Abstract
Plastid proteins that are encoded by the nuclear genome and synthesized in the cytosol undergo posttranslational targeting to plastids. Ankyrin repeat protein 2A (AKR2A) and AKR2B were recently shown to be involved in the targeting of proteins to the plastid outer envelope. However, it remains unknown whether other factors are involved in this process. In this study, we investigated a factor involved in AKR2A-mediated protein targeting to chloroplasts in Arabidopsis (Arabidopsis thaliana). Hsp17.8, a member of the class I (CI) cytosolic small heat shock proteins (sHsps), was identified in interactions with AKR2A. The interaction between Hsp17.8 and AKR2A was further confirmed by coimmunoprecipitation experiments. The carboxyl-terminal ankyrin repeat domain of AKR2A was responsible for AKR2A binding to Hsp17.8. Other CI cytosolic sHsps also interact with AKR2A to varying degrees. Additionally, Hsp17.8 binds to chloroplasts in vitro and enhances AKR2A binding to chloroplasts. HSP17.8 was expressed under normal growth conditions, and its expression increased after heat shock. Hsp17.8 exists as a dimer under normal physiological conditions, and it is converted to high oligomeric complexes, ranging from 240 kD to greater than 480 kD, after heat shock. High levels of Hsp17.8 together with AKR2A resulted in increased plastid targeting of Outer Envelope Protein7 (OEP7), a plastid outer envelope protein expressed as a green fluorescent protein fusion protein. In contrast, artificial microRNA suppression of HSP17.8 and closely related CI cytosolic sHSPs in protoplasts resulted in a reduction of OEP7:green fluorescent protein targeting to plastids. Based on these data, we propose that Hsp17.8 functions as an AKR2A cofactor in targeting membrane proteins to plastid outer membranes under normal physiological conditions.
Highlights
Plastid proteins that are encoded by the nuclear genome and synthesized in the cytosol undergo posttranslational targeting to plastids
One protein was identified as Hsp17.8, a protein belonging to the class I (CI) small heat shock proteins (sHsps) (Scharf et al, 2001; Sun et al, 2002; Basha et al, 2010)
In protoplasts transformed with three plasmids, Outer Envelope Protein7 (OEP7):GFP, HA:Ankyrin repeat protein 2A (AKR2A), and HSP17.8:HA, the amount of OEP7: GFP was increased to 143% compared with transformation with OEP7:GFP alone (Fig. 9, A and B). These results suggest that a portion of OEP7:GFP is subject to proteolytic degradation in protoplasts and that coexpression of both proteins, HA:AKR2A and Hsp17.8:HA, prevents OEP7:GFP degradation (Fig. 9, C and D)
Summary
Plastid proteins that are encoded by the nuclear genome and synthesized in the cytosol undergo posttranslational targeting to plastids. Artificial microRNA suppression of HSP17.8 and closely related CI cytosolic sHSPs in protoplasts resulted in a reduction of OEP7:green fluorescent protein targeting to plastids. Based on these data, we propose that Hsp17.8 functions as an AKR2A cofactor in targeting membrane proteins to plastid outer membranes under normal physiological conditions. In addition to acting as molecular chaperones for unfolded proteins, sHsps have been reported to interact with lipids and to function in membrane quality control (Coucheney et al, 2005; Chowdary et al, 2007; Nakamoto and Vıgh, 2007; Balogi et al, 2008) These findings expand the physiological roles of sHsps. AKR2A and AKR2B recognize the chloroplast outer membrane proteintargeting signals and deliver them to the chloroplast outer membranes (Bae et al, 2008)
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