Immunophilin AtFKBP13 Sustains All Peptidyl−Prolyl Isomerase Activity in the Thylakoid Lumen fromArabidopsis thalianaDeficient in AtCYP20-2

Abstract

The physiological roles of immunophilins are unclear, but many possess peptidyl-prolyl isomerase (PPIase) activity, and they have been found in all organisms examined to date, implying that they are involved in fundamental, protein-folding processes. The chloroplast thylakoid lumen of the higher plant Arabidopsis thaliana contains up to 16 immunophilins (five cyclophilins and 11 FKBPs), but only two of them, AtCYP20-2 and AtFKBP13, have been found to be active PPIases, indicating that the other immunophilins in this cellular compartment may have lost their putative PPIase activities. To assess this possibility, we characterized two independent Arabidopsis knockout lines lacking AtCYP20-2 in enzymological and quantitative proteomic analyses. The PPIase activity in thylakoid lumen preparations of both mutants was equal to that of corresponding wild-type preparations, and comparative two-dimensional difference gel electrophoresis analyses of the lumenal proteins of the mutants and wild type showed that none of the potential PPIases was more abundant in the AtCYP20-2 deficient plants. Enzymatic analyses established that all PPIase activity in the mutant thylakoid lumen was attributable to AtFKBP13, and oxidative activation of this enzyme compensated for the lack of AtCYP20-2. Accordingly, sequence analyses of the potential catalytic domains of lumenal cyclophilins and FKBPs demonstrated that only AtCYP20-2 and AtFKBP13 possess all of the amino acid residues found to be essential for PPIase activity in earlier studies of human cyclophilin A and FKBP12. Thus, none of the immunophilins in the chloroplast thylakoid lumen of Arabidopsis except AtCYP20-2 and AtFKBP13 appear to possess prolyl isomerase activity toward peptide substrates.

The FKBP families of higher plants: Exploring the structures and functions of protein interaction specialists

“…At (ROF2) [19] Ta (wFKBP77) [18] Os (OsFKBP65) [28] Zm (ZmFKBP62b) [29] Yes [25] 3Â FKBd, TRP, CaMBd Expressed in response to heat treatment [18] Interacts with ROF1, antagonises ROF1 activity for acquired thermotolerance [25] Regulates intracellular pH [26] Plants resistant to heat treatment following acclimation and prolonged period of recovery [25] D: AtFKBP62 (ROF1); wFKBP73; OsFKBP64; ZmFKBP62a FKBP72 At(PAS1) [49] Os [8] Zm [29] Minor At [7] Os [8] Zm [29] No [60] FKBd Unknown ND S FKBP19 At [7] Os [8] Zm [29] No [60] FKBd Unknown ND S FKBP20-2 At [7] Os [8] Zm [29] No [60,62] FKBd Vital of PSII supercomplex assembly [62] Stunted growth, pale [ influx [26], and the collaborative functionality of this pair was reiterated in higher susceptivity of the rof1/rof2 double knockout mutant to acid inhibition than single rof1 or rof2 knockouts [26]. Orthologous ROF1/ROF2 pairs exist in wheat [18,27], rice [28] and maize [29], and their domain structures and expression profiles are homologous to their Arabidopsis counterparts, indicating that the antagonistic behaviour of this FKPB pair in acquired thermotolerance is a conserved mechanism in higher plants.…”

Consequences of C4 Differentiation for Chloroplast Membrane Proteomes in Maize Mesophyll and Bundle Sheath Cells

“…Two of these Arabidopsis proteins, TLP20 and TLP40, are believed to hold most of the isomerase activity (97). In particular the TLP20 homologues appear to be relatively abundant in the maize thylakoid proteome based on their high cumulative Mowse scores (up to 14,000) and spectral counts (up to 600).…”

Crystal Structure of Arabidopsis Cyclophilin38 Reveals a Previously Uncharacterized Immunophilin Fold and a Possible Autoinhibitory Mechanism

“…In Arabidopsis, up to five cyclophilins and 11 FKBPs are predicted to reside in the thylakoid lumen (Edvardsson et al, 2007). The immunophilin with the highest PPIase activity in the thylakoid lumen is FKBP13, whose crystal structure has already been reported (Gopalan et al, 2004(Gopalan et al, , 2006, followed by CYP20-2.…”

“…The spinach thylakoid lumen has TLP20 (a homolog of At-CYP20-2) and TLP40 as the active PPIases. However, PPIase activity of the Arabidopsis thylakoid lumen has been shown to be restricted to FKBP13 and CYP20-2 (Shapiguzov et al, 2006;Edvardsson et al, 2007;Ingelsson et al, 2009). Hence, it is very likely that the remaining 14 immunophilins in the thylakoid lumen have developed functions other than PPIase activity.…”