Chloroplast envelope protein targeting fidelity is independent of cytosolic components in dual organelle assays

The general mechanisms of intracellular protein targeting are well established, and depend on a targeting sequence in the protein, which is recognized by a targeting factor. Once a membrane protein is delivered to the correct organelle its targeting sequence can be recognized by receptors and a translocase, leading to membrane insertion. However, the relative contribution of each step for generating fidelity and efficiency of the overall process has not been systematically addressed. Here, we use tail-anchored (TA) membrane proteins in cell-free competitive targeting assays to chloroplasts to show that targeting can occur efficiently and with high fidelity in the absence of all cytosolic components, suggesting that chloroplast envelope protein targeting is primarily dependent on events at the outer envelope. Efficiency of targeting was increased by the addition of complete cytosol, and by Hsp70 or Hsp90, depending on the protein, but none of these cytosolic components influenced the fidelity of targeting. Our results suggest that the main role of targeting factors in chloroplast localization is to increase targeting efficiency by maintaining recognition competency at the outer envelope.