Abstract Detail

Biochemistry, metabolism, carbon flux

Sieben, Nicolas [1], Uhrig, Glen [2], Tang, Lay Yin [3], Moorhead, Greg [1].

A.thaliana Shewnella-Like-Phosphatase 2 Activity Mediated by Internal Disulfide Bridge Formation via Oxido-Reductase MIA40.

Reversible phosphorylation, mediated by protein kinases and phosphatases is a key event that regulates many cellular signalling pathways. Two protein phosphatases (SLP1/SLP2, A. thaliana) have been discovered through a bioinformatics approach to be found in plants, some protists and bacteria, but strictly absent from mammalian systems. Annotated as novel phospho-protein phosphatases (PPP), SLP1 and 2 have no known regulatory subunits to dictate phosphatase control and specificity. Initial experiments using tandem affinity purification (TAP) found that the novel phosphatase SLP2 had a specific interaction with the oxido-reductase MIA40. Further interaction experiments including reciprocal TAP experiments and recombinant affinity purification and native PAGE confirmed the interaction between SLP2 and MIA40.Both MIA40 and SLP2 were shown to have mitochondrial localization.Artifical phosphatase assays using pNPP with recombinant SLP2 and MIA40 show that SLP2 activity is increased in the presence of MIA40 and SLP2 has specific activity for phospho-serine/threonine residues.MIA40 has been shown to confer disulfide bond formation via a cysteine transfer mechanism within the mitochondria. SLP2 was hypothesized to be a target for this transfer. SLP2 constructs were mutated at conserved cysteine residues and assayed to show that SLP2 activity is mediated by MIA40 internal disulfide bridge formation. The results of this assay showed that SLP2 activity was mediated by a disulfide bridge formation between C288 and C336. MIA40 promoted disulfide formation revealed a novel PPP regulatory mechanism.

1 - The University of Calgary, Department of BIological Sciences, 2500 University Drive NW, Calgary, AB, T2N 1N4, Canada
2 - Group of Plant Biotechnology, ETH Zurich
3 - 2500 University Drive NW, Calgary, AB, T2N 1N4, Canada

Keywords:
Phosphatase
Arabidopsis
Novel
Oxido-Reductase
MIA40
SLP2.

Presentation Type: Poster:Posters for Topics
Session: P
Location: Hall D/The Shaw Conference Centre
Date: Monday, July 27th, 2015
Time: 5:30 PM
Number: PBC004
Abstract ID:323
Candidate for Awards:CSPB President's Award for Best Student Presentation,CSPB Travel Bursary