Chloroplast Outer Membrane β-Barrel Proteins Use Components of the General Import Apparatus

Abstract

Chloroplasts evolved from a cyanobacterial endosymbiont that resided within a eukaryotic cell. Due to their prokaryotic heritage, chloroplast outer membranes contain transmembrane β-barrel proteins. While most chloroplast proteins use N-terminal transit peptides to enter the chloroplasts through the translocons at the outer and inner chloroplast envelope membranes (TOC/TIC), only one β-barrel protein, Toc75, has been shown to use this pathway. The route other β-barrel proteins use has remained unresolved. Here we use in vitro pea (Pisum sativum) chloroplast import assays and transient expression in Nicotiana benthamiana to address this. We show that a paralog of Toc75, outer envelope protein 80 kD (OEP80), also uses a transit peptide but has a distinct envelope sorting signal. Our results additionally indicate that β-barrels that do not use transit peptides also enter the chloroplast using components of the general import pathway.

Figure 1.

OEP80 Is Processed at the N Terminus. (A) In vitro-translated, radiolabeled AtOEP80 ± a C-terminal T7 tag were incubated with isolated chloroplasts (import). After 20 min the chloroplasts were reisolated and run on SDS-PAGE next to 10% of the translation product input (TL). Radioactive bands were detected with autoradiography. (B) In vitro-translated, radiolabeled AtOEP80 ± the C-terminal transmembrane β-barrel were incubated with isolated chloroplasts. After 20 min, the chloroplasts were reisolated, then treated with or without thermolysin or solubilized with 1% (v/v) Triton X100 (TX100) and treated with thermolysin. The protease was stopped with 10-mM EDTA. Intact chloroplasts were reisolated. Proteins in the solubilized sample were precipitated in 80% (v/v) acetone. Samples were run on SDS-PAGE and visualized as in (A). In (A) and (B), the asterisks (*) near the TL lane indicate bands resulting from translation starting at the second and/or third methionines in the coding sequence. The pound sign (#) indicates additional processed bands, other than the mature-sized one. (C) In vitro-translated, radiolabeled OEP80 orthologs from pea (P. sativum) and barley (H. vulgare) were incubated with isolated chloroplasts. After 20 min, the chloroplasts were reisolated and half were lysed in 10-mM HEPES at pH 8.0 and 10-mM MgCl₂. The soluble (S1) and membrane fractions were separated by centrifugation. The membranes were washed with 0.1-M sodium carbonate. The wash (S2) and pellet (P) were separated by centrifugation. Samples were run on SDS-PAGE and visualized as in (A). pr, precursor; m, mature.

Figure 2.

OEP80 Is Processed by the SPP. (A) In vitro-translated, radiolabeled precursors were incubated in lysis buffer or chloroplast soluble extract with or without 50-mM EDTA. Samples were run on SDS-PAGE and radioactive bands were detected with autoradiography. (B) The same precursors were incubated with recombinant Plsp1, Boiled Plsp1, or empty buffer. Results were visualized as in (A). (C) Precursors were incubated in chloroplast soluble extract with or without urea-solubilized recombinant precursor (prRSSU) or mRSSU. Results were visualized as in (A). i, intermediate.

Figure 3.

The N Terminus of OEP80 Is Necessary for the Import of the IMS-Localized POTRA Domains But Not the Transmembrane Domain. (A) In vitro-translated, radiolabeled sections of AtOEP80 (illustrated below the gels) were incubated with isolated chloroplasts (import). Chloroplasts were reisolated, then treated with or without thermolysin or solubilized with 1% (v/v) Triton X100 (TX100) and treated with thermolysin. The protease was stopped with 10-mM EDTA, and intact chloroplasts were reisolated. Proteins in the solubilized sample were precipitated in 80% (v/v) acetone. Samples were separated by SDS-PAGE next to 10% of the input translation product (TL). Radioactive bands were detected with autoradiography. 80TP, transit peptide of OEP80; u, uncharacterized region between the transit peptide and the first POTRA. The three POTRA domains are labeled as “1,” “2,” and “3.” The pound sign (#) indicates additional processed bands, other than the mature-sized one. (B) In vitro-translated, radiolabeled OEP24 and OEP80-OEP24 chimeras were incubated with isolated chloroplasts, then treated as in (A). pr, precursor; m, mature. In both (A) and (B), the asterisks (*) near the TL lane indicates bands resulting from translation starting at the second and/or third methionines in the coding sequence.

