AtPreP (Arabidopsis thaliana Presequence Protease) is a zink metallooligopeptidase that is dually targeted to both mitochondria and chloroplasts. In these organelles it functions as a peptidasome that degrades the N-terminal targeting peptides that are cleaved off from the mature protein after protein import, as well as other unstructured peptides. In A. thaliana there are two isoforms of PreP, AtPreP1 and AtPreP2.  We have performed characterization studies of single and double prep knockout plants. Immunoblot analysis revealed that both PreP isoforms are expressed in all tissues with highest expression levels in flowers and siliques. Furthermore, AtPreP1 was shown to be the most abundant isoform of the two. When comparing phenotype, the atprep2 mutant was similar to wild type, whereas the atprep1 mutant had a slight pale-green phenotype in the early developmental stages. The atprep1 atprep2 double knockout plants showed a chlorotic phenotype in true leaves, especially prominent during the early developmental stages. When analysing the first true leaves of double knockout plants, we found a significant decrease in chlorophyll a and b content. Mitochondrial respiratory rates measurements showed partially uncoupled mitochondria. Ultrastructure analysis using electron microscopy on double knockout plants showed aberrant chloroplasts with altered grana stacking and clearly fewer starch granules. Older plants showed less altered  phenotype, although there was a significant decrease in the accumulated biomass of about 40% compared to wild type. Peptidolytic activity studies showed no sign of compensatory mechanisms in the absence of AtPreP in mitochondria; in contrast we found a peptidolytic activity in the chloroplast membranes not related to AtPreP. In addition to zinc located in the catalytic site, crystallographic data revealed two Mg-binding sites in the AtPreP structure. To further investigate the role of these Mg-binding sites, we have made AtPreP variants that are unable to bind metal ions. Our data shows that one of these sites located close to the catalytic site is important for the activity of AtPreP. We also measured proteolytic activity of four human PreP-SNP variants and observed that the activity of all the hPreP-SNPs variants was lower; especially the hPreP-SNP (A525D) variant that displayed only 20-30 % of wild type activity. Interestingly, the activity was fully restored for all SNP-variants by addition of Mg2+.