Calmodulin-like protein AtCML3 mediates dimerization of peroxisomal processing protease AtDEG15 and contributes to normal peroxisome metabolism

Abstract

Matrix enzymes are imported into peroxisomes and glyoxysomes, a subclass of peroxisomes involved in lipid mobilization. Two peroxisomal targeting signals (PTS), the C-terminal PTS1 and the N-terminal PTS2, mediate the translocation of proteins into the organelle. PTS2 processing upon import is conserved in higher eukaryotes, and in watermelon the glyoxysomal processing protease (GPP) was shown to catalyse PTS2 processing. GPP and its ortholog, the peroxisomal DEG protease from Arabidopsis thaliana (AtDEG15), belong to the Deg/HtrA family of ATP-independent serine proteases with Escherichia coli DegP as their prototype. GPP existes in monomeric and dimeric forms. Their equilibrium is shifted towards the monomer upon Ca2+-removal and towards the dimer upon Ca2+-addition, which is accompanied by a change in substrate specificity from a general protease (monomer) to the specific cleavage of the PTS2 (dimer). We describe the Ca2+/calmodulin (CaM) mediated dimerization of AtDEG15. Dimerization is mediated by the CaM-like protein AtCML3 as shown by yeast two and three hybrid analyses. The binding of AtCML3 occurs within the first 25 N-terminal amino acids of AtDEG15, a domain containing a predicted CaM-binding motif. Biochemical analysis of AtDEG15 deletion constructs in planta support the requirement of the CaM-binding domain for PTS2 processing. Phylogenetic analyses indicate that the CaM-binding site is conserved in peroxisomal processing proteases of higher plants (dicots, monocots) but not present in orthologs of animals or cellular slime molds. Despite normal PTS2 processing activity, an atcml3 mutant exhibited reduced 2,4-DB sensitivity, a phenotype previously reported for the atdeg15 mutant, indicating similarly impaired peroxisome metabolism. Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-013-0112-6) contains supplementary material, which is available to authorized users.

Plant peroxisomes: recent discoveries in functional complexity, organelle homeostasis, and morphological dynamics

“…For example, calcium regulates in vitro dimerization and substrate specificity of the DEG15 protease that removes the N-terminal PTS2 from matrix proteins after entry into the peroxisome [22]. DEG15 dimerization is mediated by the calmodulin-like protein CML3 [23], a peroxisomal protein [24] that contributes to peroxisome metabolism, as evidenced by the slight β-oxidation defects of a cml3 mutant [23]. However, unlike deg15 mutants, which exhibit a complete block in PTS2 processing [25,26], cml3 mutants process PTS2 proteins like wild type [23], suggesting that the β-oxidation defects in cml3 might stem from additional to-be-discovered roles for CML3 (and presumably calcium) in the peroxisome.…”

Calmodulin antagonist affects peroxisomal functionality by disrupting both peroxisomal Ca2+ and protein import

“…For instance, in the Arabidopsis genome, seven distinct CaM and 50 CMLs genes have been predicted (McCormack et al, 2005). Recently, Arabidopsis thaliana (At)CML3 (also known as AGD11) has been demonstrated to be targeted to peroxisomes (Chigri et al, 2012), where it appears to mediate the dimerization of peroxisomal processing protease AtDEG15 (Dolze et al, 2013). Moreover, there is another family of proteins which act as Ca 2+ sensors called Ca 2+dependent protein kinases (CDPKs) (Boudsocq and Sheen, 2013) and in Arabidopsis the isoform AtCPK1 is targeted to peroxisomes (Dammann et al, 2003) and seems to mediate pathogen resistance (Coca and San Segundo, 2010).…”

Peroxisomes: versatile organelles with diverse roles in plants

“…In plant peroxisomes, Ca 2+ and calmodulin (CaM) are important for protein import and function of peroxisomal enzymes involved in detoxification, photorespiration and NO production (Corpas & Barroso, 2018). Arabidopsis calcium-dependent protein kinase 1 (CPK1) is involved in SA signaling and pathogen resistance (Coca & San Segundo, 2010), and the calmodulin-like protein CML3 is involved in DEG15 dimerization and peroxisomal protein import (Dolze et al, 2013). In Petunia inflata, peroxisomal Ca 2+ -dependent protein kinase 2 (CDPK2) and small CDPK-interacting protein 1 (SCP1) regulate pollen tube growth (Guo et al, 2013).…”