Let's Talk about—Chloroplast Import

Toc64 - A Preprotein-receptor at the Outer Membrane with Bipartide Function

“…[1][2][3][4][5] The subsequent transfer across the intermembrane space is thought to be assisted for instance by Toc64 7 (Figure 1). To understand the function of Toc64 at both sides of the outer envelope we first investigated the topology of the protein.…”

“…(c) From three independent experiments the amount of pSSU precipitated by Toc64, Tic22 or Toc34 antibodies was quantified and the ratio of Toc64 or Toc22 to Toc34 precipitation is shown. (d) In vitro translated 35 S-labeled Toc64 (lanes 1-5) was incubated with chloroplasts (lanes 1-5) followed by re-isolation, lyses and solubilization of the membranes by addition of 1% decylmaltoside (lanes [2][3][4][5]. From this fraction Tic22 (lanes 2 and 3) or Tic110 (lanes 4 and 5) were immunoprecipitated, the precipitate was subjected to SDS-PAGE and the amount of coprecipitated Toc64 was analyzed by autoradiography.…”

“…From this fraction Tic22 (lanes 2 and 3) or Tic110 (lanes 4 and 5) were immunoprecipitated, the precipitate was subjected to SDS-PAGE and the amount of coprecipitated Toc64 was analyzed by autoradiography. (e) In vitro translated 35 S-labeled pOE33 or NTT1 was incubated with chloroplasts (lanes [1][2][3][4][5][6][7][8][9][10][11][12] in the presence of ATP (lanes [1][2][3][4][5][6][7][8][9][10][11][12] and in the presence of Ophiobolin A (lanes 7-12). Chloroplasts were re-isolated, washed, lysed, membrane fractions solubilized by addition of 1% decylmaltoside, incubated with preimmune serume (αpre; lanes 1, 2, 7 and 8) or antibodies against Toc64 (α64; lanes 3, 4, 9 and 10) or Toc34 (α34; lanes 5, 6, 11 and 12) and proteins immunoprecipitated.…”

“…23,24 To assign functional properties to single domains of Toc64 we aimed to determine the topology of the protein in chloroplast membranes. We first analyzed the stability of the protein in the outer envelope [1][2][3][4][5][6][7][8][9][10][11][12] in the presence (lanes 1-6) or absence (lanes 7-12) of ATP. Chloroplasts were re-isolated, washed, lysed and membrane fractions solubilized by addition of 1% decylmaltoside.…”

“…The majority of these preproteins contain an N-terminal transit peptide, which is cleaved off upon translocation. [2][3][4][5] The machinery facilitating recognition and translocation of preproteins across the outer envelope membrane of the plastid is constituted of Toc75, Toc159 and Toc34 assembling the Toc core-complex 6 and the dynamically associated Toc64 and Toc12. 7 Toc159 and Toc34 expose a GTPases domain into the cytosol, which is involved in preprotein recognition and translocation.…”