Subunits CFo-I and CFo-II of ATP synthase in chloroplast thylakoid membranes are two structurally and functionally closely related proteins of bitopic membrane topology which evolved from a common ancestral gene. In higher plants, CFo-I still originates in plastid chromosomes (gene: atpF), while the gene for CFo-II (atpG) was phylogenetically transferred to the nucleus. This gene transfer was accompanied by the reorganization of the topogenic signals and the mechanism of membrane insertion. CFo-I is capable of integrating correctly as the mature protein into the thylakoid membrane, whereas membrane insertion of CFo-II strictly depends on a hydrophobic targeting signal in the transit peptide. This requirement is caused by three negatively charged residues at the N-terminus of mature CFo-II which are lacking from CFo-I and which have apparently been added to the protein only after gene transfer has occurred. Accordingly, the CFo-II transit peptide is structurally and functionally equivalent to typical bipartite transit peptides, capable of also translocating hydrophilic lumenal proteins across the thylakoid membrane. In this case, transport takes place by the Sec-dependent pathway, despite the fact that membrane integration of CFo-II is a Sec-independent, and presumably spontaneous, process.