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

Verena Kriechbaumer,Ben M Abell

doi:10.3389/fpls.2012.00148

Abstract

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.

Highlights

Protein localization to organelles is a vital aspect of cellular organisation, and we have an excellent understanding of many the molecular mechanisms underlying each pathway
To enable control of the targeting environment, we translated proteins lacking a stop codon, which locks the protein on the ribosome, and separated the ribosome-nascent chain (RNC) complexes from cytosolic components by centrifugation
This approach is effective for TA membrane proteins because their targeting information is primarily contained within the C-terminal transmembrane domain, which is contained within the ribosomal exit tunnel until puromycin release, and unavailable to targeting factors in the translation lysate (Abell et al, 2007)

Summary

Introduction

Protein localization to organelles is a vital aspect of cellular organisation, and we have an excellent understanding of many the molecular mechanisms underlying each pathway. The central concept is the signal hypothesis (Blobel and Sabatini, 1971), which involves sequence information in the nascent protein promoting engagement with membrane receptors, leading to the protein entering or crossing the membrane This hypothesis is supported by a vast number of studies involving diverse proteins and compartments (Wickner and Schekman, 2005), but most of our understanding is based on viewing the process as a series of essential steps, without quantifying the importance of each stage or the potential for bypassing some of the events. Targeting processes can be split into four key stages: (1) Ribosome coordination, in which targeting sequences can be recognized more efficiently if targeting factors are present close to the ribosomal exit tunnel, exemplified by SRP (reviewed by Grudnik et al, 2009). Association between the receptor and the translocase can be either stable or dynamic; for example Toc and Toc159 are permanent components of the chloroplast Toc complex, whereas Toc is more transiently associated (Soll, 2002; Qbadou et al, 2006). (4) Membrane translocases promote insertion into the membrane or transport across the membrane, direct membrane insertion is possible in some cases, such as the insertion of cytochrome b5 into ER membranes (Brambillasca et al, 2005)

Methods

Results

Conclusion