Functional Investigation Of Chloroplast Inner Membrane Transport Protein HP30

Chloroplasts serve as the major place of photosynthesis,lipid metabolism,starch synthesis and amino acids synthesis,meanwhile,the functional chloroplasts are necessary for these processes.Chloroplast biosynthes is involved in many biological processes,such as structural development,expression of chloroplast genome,regulation of nuclear genome,import of nuclear-encoded precursor proteins from cytoplasm and so on.A total of 97%chloroplast proteins are encoded by nuclear genome,synthesized in cytoplasm and then transported into chloroplast.The known protein transport complexes in chloroplast are TOC complex in the outer membrane and TIC complex in the inner membrane,both of them are responsible for importing N-terminal transit peptides-contained precursor proteins.Another protein transport complex is composed of outer membrane protein HP20 and inner membrane proteins HP30 as well as HP30-2,which is proposed for transporting N-terminal transit peptide-less precursor proteins,such as quinone oxidoreductase ce QORH and HP30.But we don’t know whether HP30-HP30-2could also transport N-teiminal transit peptide-contained precursor proteins.PPR proteins play an important role in chloroplast function,such as RNA editing,RNA transcription and RNA stability.The transport mechanism of PPR proteins is still unclear.Our study is aimed at exploring the function and transport mechanism of HP30-HP30-2 complex and how the PPR proteins be imported into chloroplast.In this study,by screening the growth status of arabidopsis mutant library with redundant genes targeted by ami RNA under different CO₂ conditions,several mutants whose growth were severely inhibited under normal and high CO₂ growth conditions were screened out.Among them,ami RNA sequence of the mutant 10-67 showed that the transferred ami RNA targeted two genes,HP30 and HP30-2,whose expression were down-regulated.T-DNA insertion double mutant of these two genes also displayed albino and dwarf phenotypes.A positive correlation between phenotype and expression was assured.Photosynthetic efficiency and chloroplast development were impaired in hp30hp30-2 double mutant while sucrose could restore the albino and dwarf phenotypes of hp30hp30-2.By detecting the RNA editing efficiency of chloroplast genes in hp30hp30-2 double mutant,we found that the RNA editing efficiency of ndh G,ndh F and acc D in hp30hp30-2 was significantly decreased.The expression levels of PPR protein,OTP82,OTP84 and ECB2,which were responsible for RNA editing of the three genes,were significantly increased.By analyzing the subcellular localization of these three PPR proteins in the protoplasts of Col-0 and hp30hp30-2,we found that OTP82-YFP,OTP84-YFP and ECB2-GFP could not be successfully transported into the chloroplast in hp30hp30-2,and thus were degraded in cytoplasm.Replacing the transit peptides of the three proteins with that of FD1 could guide these three proteins importing into chloroplast sucessfully,indicating that OTP82,OTP84 and ECB2 are transported into the chloroplast through different protein transport channels as FD1.Meanwhile,we found that the RNA editing efficiency of ndh G and ndh D as well as the albino and dwarf phenotypes in hp30hp30-2were partially restored by OTP82 or OTP84 import into the chloroplasts.Together,these results demonstrate that PPR proteins with N-terminal signal peptides are transported through the HP30-HP30-2 complex.we also found that HP30 interacted with Tic40 and OTP82 and OTP84 were imported into chloroplast with the help of stroma molecular chaperone,HSC70-2.Our study showed that the complex composed of HP30 and Tic40forms a new transporter,which can transport at least three PPR proteins with N-terminal transit peptides.