Figure 4.

The N Terminus of OEP80 Is Sufficient for Targeting to the Chloroplast But the C Terminus is Required for Envelope Sorting. (A) Diagram of constructs tested. 80TP, transit peptide of OEP80; u, uncharacterized region between the transit peptide and first POTRA. The three POTRA domains are labeled as “1,” “2,” and “3.” (B) Chloroplasts were isolated from plants transiently expressing a T7-tagged version of OEP80. Chloroplasts were treated with no protease, trypsin, or thermolysin. Proteases were quenched, then the chloroplasts were reisolated. Chloroplasts were fractionated into soluble (S) and membrane (M). T, Total chloroplasts. Results were visualized by immunoblot with an anti-T7 antibody. (C) Chloroplasts were isolated from plants transiently expressing YFP-tagged sections of OEP80. The chloroplasts were treated with or without thermolysin or solubilized with 1% (v/v) Triton X100 (TX100) and treated with thermolysin. After 30 min, the protease was quenched, and intact chloroplasts were reisolated. Proteins in the solubilized sample were precipitated in 80% (v/v) acetone. Samples were visualized by immunoblotting with anti-GFP antibodies. A nonspecific band recognized by the GFP antibody is indicated with an asterisks (*). (D) The same experiment was performed as in (C), but with trypsin rather than thermolysin. Immunoblots of endogenous Toc75 and Tic110 are included as trypsin-sensitive and -insensitive controls, respectively. In (C) and (D), the identities of bands are indicated by their number in (A) as well as “pr” to indicate the precursor form, “m” to indicate the processed form, or “d” to indicate degradation products. Free YFP that is the result of degradation of the OEP80 sections is indicated as “YFP.”

Figure 5.

Import OM β-Barrels With or Without Cleavable Targeting Information Competes with Import of a Stromal Protein. (A) In vitro-translated, radiolabeled precursors were incubated with isolated pea chloroplasts in the presence or absence of guanidine-solubilized recombinant prRSSU or mRSSU. After 20 min, chloroplasts were reisolated through 40% (v/v) Percoll. (B) In vitro-translated, radiolabeled precursors were incubated with isolated chloroplasts in the presence or absence of recombinant prRSSU or mRSSU. The radiolabeled PsOEP80 barrel, AtOEP24, and prRSSU were imported into the same chloroplasts. After 20 min, chloroplasts were reisolated through 40% (v/v) Percoll, treated with thermolysin, then reisolated again. In both (A) and (B), samples were separated by SDS-PAGE next to 10% (AtOEP80, AtToc75, PsOEP80) or 3.3% (PsOEP80 barrel, AtOEP24, and PsprRSSU) of the input translation product (TL). Radioactive bands were detected with autoradiography. pr, precursor; i, intermediate; m, mature.

Figure 6.

Transiently Expressed Sections of OEP80 and OEP24 Pull Down TOC Components. (A) Diagram of constructs used (left). Chloroplasts from plants transiently expressing YFP-tagged constructs were solubilized with 1% (w/v) dodecyl-maltoside (input) and mixed with GFP affinity beads. Beads were washed, then bound proteins eluted. The inputs and eluates were subjected to immunoblotting with antibodies against GFP, Toc159, Tic40, and Toc75. The blot for GFP, Toc159, and Tic40 are from one membrane each; however, some regions were removed for clarity (indicated by dotted white and black lines). The asterisks indicate bands from the GFP affinity beads recognized by the Toc75 antibody. One of these comigrated with Toc75, but eluates 1, 2, and 3 showed a higher signal in this region. 80TP, transit peptide of OEP80; Plsp1TP, transit peptide of Plsp1; u, uncharacterized region between the transit peptide and the first POTRA. The three POTRA domains are labeled as 1, 2, and 3. (B) Chloroplasts from uninfiltrated (UI) plants or plants transiently expressing T7-tagged OEP24 (24T7) or OEP40 (40T7) were solubilized with 1% (w/v) dodecyl-maltoside (input) and mixed with T7 affinity beads. The inputs and eluates were subjected to immunoblotting with antibodies against T7, Toc75, Toc159, and Tic40.