Crystal structure of pea Toc34, a novel GTPase of the chloroplast protein translocon

Abstract

Toc34, a 34-kDa integral membrane protein, is a member of the Toc (translocon at the outer-envelope membrane of chloroplasts) complex, which associates with precursor proteins during protein transport across the chloroplast outer membrane. Here we report the 2.0 A resolution crystal structure of the cytosolic part of pea Toc34 in complex with GDP and Mg2+. In the crystal, Toc34 molecules exist as dimers with features resembling those found in a small GTPase in complex with a GTPase activating protein (GAP). However, gel filtration experiments revealed that dimeric and monomeric forms of Toc34 coexisted in phosphate saline buffer solution at pH 7.2. Mutation of Arg 128, an essential residue for dimerization, to an Ala residue led to the formation of an exclusively monomeric species whose GTPase activity is significantly reduced compared to that of wild type Toc34. These results, together with a number of structural features unique to Toc34, suggest that each monomer acts as a GAP on the other interacting monomer.

The Arabidopsisppi1Mutant Is Specifically Defective in the Expression, Chloroplast Import, and Accumulation of Photosynthetic Proteins[W]

“…In support of this model, an abundant cytosolic form of atToc159 has been observed (Hiltbrunner et al, 2001b), and the crystal structure of psToc34 suggests that heterodimerization between Toc34 and Toc159 may be possible (Sun et al, 2002). This model is reminiscent of signal recognition particle (SRP)-dependent protein targeting, in which the GTP binding protein SRP54 initiates preprotein translocation by docking at receptors that are themselves GTP binding proteins (Keenan et al, 2001).…”

Structural basis of oligomerization in septin-like GTPase of immunity-associated protein 2 (GIMAP2)

“…Also the Toc GTPases, septins, and members of the dynamin-like clade bear a C-terminal helix comparable to α6 (Fig. S1) (23)(24)(25). However, the GIMAP2 structure is distinguished by the amphipathic helix α7 that is located on the opposite face of the nucleotide-binding site.…”

The gateway to chloroplast: re-defining the function of chloroplast receptor proteins

“…The unifying principle of the TOC receptors is that psToc34 or Arabidopsis Toc33/34 (atToc33/34) dimerizes in a nucleotide-dependent manner and GDP binding moderates the interaction between the G-domains of the respective monomer. Dimerization has henceforth been proposed as an important regulatory aspect that controls the entry of preproteins to the membrane translocation channel of the TOC translocon (Sun et al , 2002 ). This structural observation does not allow any conclusion to be drawn concerning the underlying mechanism of dimerization of the TOC GTPase cycle and its impact on the translocation process.…”

“…The three-dimensional structure from pea Toc34 (psToc34) disclosed a homodimeric configuration with bound GDP at the dimerization interface (Sun et al , 2002 ). Such structural arrangement is strikingly reminiscent of a GTPase with its corresponding GAP (Gasper et al , 2009 ).…”

“…In this context, several possibilities have been proposed. An early hypothesis is that each monomer of the psToc34 or atToc33/34 receptor acts as GAP for its respective partner, thus activating its cognate GTPase activity (Sun et al , 2002 ). An arginine residue (also designated as arginine finger) is often found at the dimerization cleft of these GAPs.…”