Characterization of intermediates in the process of plant peroxisomal protein import

A hybrid protein in which the immunoglobulin G-binding domain of Staphyloco ccus aureus protein A replaced the N-terminal 43 amino acids of glycolate o xidase (a peroxisomal protein) was affinity purified after expression in Es cherichia coli and used to study peroxisomal protein import in vitro. The f usion protein, which co-purifies with the bacterial chaperones dnaK and gro EL, binds to glyoxysomes and is partially translocated in an ATP-dependent reaction which is independent of eukaryotic cytosol. Both binding and trans location are dependent upon the amount of glyoxysomes present. The partiall y translocated species has a transmembrane location and is extractable by s alt, indicating that it is held in the membrane by ionic interactions. In t he absence of ATP, the fusion protein binds to the surface of the glyoxysom es and competes the binding of authentic matrix proteins. The surface-bound protein can be chased to the transmembrane species upon the addition of AT P, These results indicate that the surface-bound form is a true translocati on intermediate. The availability of this fusion protein in milligram quant ities offers the possibility to use the intermediate formed in the absence of ATP and the transmembrane species to probe interactions with the peroxis ome import machinery.