Hansenula polymorpha Pex1p and Pex6p are peroxisome-associated AAA proteins that functionally and physically interact

We have cloned the Hansenula polymorpha PEX1 and PEX6 genes by functional c omplementation of the corresponding peroxisome-deficient (pex) mutants. The gene products, HpPex1p and HpPex6p, are ATPases which both belong to the A AA protein family. Cells deleted for either gene (Delta pex1 or Delta pex6) were characterized by the presence of small peroxisomal remnants which con tained peroxisomal membrane proteins and minor amounts of matrix proteins. The bulk of the matrix proteins, however, resided in the cytosol. In cell f ractionation studies HpPex1p and HpPex6p co-sedimented with the peroxisomal membrane protein HpPex3p in both wild-type cells and in Delta pex4, Delta pex8 or Delta pex14 cells. Both proteins are loosely membrane-bound and fac e the cytosol. Furthermore, HpPex1p and HpPex6p physically and functionally interact in vivo. Overexpression of PEX6 resulted in defects in peroxisoma l matrix protein import. By contrast, overexpression of PEX1 was not detrim ental to the cells. Interestingly, co-overproduction of HpPex1p rescued the protein import defect caused by HpPex6p overproduction. Overproduced HpPex 1p and HpPex6p remained predominantly membrane-bound, but only partially co -localized with the peroxisomal membrane protein HpPex3p. Our data indicate that HpPex1p and HpPex6p function in a protein complex associated with the peroxisomal membrane and that overproduced, mislocalized HpPex6p prevents HpPex1p from reaching its site of activity. Copyright (C) 1999 John Wiley & Sons, Ltd.