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

doi:10.1083/jcb.148.1.29

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© The Rockefeller University Press, 0021-9525/2000//29 $5.00
The Journal of Cell Biology, Volume 148, Number 1, , 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. Titorenko^a, Honey Chan^a, and Richard A. Rachubinski^a

^a Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada
Department of Cell Biology, University of Alberta, Medical Sciences Building 5-14, Edmonton, Alberta T6G 2H7, Canada.(780) 492-9278(780) 492-9868

rick.rachubinski{at}ualberta.ca

We have identified and purified six subforms of peroxisomes,designated P1 to P6, from the yeast, Yarrowia lipolytica. Ananalysis of trafficking of peroxisomal proteins in vivo suggeststhe existence of a multistep peroxisome assembly pathway inY. lipolytica. This pathway operates by conversion of peroxisomalsubforms in the direction P1, P2 $->$ P3 $->$ P4 $->$ P5 $->$ P6 and involves the importof various peroxisomal proteins into distinct vesicular intermediates.We have also reconstituted in vitro the fusion of the earliestintermediates in the pathway, small peroxisomal vesicles P1and P2. Their fusion leads to the formation of a larger andmore dense peroxisomal vesicle, P3. Fusion of P1 and P2 in vitrorequires cytosol and ATP hydrolysis and is inhibited by antibodiesto two membrane-associated ATPases of the AAA family, Pex1pand Pex6p. We provide evidence that the fusion in vitro of P1and P2 peroxisomes reconstructs an actual early step in theperoxisome assembly pathway operating in vivo in Y. lipolytica.

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

Abbreviations used in this paper: AOX, acyl-CoA oxidase; HSP,high-speed pelletable peroxisomes; NSF, N-ethylmaleimide-sensitivefactor; SNAPs, soluble NSF attachment proteins; SNAREs, SNAPreceptors; v-SNAREs and t-SNAREs, vesicle- and target-specificintegral membrane proteins, respectively.

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