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.
Highlights
In plants, numerous matrix enzymes have to be imported post-translationally from the cytosol into peroxisomes, including the enzymes for seed storage oil mobilization into glyoxysomes and the components for photorespiration into leaf peroxisomes
Previous work of our group has shown that the peroxisomal processing protease DEG15/glyoxysomal processing protease (GPP) is present in two conformations, monomeric and dimeric, with different substrate specificities (Helm et al 2007)
We could show recently that the CaM-like protein AtCML3 is localized in peroxisomes (Chigri et al 2012), making it a potential mediator of calcium-dependent dimerization of AtDEG15
Summary
Numerous matrix enzymes have to be imported post-translationally from the cytosol into peroxisomes, including the enzymes for seed storage oil mobilization into glyoxysomes and the components for photorespiration into leaf peroxisomes The majority of these enzymes are imported in their mature form targeted by a C-terminal (S/ A/P/C)-(K/R/S/N)-(L/M/I) tripeptide designated as peroxisomal targeting signal 1 (PTS1) (Lingner et al 2011). The type 2 peroxisomal targeting signal (PTS2) with the consensus sequence (R/K)-(L/I/V)X5-(H/Q)-(L/A) is located in the N-terminal 30 to 50 amino acids of the pre-protein (Flynn et al 1998; Reumann 2004; Lazarow 2006) These include glyoxysomal malate dehydrogenase (gMDH), glyoxysomal citrate synthase (gCS), and acyl-CoA oxidase and thiolase in plants, as well as alkyl-dihydroxyacetone-phosphate (DHAP) synthase, phytanoyl-CoA hydroxylase and thiolase in mammals. Processing of PTS2 is not necessary for enzyme activity in higher eukaryotes (Gietl et al 1996), and it does not take place in lower eukaryotes, where non-cleaved PTS2 sequences are present in the N-terminus of the mature form of thiolase and amine oxidase (Flynn et al 1998; Helm et al 2007; Schuhmann et al 2008)
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