Accumulation of Newly Synthesized F1 in Vivo in Arabidopsis Mitochondria Provides Evidence for Modular Assembly of the Plant F1Fo ATP Synthase

F-1 subcomplex in mitochondrial samples is often considered to be a breakage product of the F1FO ATP synthase during sample handling and electrophoresis. We have used a progressive N-15 incorporation strategy to investigate the plant F1FO ATP synthase assembly model and the apparently free F-1 in plant mitochondria which is found in both the inner membrane and matrix. We show that subunits within F-1 in the inner membrane and matrix had a relatively higher N-15 incorporation rate than corresponding subunits in intact membrane F1FO. This demonstrates that free F-1 was a newer pool with a faster turnover rate consistent with it being an assembly intermediate in vivo. Import of [S-35] Met-labeled F-1 subunit precursors into Arabidopsis mitochondria showed the rapid accumulation of F-1 assembly intermediates. The different N-15 incorporation rate in matrix F-1, inner membrane F-1 and intact F1FO demonstrates these three represent different protein populations and are likely step by step intermediates during the assembly process of plant mitochondrial ATP synthase. The potential biological implications of in vivo accumulation of enzymatically active F-1 in mitochondria are discussed.