Figure 1.
Protein complexes in spinach chloroplasts analyzed by blue-native PAGE (BN-PAGE) and SDS-PAGE.
Upper panel; CBB-staining (A) and western blots (B–E) of BN-PAGE analysis of thylakoid (T) and stroma (S) proteins extracted from spinach chloroplasts. Lower panel; western blots (F–I) and CBB-staining (J) of 2D SDS-PAGE after separation by BN-PAGE of stromal proteins (F and G) and whole chloroplast proteins (H, I and 2D SDS-PAGE of J). Western blots were probed with polyclonal antibodies against each protein indicated. The numbers beside each band stand for the estimated molecular weights. All the samples loaded were derived from 10 µg on a chlorophyll basis of chloroplasts whose proteins were extracted.
Figure 2.
Protein distribution in response to the changes in solvent conditions of chloroplasts.
Stroma and thylakoid proteins extracted from spinach chloroplasts under different solvent conditions of potassium phosphate buffer at pH 6 (lane 1) or pH 8 (lane 5) containing 0.1 M NaCl (lanes 2 and 6), 10 mM DTT (lanes 3 and 7) or 10 mM ascorbic acid (lanes 4 and 8) were analyzed by western blots after BN-PAGE. Results of stromal proteins for GOGAT (A) and NiR (B), and stroma and thylakoid proteins for GS (C) were presented. The numbers beside each band stand for the estimated molecular weights.
Figure 3.
Protein distribution in response to the pH shift of stroma and whole chloroplasts.
Stromal proteins extracted by using Hepes-KOH buffer at pH 7.5 (lane 1) were four-fold diluted with the same buffer (lane 2), potassium phosphate buffer at pH 6 (lane 3) or pH 8 (lane 4), incubated for 30 minutes at room temperature and analyzed by western blots for GOGAT (A) and NiR (B) after BN-PAGE. Stromal proteins extracted from whole chloroplasts treated with above three conditions were also analyzed (whole; lanes 5–7).
Figure 4.
Western analysis of GS distribution in thylakoid proteins treated with DTT.
Thylakoid proteins treated with DTT were analyzed by western blots of BN-PAGE (right) and 2D SDS-PAGE after BN-PAGE (left) using the GS polyclonal antibody. In the pattern of 2D SDS-PAGE (left), a faint spot at about 45 kDa is probably derived from the contamination of the stromal GS holoenzyme which is fairly abundant in the stromal fraction.
Figure 5.
Protein complexes in spinach chloroplasts analyzed by using Native PAGE.
CBB staining (A), western blots (B–D) and in-gel GS activity staining (E) after Native PAGE analysis of thylakoid (T) and stroma (S) proteins extracted from spinach chloroplasts. For in-gel GS activity assay, proteins extracted from whole chloroplasts (W) were also used. Lane M stands for the protein standard markers, but the migration distance does not necessarily correlate with the molecular mass in this Native PAGE. Samples loaded were derived from 10 µg (A–D) and 20 µg (E) on a chlorophyll basis of chloroplasts whose proteins were extracted.
Figure 6.
Fd-binding assay of protein complexes in the stromal fraction of spinach chloroplasts.
Stromal proteins extracted from spinach chloroplasts by using potassium phosphate buffer at pH 6 (lane 1) or pH 8 (lane 6) were mixed with Fd resin, and the resulting unbound fractions (lanes 2&7), washed fraction (lanes 3&8) and fractions eluted with first (lanes 4&9) and second (lanes 5&10) one-column volume of the same buffer containing 0.2 M NaCl were collected and analyzed by western blots after BN-PAGE.
Figure 7.
Models of pH-dependent complex formation and relocation of carbon- and nitrogen assimilatory enzymes in chloroplasts.
Complex formation (possibly oligomerization) of GOGAT and NiR and localization of GS signals observed in this study are depicted together with a previously presented model for FNR [7]. GOGAT and NiR display pH-dependent conversion of monomers and oligomers (or protein complexes with unknown proteins) in the stromal fraction, which appears to be accompanied by differential binding to Fd (Fig. 6) and possibly by differential enzyme activity or protein stability. GS is postulated to exist both as an active enzyme of decamer in stroma and as a modified, probably inactive form (depicted with black dots) in the plastid membranes (such as thylakoids and envelop), which may interconvert by an unknown mechanism. FNR is known to relocate between stroma and thylakoids (possibly as a dimer for the stabilization of this enzyme) in the light, redox and pH-dependent manners [7], [8].