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

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.

Keywords: competitive targeting; molecular chaperones; protein localization; tail-anchored protein; targeting factors.

Figure 1

Protein import from RNCs to chloroplasts is efficient and ribosome independent. (A) Radio-labeled proteins (Toc33, Tic22, Toc64), either directly after translation in lysate or after RNC purification, were incubated with chloroplasts, and their import efficiency calculated as a percentage of the total protein input. Mean and standard error are shown (n = 3). (B) Toc64 was imported into chloroplasts with (RNC) or without (prespun) ribosomes present. Toc64 import was normalized to the RNC import, and mean and standard error are shown (n = 3). (C) Radio-labeled proteins (Toc33 or Tic22) with (+) and without (−) addition of WGE were fractionated by 50kDa molecular mass cutoff columns. Flow through (cutoff <), containing proteins smaller than 50 kDa, and retentate (cutoff >), with proteins and protein complexes of higher molecular weight than 50 kDa, were analysed by SDS-PAGE. (D) Radio-labeled proteins (Toc33 or Toc64) were released from RNCs in the presence of Hsp70 or Hsp90, either with or without a His-tag as indicated. After incubation, and apyrase treatment to stabilize interactions, complexes with His-tagged chaperone were pulled down using NiNTA agarose beads, and analysed by SDS-PAGE. 10% of protein input is shown.

Figure 2

Toc33 targets to chloroplasts efficiently and selectively under a wide range of cytosolic conditions. (A) Radio-labeled Toc33 protein derived from RNCs was incubated in a competitive targeting assay with chloroplasts (CP) and ER, with the addition of cytosolic components as indicated; control is buffer only, WGE is wheat germ extract, chaperones (Hsp) are indicated by their numbers (70/40 or 90), and AP represents apyrase treatment. Membranes were refractionated and washed with carbonate prior to analysis by SDS-PAGE. 10% of protein input is shown, and the protein molecular weight markers are indicated in kDa. (B) Quantification of protein import in part A. Mean and standard error are shown, normalized to the control import into chloroplasts (n = 3), and significant difference from the control import is indicated by one star (p = 0.05) or two stars (p = 0.01). (C) Competitive targeting assay for Toc33 import into chloroplasts (CP) and mitochondria (MT), conducted as described in part A. (D) Quantification of protein import in part C. Mean and standard error are shown, normalized to the control import into chloroplasts (n = 3), and significant difference from the control import is indicated by one star (p = 0.05) or two stars (p = 0.01). (E) Competitive targeting assay for Arabidopsis Sec61β import into ER and chloroplasts (CP), and for At3g58 import into mitochondria (MT) and chloroplasts (CP), conducted as described in part A.

Figure 3

At1g17 targets to chloroplasts efficiently and selectively under a wide range of cytosolic conditions. (A) Radio-labeled At1g17 protein derived from RNCs was incubated in a competitive targeting assay with chloroplasts (CP) and ER, with the addition of cytosolic components as indicated; control is buffer only, WGE is wheat germ extract, chaperones (Hsp) are indicated by their numbers (70/40 or 90), and AP represents apyrase treatment. Membranes were refractionated and washed with carbonate for analysis by SDS-PAGE. 10% of protein input is shown, and the protein molecular weight markers are indicated in kDa. (B) Quantification of protein import in part A. Mean and standard error are shown, normalized to the control import into chloroplasts (n = 3), and significant difference from the control import is indicated by one star (p = 0.05) or two stars (p = 0.01). (C) Competitive targeting assay for At1g17 import into chloroplasts (CP) and mitochondria (MT), conducted as described in part A. (D) Quantification of protein import in part C. Mean and standard error are shown, normalized to the control import into chloroplasts (n = 3), and significant difference from the control import is indicated by one star (p = 0.05) or two stars (p = 0.01).

