Dimerization is important for the GTPase activity of chloroplast translocon components atToc33 and psToc159

J Biol Chem. 2007 May 4;282(18):13845-53. doi: 10.1074/jbc.M608385200. Epub 2007 Mar 1.

Abstract

Arabidopsis Toc33 (atToc33) is a GTPase and a member of the Toc (translocon at the outer-envelope membrane of chloroplasts) complex that associates with precursor proteins during protein import into chloroplasts. By inference from the crystal structure of psToc34, a homologue in pea, the arginine at residue 130 (Arg(130)) has been implicated in the formation of the atToc33 dimer and in intermolecular GTPase activation within the dimer. Here we report the crystal structure at 3.2-A resolution of an atToc33 mutant, atToc33(R130A), in which Arg(130) was mutated to alanine. Both in solution and in crystals, atToc33(R130A) was present in its monomeric form. In contrast, both wild-type atToc33 and another pea Toc GTPase homologue, pea Toc159 (psToc159), were able to form dimers in solution. Dimeric atToc33 and psToc159 had significantly higher GTPase activity than monomeric atToc33, psToc159, and atToc33(R130A). Molecular modeling using the structures of psToc34 and atToc33(R130A) suggests that, in an architectural dimer of atToc33, Arg(130) from one monomer interacts with the beta-phosphate of GDP and several other amino acids of the other monomer. These results indicate that Arg(130) is critical for dimer formation, which is itself important for GTPase activity. Activation of GTPase activity by dimer formation is likely to be a critical regulatory step in protein import into chloroplasts.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Substitution
  • Arabidopsis / enzymology*
  • Arabidopsis / genetics
  • Arabidopsis Proteins / chemistry*
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism
  • Binding Sites
  • Chloroplasts / enzymology*
  • Chloroplasts / genetics
  • Crystallography, X-Ray
  • Dimerization
  • GTP Phosphohydrolases / chemistry
  • GTP Phosphohydrolases / genetics
  • GTP Phosphohydrolases / metabolism
  • Guanosine Diphosphate / chemistry*
  • Guanosine Diphosphate / metabolism
  • Membrane Proteins / chemistry*
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism
  • Mutation, Missense
  • Pisum sativum / enzymology
  • Pisum sativum / genetics
  • Protein Structure, Quaternary
  • Protein Transport / physiology
  • Sequence Homology, Amino Acid

Substances

  • Arabidopsis Proteins
  • Membrane Proteins
  • TOC159 protein, Arabidopsis
  • Toc33 protein, Arabidopsis
  • Guanosine Diphosphate
  • GTP Phosphohydrolases

Associated data

  • PDB/2J3E