Arabidopsis AtCYP20-2 is a light-regulated cyclophilin-type peptidyl-prolyl cis-trans isomerase associated with the photosynthetic membranes.

This work describes additional characterization of the localization of AtCYP20-2: this single domain cyclophilin was found to specifically associate with membrane regions enriched in photosystem II supercomplexes. In addition to the FK506 binding proteins (FKBPs), cyclophilins are defined as immunophilins, a ubiquitous protein family originally identified as the intracellular receptors of immunosuppressant drugs. Immunophilins are known to be localized in all plant subcellular compartments, and proteomic analysis has revealed the presence of 15 isoforms that are targeted to the chloroplast: one in the stroma (Lippuner et al., 1994), and up to 14 in the thylakoid lumen (Peltier et al., 2002; Schubert et al., 2002; Friso et al., 2004). Immunophilins exist as both single and multidomain isoforms, which have been shown to function both as active protein foldases and as regulatory components of thylakoidmembrane complexes. TLP20, the spinach (Spinacia oleracca) homolog of AtCYP20-2, has been shown to be responsible for the major component of peptidyl-prolyl cis-trans isomerase activity within the thylakoid lumen (Edvardsson et al., 2003). In vitro studies have shown that the multidomain spinach cyclophilin TLP40 associates with and regulates the activity of a PSII-specific protein phosphatase within the thylakoid membrane, an essential process in the coordination of proteolysis and integration of newly synthesized D1 subunits during PSII protein turnover (Fulgosi et al., 1998; Vener et al., 1999). As an example of the specificity of single domain chloroplast immunophilin function, the precursor of the lumenal AtFKBP13 has been shown to interact with the Rieske Fe-S protein, a component of the cytochrome b6f complex, suggesting association occurs along the import pathway. Antisense AtFKBP13 lines have increased Rieske levels, suggesting it may act as a suppressor of Rieske accumulation (Gupta et al., 2002). Given the abundance of immunophilin-like proteins in the thylakoid membrane system, it is likely that PPIase activity may be an integral component of the photosynthetic acclimation response; in particular, these proteins may play an important role in catalyzing correct folding and integration of proteins in and around the thylakoid membrane system. Using chromatographic and immunohistochemical approaches, we sought to determine the specific membrane location of AtCYP20-2. A specific antibody was obtained by using the C-terminal motif CGQLPMSEA as an antigen. The antibody reacted specifically with recombinant AtCYP20-2 and did not react with any proteins extracted from knockout plants lacking the AtCYP20-2 (data not shown). Thus it was concluded the antibody is highly specific for the AtCYP20-2 protein. To determine the precise localization of AtCYP20-2, chloroplast fractionation was carried out in conjunction with western blotting using the specific AtCYP20-2 antibody (Fig. 1). Control blots carried out using the integral thylakoid membrane protein LHCII, the 33-kD subunit of the oxygen-evolving complex (OEC33), and the small subunit of Rubisco (RbcS) allowed verification of the purity and composition of the fractions. As expected, LHCII was found to be exclusively located within the thylakoid membrane, whereas the RbcS was present only in the chloroplast stroma. AtCYP20-2 was localized to the thylakoid lumen, however, a significant portion of this cyclophilin was also bound to the thylakoid membrane fraction (Fig. 1). This finding was unexpected because AtCYP20-2 is a hydrophilic and soluble protein and, in contrast to the multidomain lumenal cyclophilin TLP40 (Fulgosi et al., 1998; Vener et al., 1999), has no specific membrane-binding domains. The membrane association of TLP20, the spinach homolog of AtCYP20-2, has not been reported (Edvardsson et al., 1 This work was supported by the Natural Environment Research Council, the Biotechnology and Biological Sciences Research Council (grant no. 50/P16446), the Swedish Research Council, the Swedish Research Council for Environment, Agriculture, and Spatial Planning (Formas), and Nordic Joint Committee for Agricultural Research (NKJ). 2 Present address: Department of Plant and Microbial Biology, University of California, Berkeley, CA 94720–3102. * Corresponding author; e-mail [email protected]; fax 510–642–4995. www.plantphysiol.org/cgi/doi/10.1104/pp.104.041186.