2 AAA FAMILY PEROXINS, PPPEX1P AND PPPEX6P, INTERACT WITH EACH OTHER IN AN ATP-DEPENDENT MANNER AND ARE ASSOCIATED WITH DIFFERENT SUBCELLULAR MEMBRANOUS STRUCTURES DISTINCT FROM PEROXISOMES

Two peroxins of the AAA family, PpPex1p and PpPex6p, are required for peroxisome biogenesis in the yeast Pichia pastoris, Cells from the cor responding deletion strains (Pp Delta pex1 and Pp Delta pex6) contain only small vesicular remnants of peroxisomes, the bulk of peroxisomal matrix proteins is mislocalized to the cytosol, and these cells cannot grow in peroxisome-requiring media (J.A. Heyman, E. Monosov, and S. S ubramani, J. Cell Biol, 127:1259-1273, 1994; A. P. Spong and S, Subram ani, J, Cell Biol. 123:535-548, 1993), We demonstrate that PpPex1p and PpPex6p interact in an ATP-dependent manner, Genetically, the interac tion was observed in a suppressor screen with a strain harboring a tem perature-sensitive allele of PpPEX1 and in the yeast two-hybrid system , Biochemially, these proteins were coimmunoprecipitated with antibodi es raised against either of the proteins, but only in the presence of ATP. The protein complex formed under these conditions was 320 to 400 kDa in size, consistent with the formation of a heterodimeric PpPex1p- PpPex6p complex, Subcellular fractionation revealed PpPex1p and PpPex6 p to he predominantly associated with membranous subcellular structure s distinct from peroxisomes. Based on their behavior in subcellular fr actionation experiments including flotation gradients and on the fact that these structures are also present in a Pp Delta ex3 strain in whi ch no morphologically detectable proxisomal remnants have been observe d, we propose that these structures are small vesicles, The identifica tion of vesicle-associated peroxins is novel and implies a role for th ese vesicles in peroxisome biogenesis. We discuss the possible role of the ATP-dependent interaction between PpPex1p and PpPex6p in regulati ng peroxisome biogenesis events.