Institute of Molecular Biology, Academia Sinica
○Chwan-Deng Hsiao Yuh-Ju Sun Farhad Forouhar Hsou-min Li
Protein import into chloroplasts is regulated by the binding and hydrolysis of GTP at two homologous GTPase, Toc34 and Toc159. Toc34, a 34-kDa integral membrane protein, is a member of the Toc (translocon at the outer-envelope membrane of chloroplasts) complex that associates with precursor proteins during protein transport across the chloroplasts outer membrane. Here we report the crystal structure of the cytosolic part of pea Toc34 complexed with GDP and Mg2+ at 2.0 Å resolution. In the crystal, the Toc34 molecules exist as dimers with features resembling the ones found in a small GTPase complexed with a GTPase activating protein (GAP). Gel-filtration, however, revealed that dimeric and monomeric forms of Toc34 coexisted in phosphate saline buffer (pH 7.2) solution. Mutation of Arg128, an essential residue for dimerization, to alanine led to the formation of only a monomeric form whose GTPase activity is significantly reduced compared to that of the wide-type Toc34. These results together with a number of the structural features unique to Toc34, suggest that each monomer acts as a GAP on the other interacting monomer. In addition, the crystal structure of the Toc34 GTP-binding domain suggests that GTP-regulated dimerization of the Toc GTPase domains controls the targeting and translocation of preproteins at the chloroplast envelope.