Supporting Figures

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Supporting Figure 8
Supporting Figure 9

Fig. 8. Analytical test on the substrate specificity of CAO. (A) AtCAO protein was produced as in Fig. 1A and an aliquot was incubated with either synthetic Chlide a or Pchlide a under the same conditions as those described in Fig. 1B. The different curves show pigments formed in either the presence (dotted lines) or absence (solid lines) of AtCAO protein in incubation mixtures supplemented with Chlide a (A) or Pchlide a (B). Pigment detection was made at 435 nm (A) or 455 nm (B). Note that the absorption coefficient of Pchlide b is 5-fold higher than that of Pchlide a at 455 nm and that only minor traces of Pchlide b were produced in the CAO reaction, corresponding to ≈4% of the initial Pchlide a level.

Fig. 9. In vitro import of ³⁵S-pCAO and ³⁵S-pSSU into isolated barley chloroplasts. (A) ³⁵S-AtpCAO was produced as described in Fig. 1A and incubated with isolated, energy-depleted barley chloroplasts that had been pretreated with (+Thl_pre) or without (–Thl_pre) thermolysin and repurified. Import reactions were carried out in the presence of the indicated, millimolar concentrations of Mg-GTP and Mg-ATP for 15 min darkness. After import, intact plastids were repurified on Percoll and subsequently treated with or without thermolysin (Thl_post) and trypsin (Trp) as specified. Protein was extracted with trichloroacetic acid, electrophoresed on a 10-20% polyacrylamide gradient containing SDS and detected by autoradiography. (B) Import of wheat germ-translated small subunit precursor of ribulose-1,5-bisphosphate carboxylase/oxygenase (pSSU) used as control. P, precursors; m, mature proteins. Std defines ³⁵S-input standards. (C) Plastid sublocalization and topology of imported ³⁵S-AtCAO. ³⁵S-AtpCAO was imported into isolated barley chloroplasts in the presence of 0.1 mM Mg-GTP and 2 mM Mg-ATP. Intact plastids were reisolated on Percoll, lysed, and subsequently fractionated into mixed envelopes (ME), outer (OM) and inner (IM) plastid envelope membranes, thylakoids (Th), and stroma (St). From each of these fractions protein was extracted with trichloroacetic acid and resolved on 10-20% SDS-polyacrylamide gradient and detected by autoradiography (³⁵S-atCAO) or transferred onto nitrocellulose membranes. The resulting filters then were probed with antisera against Tic55, Toc75, and a mixed antiserum against the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (SSU) and light-harvesting chlorophyll a/b binding protein of photosystem II (LHCII). In the case of the latter, protein was separated on a 10% polyacrylamide minigel before protein gel blot analysis. Two faint protein bands cross-reacted with the mixed anti-LHCII and anti-SSU antisera in the outer envelope membrane fraction of which the higher molecular mass band may be due to trace amounts of unimported pLHCII or pSSU. It has been reported that plastid envelope membranes contain small pools of unimported chloroplast precursors (1). (D) Inner plastid envelope membranes containing imported ^3SS-CAO were isolated from thermolysin-treated plastids and extracted with 0.1 M Na₂CO₃, pH 11, or 1 M NaCl. After a step of centrifugation, protein found in the membrane pellets (P) and supernatants (S) was resolved by SDS/PAGE and detected by autoradiography. The lack of ³⁵S-CAO in the supernatants is indicative of its tight membrane binding. (E) Import reactions were conducted as described. Inner envelope membranes were purified from trypsin-treated (+Try, lane 1) or mock-incubated (–Try, lane 2) chloroplasts and extracted. Protein was detected by SDS/PAGE and autoradiography. To trace degradation products of imported ³⁵S-CAO, »150-fold higher amounts of radioactivity were loaded in lane 2 versus lane 1.

1. Bovet, L., Müller, M. O. & Siegenthaler, P. A. (1995) Planta 195, 563–569.