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J. Cell Biol.,
Volume 141, Number 4, May 18, 1998 895-904
Botanisches Institut, Christian-Albrechts-Universität Kiel, D-24118 Kiel, Germany
The chloroplastic outer envelope protein
Toc34 is inserted into the membrane by a COOH-terminal membrane anchor domain in the orientation
Ncyto-Cin. The insertion is independent of ATP and a
cleavable transit sequence. The cytosolic domain of
Toc34 does not influence the insertion process and can
be replaced by a different hydrophilic reporter peptide.
Inversion of the COOH-terminal, 45-residue segment,
including the membrane anchor domain (Toc34Cinv), resulted in an inverted topology of the protein, i.e., Nin-Ccyto. A mutual exchange of the charged amino acid
residues NH2- and COOH-proximal of the hydrophobic 
-helix indicates that a double-positive charge at
the cytosolic side of the transmembrane -helix is the sole determinant for its topology. When 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. The inverted peptide domain of Toc34 functions as a stop transfer signal
and is released out of the outer envelope protein translocation machinery into the lipid phase. Simultaneously, the NH2-terminal part of the hybrid precursor
remained engaged in the inner envelope protein translocon, 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.
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