Chloroplast import motor subunits FtsHi1 and FtsHi2 are located on opposite sides of the inner envelope membrane

Protein import into chloroplasts is powered by ATP hydrolysis in the stroma. Establishing the identity and functional mechanism of the stromal ATPase motor that drives import is critical for understanding chloroplast biogenesis. Recently, a complex consisting of Ycf2, FtsHi1, FtsHi2, FtsHi4, FtsHi5, FtsH12, and malate dehydrogenase was shown to be important for chloroplast protein import, and it has been proposed to act as the motor driving protein translocation across the chloroplast envelope into the stroma. To gain further mechanistic understanding of how the motor functions, we performed membrane association and topology analyses on two of its subunits, FtsHi1 and FtsHi2. We isolated cDNA clones encoding FtsHi1 and FtsHi2 preproteins to perform in vitro import experiments in order to determine the exact size of each mature protein. We also generated antibodies against the C-termini of the proteins, i.e., where their ATPase domains reside. Protease treatments and alkaline and high-salt extractions of chloroplasts with imported and endogenous proteins revealed that FtsHi1 is an integral membrane protein with its C-terminal portion located in the intermembrane space of the envelope, not the stroma, whereas FtsHi2 is a soluble protein in the stroma. We further complemented an FtsHi1-knockout mutant with a C-terminally tagged FtsHi1 and obtained identical results for topological analyses. Our data indicate that the model of a single membrane-anchored pulling motor at the stromal side of the inner membrane needs to be revised and suggest that the Ycf2-FtsHi complex may have additional functions.