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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.

Original publication

DOI

10.1074/jbc.M115.686501

Type

Journal article

Journal

J Biol Chem

Publication Date

2016

Volume

291

Pages

2460 - 2468

Keywords

Arabidopsis/*metabolism Arabidopsis Proteins/*metabolism Biotin/chemistry Biotinylation Carrier Proteins/metabolism Cell Membrane/metabolism Cytosol/metabolism Helianthus Malate Dehydrogenase/metabolism Membrane Proteins/*metabolism Peptide Hydrolases/metabolism Peroxisome-Targeting Signal 1 Receptor Peroxisomes/*metabolism Protein Binding Protein Interaction Mapping Protein Structure, Tertiary Protein Transport Receptors, Cytoplasmic and Nuclear/*metabolism Repressor Proteins/*metabolism Arabidopsis Pex14 Pex5 peroxisome protein chemical modification protein import protein-protein interaction