperfluidization of membranes [1]. To adapt to elevated temperatures, plants have developed a wide range of strategies at the cellular and molecular levels. When plants are exposed to heat stress, many heat-inducible genes are upregulated. The transcriptional response to heat stress has been extensively characterized in many model organisms [2]. The heat shock transcription factors (HSFs) are the most well documented protein family involved in the heat stress response [3]. HSFs bind to heat shock elements (HSEs) found in the promoter regions of heat-inducible genes to activate their expression [3].

The expression of heat shock proteins (HSPs) is the most highly conserved mechanism for protecting cells from heat shock damage. HSPs interact with damaged proteins and assist in properly refolding them [2]. HSPs can be classified into several groups based on their protein weight (small, 60, 70, 90, and 100 kDa HSPs), and they are involved in many cellular processes [2]. The HSP70 protein family is highly conserved in eukaryotes, and its biological functions are diverse. Because protein misfolding and aggregation disrupt cellular homeostasis, the HSP70 chaperone system is crucial during both the stress response and development, and it has therefore been a therapeutic target in humans [4], [5], [6]. Five cytosolic/nuclear HSP70s have been identified in Arabidopsis, and their amino acid sequences are highly conserved [7], [8]. Because cytosolic/nuclear HSP70 isoforms most likely function in a redundant manner, loss-of-function analyses of cytosolic/nuclear HSP70 may be difficult [9]. Consequently, little is known about HSP70-mediated cellular stress responses in plants despite their essential role in thermotolerance [9].

Among all organelles, the nucleus is the most essential compartment. Protecting nuclear proteins from heat shock damage is likely an important mechanism for survival under heat stress conditions [6], [10]. Recently, an HSP70 nuclear import carrier protein named Hikeshi was identified in human cells [11]. Hikeshi plays an important role in cellular protection against heat-induced damage via the nuclear translocation of HSP70 in human cells. Because this Hikeshi-mediated HSP70 nuclear import pathway appeared to be a fundamental mechanism for heat stress tolerance, we searched for a Hikeshi ortholog in the Arabidopsis genome.

In this study, we identify an Arabidopsis ortholog to Hikeshi, Hikeshi-like protein (HKL), and show that it interacts with Arabidopsis HSP70 isoforms and is involved in the regulation of the subcellular localization of HSP70. Our histochemical expression analysis reveals that HKL is predominantly expressed in meristematic tissues in a heat-dependent manner. Moreover, transgenic plants overexpressing HKL showed higher thermotolerance. Our results suggest that HKL plays a positive role in the protection of cells under heat stress in cooperation with HSP70 proteins.

Section snippets

Plant materials and growth conditions

The plants (Arabidopsis thaliana Columbia ecotype) were grown on GM agar plates under a 16 h light/8 h dark cycle at 22 °C for 2 weeks. Transgenic plants and control plants were grown on GM agar plates containing kanamycin (20 mg/l) for 2 weeks.

Plant transformation

To generate the 35S:sGFP-HKL construct, full-length coding sequences were amplified using the primers listed in the Supplemental Table 1, and the fragments were cloned into the pGreen0029 El2-35S-Ω Ns-GFP vector [12]. After confirming the sequence, the

Analysis of HKL expression and tissue specificity

To identify a Hikeshi gene in Arabidopsis, we searched the Arabidopsis genome for genes that encoded proteins similar to Hikeshi. Our database search indicated that only one gene encodes a protein with high homology to Hikeshi, and the amino acid sequence of this protein was partially conserved with that of Hikeshi (Fig. S1A). We named the Hikeshi homolog Hikeshi-like protein (HKL). To determine whether this homologous protein is conserved in both animals and plants, we searched for orthologous

Acknowledgments

We thank Y. Tanaka and S. Murasaki for technical assistance and E. Toma for skillful editorial assistance. We also thank K. Kidokoro for material support and his helpful discussion, and T. Yoshida for his fruitful discussion. This work was supported by a Grant-in-Aid for Scientific Research on Innovative Areas (No. 22119004) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Program for the Promotion of Basic Research Activities for Innovative Biosciences

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