Figure 4

At3g63 targets to chloroplasts efficiently and selectively under a wide range of cytosolic conditions. (A) Radio-labeled At3g63 protein derived from RNCs was incubated in a competitive targeting assay with chloroplasts (CP) and ER, with the addition of cytosolic components as indicated; control is buffer only, WGE is wheat germ extract, chaperones (Hsp) are indicated by their numbers (70/40 or 90), and AP represents apyrase treatment. Membranes were refractionated and washed with carbonate before analysis by SDS-PAGE. 10% of protein input is shown, and the protein molecular weight markers are indicated in kDa. (B) Quantification of protein import in part A. Mean and standard error are shown, normalized to the control import into chloroplasts (n = 3), and significant difference from the control import is indicated by one star (p = 0.05) or two stars (p = 0.01). (C) Competitive targeting assay for At3g63 import into chloroplasts (CP) and mitochondria (MT), conducted as described in part A. (D) Quantification of protein import in part C. Mean and standard error are shown, normalized to the control import into chloroplasts (n = 3), and significant difference from the control import is indicated by one star (p = 0.05) or two stars (p = 0.01).

Figure 5

Tic22 targets to chloroplasts efficiently and selectively under a wide range of cytosolic conditions. (A) Radio-labeled Tic22 protein derived from RNCs was incubated in a competitive targeting assay with chloroplasts (CP) and ER, with the addition of cytosolic components as indicated; control is buffer only, WGE is wheat germ extract, chaperones (Hsp) are indicated by their numbers (70/40 or 90), and AP represents apyrase treatment. Membranes were refractionated, treated with thermolysin where indicated (Th), and washed with carbonate for analysis by SDS-PAGE. 10% of protein input is shown, and the protein molecular weight markers are indicated in kDa. (B) Quantification of protein import in part A. Mean and standard error are shown, normalized to the control import into chloroplasts (n = 3), and significant difference from the control import is indicated by one star (p = 0.05) or two stars (p = 0.01). (C) Competitive targeting assay for Tic22 import into chloroplasts (CP) and mitochondria (MT), conducted as described in part A. (D) Quantification of protein import in part C. Mean and standard error are shown, normalized to the control import into chloroplasts (n = 3), and significant difference from the control import is indicated by one star (p = 0.05) or three stars (p = 0.001). (E) Targeting assays for the small subunit of Rubisco (pSSU), Tic22, and Toc33 into chloroplasts (CP) were conducted as described in part A. Samples were treated after import with Thermolysin (Th) or thermolysin combined with Triton X-100 (Th + Triton) as indicated.

Figure 6

Toc64 targets to chloroplasts efficiently and selectively under a wide range of cytosolic conditions. (A) Radio-labeled Toc64 protein derived from RNCs was incubated in a competitive targeting assay with chloroplasts (CP) and ER, with the addition of cytosolic components as indicated; control is buffer only, WGE is wheat germ extract, chaperones (Hsp) are indicated by their numbers (70/40 or 90), and AP represents apyrase treatment. Membranes were refractionated and washed with carbonate prior to analysis by SDS-PAGE. 10% of protein input is shown, and the protein molecular weight markers are indicated in kDa. (B) Quantification of protein import in part A. Mean and standard error are shown, normalized to the control import into chloroplasts (n = 3), and significant difference from the control import is indicated by one star (p = 0.05) or two stars (p = 0.01). (C) Competitive targeting assay for Toc64 import into chloroplasts (CP) and mitochondria (MT), conducted as described in part A. (D) Quantification of protein import in part C. Mean and standard error are shown, normalized to the control import into chloroplasts (n = 3), and significant difference from the control import is indicated by one star (p = 0.05) or two stars (p = 0.01).

Figure 7

Protein mistargeting to single organelles does not occur in the absence of cytosolic components. (A) Radio-labeled Toc33 protein derived from RNCs was incubated in a targeting assay with mitochondria (MT), with the addition of cytosolic components as indicated; control is buffer only, and chaperones (Hsp) are indicated by their numbers (70/40 or 90). Membranes were refractionated and washed with carbonate for analysis by SDS-PAGE. 10% of protein input is shown, and the protein molecular weight markers are indicated in kDa. (B) Radio-labeled At3g58 protein derived from RNCs was incubated in a targeting assay with chloroplasts (CP), and otherwise processed as described for part A.