THE UBIQUITIN-CONJUGATING ENZYME PEX4P OF HANSENULA-POLYMORPHA IS REQUIRED FOR EFFICIENT FUNCTIONING OF THE PTS1 IMPORT MACHINERY

We have cloned the Hansenula polymorpha PEX4 gene by functional comple mentation of a peroxisome-deficient mutant. The PEX4 translation produ ct, Pex4p, is a member of the ubiquitin-conjugating enzyme family. In H.polymorpha, Pex4p is a constitutive, low abundance protein. Both the original mutant and the pex4 deletion strain (Delta pex4) showed a sp ecific defect in import of peroxisomal matrix proteins containing a C- terminal targeting signal (PTS1) and of malate synthase, whose targeti ng signal is not yet known. Import of the PTS2 protein amine oxidase a nd the insertion of the peroxisomal membrane proteins Pex3p and Pex14p was not disturbed in Delta pex4 cells. The PTS1 protein import defect in Delta pex4 cells could be suppressed by overproduction of the PTS1 receptor, Pex5p, in a dose-response related manner. In such cells, Pe x5p is localized in the cytosol and in peroxisomes. The peroxisome-bou nd Pex5p specifically accumulated at the inner surface of the peroxiso mal membrane and thus differed from Pex5p in wild-type peroxisomes, wh ich is localized throughout the matrix. We hypothesize that in H.polym orpha Pex4p plays an essential role for normal functioning of Pex5p, p ossibly in mediating recycling of Pex5p from the peroxisome to the cyt osol.