Two AAA family ATPases, NSF and p97, have been implicated in membrane fusio
n during assembly and inheritance of organelles of the secretory pathway. W
e have now investigated the roles of AAA ATPases in membrane fusion during
assembly of the peroxisome, an organelle outside the classical secretory sy
stem. Here, we show that peroxisomal membrane fusion in the yeast Yarrowia
lipolytica requires two AAA ATPases, Pex1p and Pex6p. Release of membrane-a
ssociated Pex1p and Pex6p drives the asymmetric priming of two fusion partn
ers. The next step, peroxisome docking, requires release of Pex1p from one
part ner. Subsequent fusion of the peroxisomal membranes is independent of
both Pex1p and Pex6p.