FORMATION OF THE PEROXISOME LUMEN IS ABOLISHED BY LOSS OF PICHIA-PASTORIS PAS7P, A ZINC-BINDING INTEGRAL MEMBRANE-PROTEIN OF THE PEROXISOME

We have cloned and sequenced PAS7, a gene required for peroxisome asse mbly in the yeast Pichia pastoris. The product of this gene, Pas7p, is a member of the C3HC4 superfamily of zinc-binding proteins. Point mut ations that alter conserved residues of the C3HC4 motif abolish P.AS7 activity and reduce zinc binding, suggesting that Pas7p binds zinc in vivo and that zinc binding is essential for PAS7 function. As with mos t pas mutants, pas7 cells exhibit a pronounced deficiency in import of peroxisomal matrix proteins that contain either the type 1 peroxisoma l targeting signal (PTS1) or the type 2 PTS (PTS2). However, while oth er yeast and mammalian pas mutants accumulate ovoid, vesicular peroxis omal intermediates, loss of Pas7p leads to accumulation of membrane sh eets and vesicles which lack a recognizable lumen. Thus, Pas7p appears to be essential for protein translocation into peroxisomes as well as formation of the lumen of the organelle. Consistent with these data, we find that Pas7p is an integral peroxisomal membrane protein which i s entirely resistant to exogenous protease and thus appears to reside completely within the peroxisome. Our observations suggest that the Fu nction of Pas7p defines a previously unrecognized step in peroxisome a ssembly: formation of the peroxisome lumen. Furthermore, because the p eroxisomal intermediates in the pas7 Delta mutant proliferate in respo nse to peroxisome-inducing environmental conditions, we conclude that Pas7p is not required for peroxisome proliferation.