Covalent Label Transfer Between Peroxisomal Importomer Components Reveals Export-Driven Import Interactions

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, owing to 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 PEX5C construct is monomeric in 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. Labelling 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.