POSITIVE CHARGES DETERMINE THE TOPOLOGY AND FUNCTIONALITY OF THE TRANSMEMBRANE DOMAIN IN THE CHLOROPLASTIC OUTER ENVELOPE PROTEIN TOC34

The chloroplastic outer envelope protein Toc34 is inserted into the me mbrane by a COOH-terminal membrane anchor domain in the orientation N- cyto-C-in. The insertion is independent of ATP and a cleavable transit sequence. The cytosolic domain of Toc34 does not influence the insert ion process and can be replaced by a different hydrophilic reporter pe ptide. Inversion of the COOH-terminal, 45-residue segment, including t he membrane anchor domain (Toc34Cinv) resulted in an inverted topology of the protein, i.e. N-in-C-cyto, A mutual exchange of the charged am ino acid residues NH2- and COOH-proximal of the hydrophobic alpha-heli x indicates that a double-positive charge at the cytosolic side of the transmembrane alpha-helix is the sole determinant for its topology. W hen the inverted COOH-terminal segment was fused to the chloroplastic precursor of the ribulose-1,5-bisphosphate carboxylase small subunit ( pS34Cinv), it engaged the transit sequence-dependent import pathway. T he inverted peptide domain of Toc34 functions as a stop transfer signa l and is released out of the outer envelope protein translocation mach inery into the lipid phase. Simultaneously, the NH2-terminal part of t he hybrid precursor remained engaged in the inner envelope protein tra nslocon, which could be reversed by the removal of ATP, demonstrating that only an energy-dependent force but no further ionic interactions kept the precursor in the import machinery.