1.2. Redox regulation of immunophilin function Redox reactions are at the heart of the complex combination of pathways which comprise photosynthesis, and as such constitute an integral part of chloroplast biology. As the foundation of the process encompassing the photochemical reactions involving both photosystem I and II and the cytochrome b6f complex, redox reactions drive the production of reducing equivalents used for carbohydrate synthesis in the Calvin cycle. In addition to its role in primary metabolism, redox has been found to be a key regulator in chloroplast biology at multiple levels. Through a set of specific signaling proteins called thioredoxins, redox can act as an indicator of the energy status of the chloroplast by regulating the activity of biosynthetic enzymes, such as members of the Calvin cycle, which are converted from a less active oxidized state in the dark to a more active reduced state by light. Regulation of both nuclear-encoded (Escoubas et al., 1995; Oswald et al., 2000) and chloroplast-encoded (Pfannschmidt, 2003) gene transcription is known to be regulated by redox state, as well as the translation of certain chloroplast encoded genes by a multiprotein complex (see the RB60 complex described below; Trebitsh et al., 2000; Trebitsh & Danon, 2001). Recent evidence suggests that immunophilins play an important role in redox regulation.
With 11 distinct isoforms ranging in size from 13 to 20 kDa, the FKBPs make up the largest portion of the lumenal immunophilins; as yet largely uncharacterized, sequence variation lying within the N-terminal region may confer substrate specificity to the different FKBPs. The smallest isoform, AtFKBP13, is the best characterized to date: a yeast 2-hybrid screen revealed the Rieske Fe–S subunit of the cytochrome b6f complex as an interacting partner of this FKBP protein. Interestingly, only the precursor (i.e. possessing both stromal and lumenal transit peptides) and partially processed (i.e. possessing only the lumenal transit peptide) forms of AtFKBP13 were able to interact with the precursor, partially processed, and lumenal (i.e. fully processed, lacking both chloroplast and stromal transit peptides) forms of the Rieske protein. Moreover, the transit peptide of AtFKBP13 alone also showed interaction, albeit reduced, with both the precursor and partially processed forms of the Rieske protein. This suggests that the mature region of the Rieske protein is sufficient for interaction, but that interaction between the two proteins can only occur in the presence of the AtFKBP13 transit peptide (Gupta et al., 2002). Coupled with the finding that mutants lacking the AtFKBP13 protein show a small increase in the levels of Rieske protein, this suggests that AtFKBP13 may be required for the regulation of cytochrome b6f levels. Further evidence for a post-transcriptional mode of Rieske regulation comes from early data showing that a 10-fold light-induced increase in Rieske transcripts only results in a 2- to 3-fold increase in protein levels (Palomares et al., 1993). A significant feature of the AtFKBP13 primary structure is the presence of two proximal pairs of cysteine residues which form two disulfide bridges. Recent evidence suggests that AtFKBP13 is likely to be oxidized under native conditions, and that disruption of either of the two disulfide bonds, or reduction of the protein by thioredoxin, leads to a reduction in PPIase activity (Gopalan et al., 2004). A key finding is that, in contrast to the stromal cyclophilin AtCYP20-3 and a number of other biosynthetic enzymes, AtFKBP13 is activated by oxidation (conversion of 2SH to S–S) rather than reduction (S–S to 2SH). These data suggest that the redox state of AtFKBP13 may play an important role in its function. One possibility we would like to propose is that AtFKBP13 has three functionally distinct forms which differ in localization and redox status (Fig. 3). Upon entry into the chloroplast, oxidized precursor AtFKBP13 may act as a chaperone for precursor Rieske protein, maintaining it in an inactive soluble form. Under conditions which up-regulate cytochrome b6f synthesis, AtFKBP13 may be reduced by thioredoxin and the AtFKBP13–Rieske complex may then be recruited to the membrane. Upon release of the Rieske subunit, AtFKBP13 may be processed to the mature form and targeted to the thylakoid lumen, where its PPIase activity may be activated by oxidation to form a catalytically active protein, which may function in folding other lumenal or thylakoid membrane proteins. A regulatory mechanism such as the one described would allow harmonization of photosynthetic activity with post-translational targeting. Redox regulation of the synthesis of photosystem components has been previously shown for the synthesis of photosystem II core protein psbA in Chlamydomonas reinhardtii, whose translation is finely tuned to electron flow by the RB60 complex, a target protein of thioredoxin (Trebitsh et al., 2000; Trebitsh & Danon, 2001). It is interesting to note that the PetC gene encoding the Rieske protein is the only nuclear encoded cytochrome b6f complex subunit; as well as being regulated at the transcriptional level (Knight et al., 2002), post-translational control of Rieske accumulation by AtFKBP13 sequestration may ensure an additional level of control, which is more directly linked to electron flow, providing ‘up to the minute’ information on cytochrome b6f requirements. The importance of the Rieske subunit is illustrated by the finding that a petC mutant of Lemna perpusilla is able to synthesize the additional, chloroplast-encoded subunits of the b6f complex, although they are rapidly degraded (Bruce & Malkin, 1991). The above hypothesis for AtFKBP13 not only provides important evidence as to the potential control mechanism for the Rieske subunit, but also suggests, for the first time, that a lumenal protein may be present in distinct functional formats which are operational in separate suborganellar compartments.
Redox regulation of chloroplast immunophilins is not only a feature of the lumenal AtFKBP13 but may also play a role in the function of one of two immunophilins present in the stroma, the conserved single-domain cyclophilin AtCYP20-3 (Motohashi et al., 2003). AtCYP20-3 may not be required in the chloroplast import process, given cyclosporin A failed to inhibit import of Rubsico small subunit protein in an in vitro import experiment (Lippuner et al., 1994). However, when compared to other plant single-domain cyclophilins, a unique feature of AtCYP20-3 is two pairs of cysteine residues, the potential importance of which has been uncovered in recent studies. AtCYP20-3 was initially identified as a putative substrate for chloroplast thioredoxin (Motohashi et al., 2001); subsequently, oxidized AtCYP20-3 was found to be catalytically inactive, and PPIase activity restored by incubation and reduction with chloroplast thioredoxin-m (Motohashi et al., 2003). AtCYP20-2, a conserved single-domain cyclophilin which partitions between the lumen and the inner thylakoid membrane, in association with photosystem II supercomplexes (Romano et al., 2004b), possesses three cysteine residues, two of which are also found in AtCYP20-3. It is tempting to speculate that, much like its stromal counterpart, the activity of this protein may be subject to redox-dependent regulation. An additional lumenal immunophilin AtFKBP16-2 shows significant homology to AtFKBP13 and shares its four cysteine residues (He et al., 2004), suggesting that it too may be a redox-regulated protein. Together with the data obtained for AtFKBP13, these findings suggest that a portion of the chloroplast immunophilins may comprise a novel target of the thioredoxin system: as cysteine-containing protein foldases, they may provide a link between protein tertiary structure formation and chloroplast redox state.