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© The Rockefeller University Press, 0021-9525/2000/1/29/ $5.00
The Journal of Cell Biology, Volume 148, Number 1, January 10, 2000 29-44

Original Article

Fusion of Small Peroxisomal Vesicles In Vitro Reconstructs an Early Step in the In Vivo Multistep Peroxisome Assembly Pathway of Yarrowia lipolytica

Vladimir I. Titorenkoa, Honey Chana, and Richard A. Rachubinskia
a Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada

Correspondence to: Richard A. Rachubinski, Department of Cell Biology, University of Alberta, Medical Sciences Building 5-14, Edmonton, Alberta T6G 2H7, Canada. Tel:(780) 492-9868 Fax:(780) 492-9278 E-mail:rick.rachubinski{at}ualberta.ca.

We have identified and purified six subforms of peroxisomes, designated P1 to P6, from the yeast, Yarrowia lipolytica. An analysis of trafficking of peroxisomal proteins in vivo suggests the existence of a multistep peroxisome assembly pathway in Y. lipolytica. This pathway operates by conversion of peroxisomal subforms in the direction P1, P2->P3->P4->P5->P6 and involves the import of various peroxisomal proteins into distinct vesicular intermediates. We have also reconstituted in vitro the fusion of the earliest intermediates in the pathway, small peroxisomal vesicles P1 and P2. Their fusion 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 is inhibited by antibodies to two membrane-associated ATPases of the AAA family, Pex1p and Pex6p. We provide evidence that the fusion in vitro of P1 and P2 peroxisomes reconstructs an actual early step in the peroxisome assembly pathway operating in vivo in Y. lipolytica.

Key Words: microbody, peroxisomal protein import, biogenesis, membrane fusion, in vitro reconstitution


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