Fusion of small peroxisomal vesicles in vitro reconstructs an early step in the in vivo multistep peroxisome assembly pathway of Yarrowia lipolytica

We have identified and purified six subforms of peroxisomes, designated P1 to P6, from the yeast, Yarrowia lipolytica. An analysis of trafficking of p eroxisomal proteins in vivo suggests the existence of a multistep peroxisom e assembly pathway in Y. lipolytica. This pathway operates by conversion of peroxisomal subforms in the direction P1, P2-->P3-->P4-->P5-->P6 and invol ves the import of various peroxisomal proteins into distinct vesicular inte rmediates. We have also reconstituted in vitro the fusion of the earliest i ntermediates in the pathway, small peroxisomal vesicles P1 and P2, Their fu sion leads to the formation of a larger and more dense peroxisomal vesicle, P3, Fusion of P1 and P2 in vitro requires cytosol and ATP hydrolysis and i s inhibited by antibodies to two membrane-associated ATPases of the AAA fam ily, Pex1p and Pex6p, We provide evidence that the fusion in vitro of P1 an d P2 peroxisomes reconstructs an actual early step in the peroxisome assemb ly pathway operating in vivo in Y. lipolytica.