Kinetic analysis of translocation into isolated chloroplasts of the purified ferredoxin precursor

Abstract

Timecourses ofthc import into isolatedchloroplasts ofthcpurified fcrredoxin precursor were measured at different proteinconcentrations. Analysis of the initial import rates indicates the presence of one saturable import system with an apparent K,,, value of approximately I00 nM and a V,,, corresponding to the uptake of approximately 2.5 IO' precursor proteins per minute per chloroplast. We conclude that the in vitro observed activity of the chloroplast protein import machinery, functioning independently Cram cytosolic factor-, p would be enough to allow chloroplast development at at physiological rate.Chloroplast protein import; Ferrcdoxin; Michsclis-Menten kinetics

The C Terminus of a Chloroplast Precursor Modulates Its Interaction with the Translocation Apparatus and PIRAC

“…The V max of prSSU import is also comparable to that reported for prLHCP. In contrast, the V max of prSSU import is significantly less than the V max reported for either prFd (ϳ22,000 molecules/chloroplast/min) (19) or prOEE23 (Ͼ40,000 molecules/chloroplast/ min) (20). These differences may reflect genuine disparities in the rate or efficiency of import for the various precursors, or alternatively, they may represent differential responses to chemical denaturation agents used for solubilization of the precursor.…”

“…These differences may reflect genuine disparities in the rate or efficiency of import for the various precursors, or alternatively, they may represent differential responses to chemical denaturation agents used for solubilization of the precursor. As discussed previously by Pilon et al (19), however, the rates of translocation measured in vitro are quite close to the rates required in vivo for normal maintenance of chloroplast biogenesis, which lends credibility to the use of in vitro import studies as a means of exploring the mechanism of chloroplast protein transport.…”

Differential Transit Peptide Recognition during Preprotein Binding and Translocation into Flowering Plant Plastids

“…Although previous studies have reported that cytosolic factors initially recognize TPs/precursors, kinetic arguments suggest that in vitro import can attain import rates without cytosolic factors to support organelle biogenesis (Pilon et al, 1992;May and Soll, 2000). This very rapid binding and translocation occurs despite a very low density distribution of TOC complexes on the plastid outer membrane (#1 TOC/13,600 nm 2 ) (Friedman and Keegstra, 1989;Schleiff et al, 2003).…”

“…However, the algorithm developed by Rüdiger et al show some disagreement in the predictions of SSF and SSR where the calculated energy contribution within the first 10 residues shows no difference. NTT1tp is the TP of Arabidopsis nucleotide transporter 1. kinetics of posttranslational preprotein import in vitro may be able to match the maximum rates predicted in vivo during greening and chloroplast development (Pilon et al, 1992) without the need for cytosolic factors or the topological facilitation provided during the cotranslational processes of protein transport.…”

Conformational Requirements of a Recombinant Ferredoxin‐NADP+ Reductase Precursor for Efficient Binding to and Import into Isolated Chloroplasts

“…Both the number of receptor siteslplastid and the Kd for precursor binding are close to those determined by Friedman and Keegstra [5], who used the in-vim-translated precursor of the Rubisco small subunit. With respect to the kinetic parameters K, and l ( they have been estimated by Pilon et al [29] for import of purified prefer- We have also examined the substrate requirements of translocation by testing the ability of different conformational forms of preFNR to be imported in the in vitro assay. The precursor holoprotein, which has a rather folded structure [15], bound efficiently to the plastid envelope.…”