Red bell pepper chromoplasts exhibit in vitro import competency and membrane targeting of passenger proteins from the thylakoidal sec and DeltapH pathways but not the chloroplast signal recognition particle pathway

Abstract

Chloroplast to chromoplast development involves new synthesis and plastid localization of nuclear-encoded proteins, as well as changes in the organization of internal plastid membrane compartments. We have demonstrated that isolated red bell pepper (Capsicum annuum) chromoplasts contain the 75-kD component of the chloroplast outer envelope translocon (Toc75) and are capable of importing chloroplast precursors in an ATP-dependent fashion, indicating a functional general import apparatus. The isolated chromoplasts were able to further localize the 33- and 17-kD subunits of the photosystem II O2-evolution complex (OE33 and OE17, respectively), lumen-targeted precursors that utilize the thylakoidal Sec and DeltapH pathways, respectively, to the lumen of an internal membrane compartment. Chromoplasts contained the thylakoid Sec component protein, cpSecA, at levels comparable to chloroplasts. Routing of OE17 to the lumen was abolished by ionophores, suggesting that routing is dependent on a transmembrane DeltapH. The chloroplast signal recognition particle pathway precursor major photosystem II light-harvesting chlorophyll a/b protein failed to associate with chromoplast membranes and instead accumulated in the stroma following import. The Pftf (plastid fusion/translocation factor), a chromoplast protein, integrated into the internal membranes of chromoplasts during in vitro assays, and immunoblot analysis indicated that endogenous plastid fusion/translocation factor was also an integral membrane protein of chromoplasts. These data demonstrate that the internal membranes of chromoplasts are functional with respect to protein translocation on the thylakoid Sec and DeltapH pathways.

Figure 1

Electron micrograph images of isolated red bell pepper chromoplasts. A, Representative chromoplast showing internal membranes (M), osmiophilic plastoglobules (P), and fibrils (F). B, Detail of chromoplast internal membrane compartments. Note the inner and outer envelope membranes (Ei and Eo) and the internal membranes organized into vesicles (V) and lamellae (L).

Figure 2

Pigment composition of chromoplasts and chloroplasts. Chlorophyll and carotenoid levels of chloroplasts and chromoplasts were analyzed by absorption spectra of 80% acetone extracts of 100-μg protein equivalents of plastids using a spectrophotometer scanning wavelengths of 350 to 750 nm. , Red bell pepper chromoplast extract; ———, green pea chloroplast extract. A₆₅₂ of chromoplasts = 0.012; A₆₅₂ of chloroplasts = 0.256

Figure 3

Protein profiles of chromoplast and chloroplast fractions. SDS-PAGE (12.5%) and Coomassie blue staining of fractionated red bell pepper fruit chromoplasts (lanes 1–4), pea seedling chloroplasts (lanes 5–7), and pepper seedling chloroplasts (lane 8). Lanes T, Total proteins; lanes S, stromal proteins; lanes TM, total membrane proteins; and lanes MN, NaOH-extracted membranes. Each lane contained 10 μg of protein except MN, which is equivalent to M prior to extraction. Positions of ChrA and ChrB are indicated.

Figure 4

Detection of photosynthetic and protein assembly-specific proteins in chromoplasts and chloroplasts. Isolated pea chloroplasts (lanes CHL) and red pepper chromoplasts containing 3 μg chlorophyll/mg protein (lanes CHR) representing total plastid (lanes T), soluble (stromal) plastid (lanes S), and membrane plastid (lanes M) fractions were subjected to immunoblot analysis (see Methods). Immunoblots were probed with antibodies against: Rbcs (A), OE23 (B), LHCP (C), Hsp70 (D), cpSecA (E), Toc75 (F), Pftf (G), and cpSRP54 (H) and visualized by enhanced chemiluminescence.

Figure 5

Import and subplastid localization of precursors. Isolated chromoplasts containing 2.0 μg chlorophyll/mg protein were incubated with radiolabeled in vitro translation products (lanes tp) in the presence (+ATP) or absence (−ATP) of ATP, as described in Methods. Following import, chromoplasts were recovered without protease posttreatment (lanes C) or with protease posttreatment (lanes CP). Untreated chromoplasts were subfractionated into stroma (lanes S) and total membranes (lanes TM). Equivalent aliquots of membranes were extracted with NaOH (lanes MN) or treated with protease (lanes MP). One microliter of radiolabeled translation product and 15 μL of each sample (representing 7.5 μL of the original radiolabeled translation product added to the import reaction) were analyzed by SDS-PAGE (A–D, 12.5%; E, 7.5%) and fluorography. Precursors utilized for import were: Rbcs (A), OE17 (B), OE33 (C), LHCP (D), and Pftf (E). p, pOE17; i, iOE17; m, mOE17.

Figure 6

Ionophores abolish the luminal localization of OE17 in both chromoplasts and chloroplasts. Import of radiolabeled pOE17 into isolated chromoplasts (containing 0.4 μg chlorophyll/mg protein) and chloroplasts was performed in the absence or presence (+N/V) of nigericin and valinomycin at final concentrations of 0.5 and 1.0 μm, respectively, as described in Methods. Following import, total plastids were recovered (lanes T) and fractionated into stromal (lanes S) and membrane fractions, and the membranes were treated with protease (lanes MP). One microliter of radiolabeled precursor (lanes tp) and 15 μL of each sample (representing 7.5 μL of the original radiolabeled precursor added to the import reaction) were analyzed by 12.5% SDS-PAGE and fluorography. p, pOE17; i, iOE17; m, mOE17.

Figure 7

Endogenous and imported Pftf is integrally associated with membranes of both pepper chromoplasts and pea chloroplasts. A, Analysis of identical samples from Figure 5E and the protease-treated thylakoid fraction following import into pea chloroplasts (lane MP^Chl) by 12.5% polyacrylamide SDS-PAGE and fluorography. The 65-kD mPftf and 13-kD protease-protected (pp) bands are indicated by arrows. B, Immunoblot analysis of chromoplast (containing 0.4 μg chlorophyll/mg protein) and chloroplast membranes. Proteins from chromoplast and chloroplast total membranes (lanes TM), NaOH-extracted membranes (lanes MN), and protease-treated membranes (lanes MP) were separated by 12.5% SDS-PAGE, transferred to nitrocellulose, probed with anti-Pftf, and visualized by enhanced chemiluminescence. The 65-kD mPftf and 13-kD protease-protected (pp) bands are indicated by arrows. C, Transmembrane prediction of the amino acid sequence of Pftf generated by the TMpred program. More positive values indicate residues more likely to occupy a transmembrane domain.