Three proteins mediate import of transit sequence-less precursors into the inner envelope of chloroplasts in Arabidopsis thaliana

A family of 17 putative preprotein and amino acid transporters designated PRAT has been identified in Arabidopsis thaliana, comprising PRAT proteins in mitochondria and chloroplasts. Although some PRAT proteins, such as the translocon of the mitochondrial inner membrane (TIM) proteins TIM22 and TIM23, play decisive roles for the translocation and import of mitochondrial inner membrane proteins, little is known about the role of the different PRAT members in chloroplasts. Here we report the identification of three distinct PRAT proteins as part of a unique protein import site. One of the identified PRAT proteins is identical with a previously characterized hypothetical protein (HP) of 20 kDa designated HP20 of the outer plastid envelope membrane. The second PRAT component is represented by HP30, and the third is identical to HP30- 2, a close relative of HP30. Both HP30 and HP30- 2 are inner plastid envelope membrane proteins of chloroplasts. Using biochemical, cell biological, and genetic approaches we demonstrate that all three PRAT proteins cooperate during import of transit sequence- less proteins, such as the quinone oxidoreductase homolog ceQORH used as model, into the inner chloroplast envelope membrane. Our data are reminiscent of findings reported for the TIM22 translocase, which is involved in the import of carrier proteins and other, hydrophobic membrane proteins lacking cleavable transit sequences into the inner mitochondrial membrane. Together our results establish the PRAT family as a widely used system of protein translocases in different membranes of endosymbiotic origin.