title: Covalent Label Transfer between Peroxisomal Importomer Components Reveals Export-driven Import Interactions.
abstract: Peroxisomes are vital metabolic organelles found in almost all eukaryotic organisms, and they rely exclusively on import of their matrix protein content from the cytosol. In vitro import of proteins into isolated peroxisomal fractions has provided a wealth of knowledge on the import process. However, the common method of protease protection garnered no information on the import of an N-terminally truncated PEX5 (PEX5C) receptor construct or peroxisomal malate dehydrogenase 1 (pMDH1) cargo protein into sunflower peroxisomes because of high degrees of protease susceptibility or resistance, respectively. Here we present a means for analysis of in vitro import through a covalent biotin label transfer and employ this method to the import of PEX5C. Label transfer demonstrates that the PEX5C construct is monomeric under the conditions of the import assay. This technique was capable of identifying the PEX5-PEX14 interaction as the first interaction of the import process through competition experiments. Labeling of the peroxisomal protein import machinery by PEX5C demonstrated that this interaction was independent of added cargo protein, and, strikingly, the interaction between PEX5C and the import machinery was shown to be ATP-dependent. These important mechanistic insights highlight the power of label transfer in studying interactions, rather than proteins, of interest and demonstrate that this technique should be applied to future studies of peroxisomal in vitro import.
authors.affiliation: From the Centre for Plant Sciences and School of Molecular and Cellular Biology and.
authors.forename: Moninder S
authors.initials: MS
authors.lastname: Bhogal
authors.affiliation: From the Centre for Plant Sciences and School of Molecular and Cellular Biology and School of Chemistry and Astbury Centre, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Thomas
authors.identifier: http://orcid.org/0000-0002-7092-8096
authors.initials: T
authors.lastname: Lanyon-Hogg
authors.affiliation: From the Centre for Plant Sciences and School of Molecular and Cellular Biology and.
authors.forename: Katherine A
authors.initials: KA
authors.lastname: Johnston
authors.affiliation: School of Chemistry and Astbury Centre, University of Leeds, Leeds LS2 9JT, United Kingdom.
authors.forename: Stuart L
authors.initials: SL
authors.lastname: Warriner
authors.affiliation: From the Centre for Plant Sciences and School of Molecular and Cellular Biology and a.baker@leeds.ac.uk.
authors.forename: Alison
authors.initials: A
authors.lastname: Baker
mesh_terms: D017360:Arabidopsis / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D002352:Carrier Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D006368:Helianthus; D008291:Malate Dehydrogenase / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D008565:Membrane Proteins; C499192:PEX14 protein, Arabidopsis; C000590561:PEX5 protein, Arabidopsis; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D012097:Repressor Proteins; D001710:Biotin; D008291:Malate Dehydrogenase; C521419:PMDH1 protein, Arabidopsis; D010447:Peptide Hydrolases
keywords: Arabidopsis; PEX14; PEX5; biotinylation; peroxisome; protein chemical modification; protein import; protein-protein interaction