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© The Rockefeller University Press,
0021-9525/1998//895 $5.00
The Journal of Cell Biology, Volume 141, Number 4,
, 1998 895-904
Articles |
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 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.
Abbreviations used in this paper: chl, chlorophyll; inv, inverse; mS, small subunit of ribulose-1,5-bisphosphate carboxylase; pS, precursor form; Tic, translocon at the inner envelope membrane of chloroplasts; TIM, translocation intermediates; Toc, translocon at the outer envelope membrane of chloroplasts.
Address all correspondence to Prof. Dr. Jürgen Soll, Botanisches Institut, Am Botanischen Garten 1-9, Christian-Albrechts-Universität, D-24118 Kiel, Germany. Tel.: (049)-431-8804210. Fax: (049)-431-8804222. E-mail: jsoll{at}bot.uni-kiel.de
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