title: Experimental validation of plant peroxisomal targeting prediction algorithms by systematic comparison of in vivo import efficiency and in vitro PTS1 binding affinity.
abstract: Most peroxisomal matrix proteins possess a C-terminal targeting signal type 1 (PTS1). Accurate prediction of functional PTS1 sequences and their relative strength by computational methods is essential for determination of peroxisomal proteomes in silico but has proved challenging due to high levels of sequence variability of non-canonical targeting signals, particularly in higher plants, and low levels of availability of experimentally validated non-canonical examples. In this study, in silico predictions were compared with in vivo targeting analyses and in vitro thermodynamic binding of mutated variants within the context of one model targeting sequence. There was broad agreement between the methods for entire PTS1 domains and position-specific single amino acid residues, including residues upstream of the PTS1 tripeptide. The hierarchy Leu>Met>Ile>Val at the C-terminal position was determined for all methods but both experimental approaches suggest that Tyr is underweighted in the prediction algorithm due to the absence of this residue in the positive training dataset. A combination of methods better defines the score range that discriminates a functional PTS1. In vitro binding to the PEX5 receptor could discriminate among strong targeting signals while in vivo targeting assays were more sensitive, allowing detection of weak functional import signals that were below the limit of detection in the binding assay. Together, the data provide a comprehensive assessment of the factors driving PTS1 efficacy and provide a framework for the more quantitative assessment of the protein import pathway in higher plants.
authors.affiliation: School of Chemistry and the Astbury Centre, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Nicola S
authors.initials: NS
authors.lastname: Skoulding
authors.affiliation: Centre for Organelle Research, Faculty of Science and Technology, University of Stavanger, Richard Johansens Gate 4, N-4021 Stavanger, Norway; KIIT School of Biotechnology, Campus XI, KIIT University, I-751024 Bhubaneswar, India.
authors.forename: Gopal
authors.initials: G
authors.lastname: Chowdhary
authors.affiliation: Centre for Organelle Research, Faculty of Science and Technology, University of Stavanger, Richard Johansens Gate 4, N-4021 Stavanger, Norway.
authors.forename: Mara J
authors.initials: MJ
authors.lastname: Deus
authors.affiliation: Centre for Plant Sciences, School of Molecular and Cellular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Alison
authors.initials: A
authors.lastname: Baker
authors.affiliation: Centre for Organelle Research, Faculty of Science and Technology, University of Stavanger, Richard Johansens Gate 4, N-4021 Stavanger, Norway; Department of Biology, Biocentre Klein Flottbek, University of Hamburg, D-22609 Hamburg, Germany.
authors.forename: Sigrun
authors.initials: S
authors.lastname: Reumann
authors.affiliation: School of Chemistry and the Astbury Centre, University of Leeds, Leeds LS2 9JT, UK. Electronic address: s.l.warriner@leeds.ac.uk.
authors.forename: Stuart L
authors.initials: SL
authors.lastname: Warriner
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D020675:Peroxisomes / Q000378:metabolism*; D010940:Plant Proteins / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology*; D021382:Protein Sorting Signals / Q000502:physiology; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology
chemical_list: D000596:Amino Acids; D010940:Plant Proteins; D021382:Protein Sorting Signals
keywords: PEX5; YFP fusion; fluorescence anisotropy; peptide; specificity