엽록체 외막 단백질의 세포질 수용체 AKR2A의 엽록체 결합 기작과 보조인자 HSP17.8의 기능에 관한 연구

Abstract

In plant cells, chloroplasts have essential roles in photosynthesis and in the mediation of biogenesis of many important primary and secondary metabolic processes. Besides the small portion of proteins encoded by the chloroplast genome, most of the ~3000 proteins, which are necessary for composing functional chloroplast, are encoded by the nuclear genome. Thus, it is of great importance to study the mechanism of how nuclear encoded proteins could target to chloroplast. In previous works, the mechanisms of protein targeting to chloroplast have been largely focused on the translocation mechanisms of the proteins containing a transit peptide which is the sorting signal for chloroplast targeting. It was also reported that the ankyrin repeat domain-containing protein AKR2A was involved in the targeting of chloroplast outer envelop membrane proteins lacking a transit peptide. However, several questions still remain. First, what is the AKR2A receptor located on the chloroplast outer membrane? Second, what other factors are involved in this process?I demonstrated that AKR2A specifically recognized MGDG (monogalactosyldiacylglycerol), which was highly concentrated in chloroplasts, thereby delivered chloroplast outer envelope membrane proteins (C-OEPs) to chloroplasts. An in vitro binding assay revealed that AKR2A is specifically bound to MGDG with the initial three C-terminal ankyrin repeat domain. In addition, in the mgd1-3 mutant that had lower amount of MGDG, AKR2A displayed reduced chloroplast binding activity and reduced chloroplast targeting efficiency of OEP7:GFP, in which the GFP was fused to the C-terminus of AtOEP7. All these data strongly suggest that AKR2A recognizes chloroplast-specific lipid MGDG for trafficking of nascent C-OEPs to chloroplast.I screened a factor involved in the AKR2A-mediated protein targeting to chloroplasts through protein pull-down experiments and MALDI-TOF analysis. Hsp17.8, a member of class I (CI) cytosolic small heat shock proteins (sHsps), was identified as an AKR2A-binding protein. The interaction between Hsp17.8 and AKR2A was further confirmed by protein pull-down and coimmunoprecipitation experiments. The C-terminal ankyrin repeat domain of AKR2A was responsible for the AKR2A binding to Hsp17.8. Other CI cytosolic sHsps also interacted with AKR2A to varying degrees. Additionally, Hsp17.8 bound to chloroplasts in vitro and enhanced AKR2A binding to chloroplasts in a dose-dependent manner. HSP17.8 was expressed under normal growth conditions and its expression increased after heat shock. Blue native polyacrylamide gel electrophoresis showed that Hsp17.8 existed as dimers under normal physiological conditions and that it was converted to high oligomeric complexes, ranging from 240 kD to greater than 480 kD, after heat shock. In protoplasts, high levels of Hsp17.8 together with AKR2A resulted in increased plastid targeting of OEP7, a plastid outer envelope protein expressed as a GFP fusion protein. In contrast, artificial microRNA suppression of HSP17.8 together with closely related CI cytosolic sHSP genes in protoplasts resulted in reduction of OEP7:GFP targeting to plastids. Based on these data, I propose that Hsp17.8 functions as an AKR2A cofactor in targeting membrane proteins to plastid outer membranes under normal physiological conditions.