The chloroplastic protein import machinery contains a Rieske-type iron-sulfur cluster and a mononuclear iron-binding protein

Abstract

Transport of precursor proteins across the chloroplastic envelope membranes requires the interaction of protein translocons localized in both the outer and inner envelope membranes. Analysis by blue native gel electrophoresis revealed that the translocon of the inner envelope membranes consisted of at least six proteins with molecular weights of 36, 45, 52, 60, 100 and 110 kDa, respectively. Tic110 and ClpC, identified as components of the protein import apparatus of the inner envelope membrane, were prominent constituents of this complex. The amino acid sequence of the 52 kDa protein, deduced from the cDNA, contains a predicted Rieske-type iron-sulfur cluster and a mononuclear iron-binding site. Diethylpyrocarbonate, a Rieske-type protein-modifying reagent, inhibits the translocation of precursor protein across the inner envelope membrane, whereas binding of the precursor to the outer envelope membrane is still possible. In another independent experimental approach, the 52 kDa protein could be copurified with a trapped precursor protein in association with the chloroplast protein translocon subunits Toc86, Toc75, Toc34 and Tic110. Together, these results strongly suggest that the 52 kDa protein, named Tic55 due to its calculated molecular weight, is a member of the chloroplastic inner envelope protein translocon.

A 1-Megadalton Translocation Complex Containing Tic20 and Tic21 Mediates Chloroplast Protein Import at the Inner Envelope Membrane

“…By contrast, the amount of radiolabeled 1-MD complex was not affected ( Figure 5B, compare lanes 1 and 2). The size of the Tic110 complex that migrated in the 200-to 300-kD range is well consistent with previous reports (Caliebe et al, 1997;Kü chler et al, 2002;Kikuchi et al, 2006). Based on observations of antibody-shift BN-PAGE and immunodepletion together with the substantial size difference between the 1-MD complex and the 200-to 300-kD Tic110 complex, we conclude that Tic110 is not involved in the 1-MD translocation complex.…”

In Situ Conversion of Protochlorophyllideb to Protochlorophyllide a in Barley

“…In the first case, we used an almost pure, nonaqueous solution of acetone containing 0.1% DEP, which was used in order to block the potential generation of Pchlide b by virtue of the previously identified Chlide a oxygenase and related enzymes (27)(28)(29)(30). This solution is referred to as 100% acetone throughout the rest of the paper.…”

“…This compound had previously been shown to inhibit bacterial and plant Rieske-type oxygenases (27,28), to which Chlide a oxygenase and related enzymes belong (29,30). To block this activity seemed particularly necessary, in order to allow accurate determination of Pchlide a and Pchlide b levels, respectively.…”

A Role for TIC55 as a Hydroxylase of Phyllobilins, the Products of Chlorophyll Breakdown during Plant Senescence

“…This finding is rather surprising because TIC55 was originally identified as a potential component of the protein import machinery at the chloroplast inner envelope (Caliebe et al, 1997). Specifically, it was proposed that TIC55 together with TIC62 and TIC32 functions as redox sensor that links protein import to the redox state of the chloroplast (Balsera et al, 2010).…”