Cytosol-dependent peroxisomal protein import in a permeabilized cell system.

Abstract

Using streptolysin-O (SLO) we have developed a permeabilized cell system retaining the competence to import proteins into peroxisomes. We used luciferase and albumin conjugated with a peptide ending in the peroxisomal targeting sequence, SKL, to monitor the import of proteins into peroxisomes. After incubation with SLO-permeabilized cells, these exogenous proteins accumulated within catalase-containing vesicles. The import was strictly signal dependent and could be blocked by a 10-fold excess of peptide containing the SKL-targeting signal, while a control peptide did not affect the import. Peroxisomal accumulation of proteins was time and temperature dependent and required ATP hydrolysis. Dissipation of the membrane potential did not alter the import efficiency. GTP-hydrolyzing proteins were not required for peroxisomal protein targeting. Depletion of endogenous cytosol from permeabilized cells abolished the competence to import proteins into peroxisomes but import was reconstituted by the addition of external cytosol. We present evidence that cytosol contains factors with SKL-specific binding sites. The activity of cytosol is insensitive to N- ethylmaleimide (NEM) treatment, while the cells contain NEM-sensitive membrane-bound or associated proteins which are involved in the import machinery. The cytosol dependence and NEM-sensitivity of peroxisomal protein import should facilitate the purification of proteins involved in the import of proteins into peroxisomes.

Quantitative Analysis of Peroxisomal Protein Import in Vitro

“…An immunofluorescence-based assay, also utilizing semipermeabilized cells, is an effective system that has led to the description of several novel properties of the import process [20]. Indeed, a variation of it is used in this study (Figs.…”

“…A431 cells were permeabilized with 0.05 U/ml streptolysin-O and incubated with 60 g/ml biotinylated luciferase for 45 min at 37°C in an in vitro import assay essentially as described by Wendland and Subramani [20]. The import system was optimized for use with A431 cells as follows.…”

“…Since the necessary confocal fluorescence microscopy could be performed only on immobilized cells, mechanical disruption was not possible. Therefore, we employed streptolysin-O to permeabilize cells-in essence, we were performing the immunofluorescence-based import assay described by Wendland and Subramani [20]. Using this approach, for the first time with human A431 cells, it was clear that biotinylated luciferase was taken up into the lumen of peroxisomes (Figs.…”

“…The assay, developed by Wendland and Subramani [20], relies on the properties of the bacterial toxin streptolysin-O, which permeabilizes plasma, but not peroxisomal, membranes. After this selective fenestration of the cell's outer membranes, import substrates (e.g., human serum albumin enzymatically coupled to a PTS1 peptide) and various biochemical factors are allowed to diffuse into the cell.…”

“…That these structures are actually peroxisomes is then confirmed by their colocalization with bona fide peroxisomal marker proteins. In addition to the properties revealed by the other approaches, this assay has been used to demonstrate a direct requirement for cytosolic factors, in general [20], and for Pex5p [21] and Pex14p [11] in particular.…”