IJUP08 - Universidade do Porto

Effect of metals on the AtPreP1 peptidasome activity
J. Pessoa 1,2 , H.G. Bäckman 2 , S. Bhushan 2 and E. Glaser 2
1 Institute for Molecular and Cell Biology, Faculty of Sciences, University of Porto, Portugal.
2 Department of Biochemistry and Biophysics, Faculty of Science, University of Stockholm,
Sweden.
In eukaryotic cells, proteins are targeted to their correct organelle by targeting peptides,
which are amino acid extensions located in the N-terminal portion of the precursor protein.
After the protein has reached its final intracellular destination, targeting peptides are
cleaved off, and because of their cell toxicity, they must be removed. A novel
metalloprotease that efficiently degrades targeting peptides in mitochondria and
chloroplasts was identified by our group, and named Presequence Protease, PreP [1]. In
Arabidopsis thaliana there are two isoforms of PreP (AtPreP1 and AtPreP2) that show an
86% homology at the amino acid sequence level [2].
AtPreP1 is composed of 995 amino acids, and contains a zinc-binding motif in its catalytic
site. It also contains two magnesium-binding sites, one located on the inner side of the
peptide binding cavity and another on the outer surface of the enzyme [3]. Magnesium (or
calcium) has been shown to be needed for catalysis. The aim of this project is to
understand the role of magnesium for the degradation activity.
For that, the activity of AtPreP1 variants (which are unable to bind magnesium) was
measured and compared to the wild-type. Three AtPreP1 variants, one in which both
magnesium-binding sites were changed, and two in which only one magnesium-binding
site was altered, were generated, overexpressed in bacteria and purified. Their activity was
tested using different concentrations of magnesium, calcium or zinc and different
substrates.
The optimal metal concentrations for catalysis were estimated. Both the single and double
AtPreP1 variants were inactive against a 54 amino acid residue peptide corresponding to
the ATPase F1β presequence. However, when the degradation was tested with five other
shorter peptides, the results showed that the activity was substrate-specific and that it may
depend on the substrate length.
References:
[1] Ståhl, A., Moberg, P., Ytterberg, J., Panfilov, O., Brockenhuus von Löwenhielm, H., Nilsson, F.
and Glaser, E. (2002), Isolation and Identification of a Novel Mitochondrial Metalloprotease
(PreP) that Degrades Targeting Presequences, J. Biol. Chem., 277 (44), 41931-41939.
[2] Ståhl, A., Nilsson, S., Lundberg, P., Bhushan, B., Biverståhl, H., Moberg, P., Morisset, M.,
Vener, A., Mäler, L., Langel, U., and Glaser, E. (2005), Two Novel Targeting Peptide Degrading
Proteases, PrePs, in Mitochondria and Chloroplasts, so Similar and Still Different, J. Mol. Biol.,
349, 847–860.
[3] Johnson, K.A., Bhushan, S., Ståhl, A., Hallberg, B.M., Frohn, A., Glaser, E. and Eneqvist, T.
(2006), The closed structure of presequence protease PreP forms a unique 10 000 Å 3 chamber for
proteolysis, EMBO J., 25 (9), 1977-1986.
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