DEVIANT PEX3P LEVELS AFFECT NORMAL PEROXISOME FORMATION IN HANSENULA-POLYMORPHA - A SHARP INCREASE OF THE PROTEIN LEVEL INDUCES THE PROLIFERATION OF NUMEROUS, SMALL PROTEIN-IMPORT COMPETENT PEROXISOMES

Pex3p has been implicated in the biosynthesis of the peroxisomal membr ane of the yeast Hansenula polymorpha. Here we show that in the initia l stages of a sharp increase in Pex3p levels, induced in batch culture s of cells of a constructed H. polymorpha strain, which contained seve n copies of PEX3 under control of the alcohol oxidase promoter (WT::P- AOX.PEX3(7x)), strongly interfered with normal peroxisome proliferatio n. Ultrastructural studies demonstrated that in such cells numerous sm all peroxisomes had developed, which were absent in wild-type controls . These organelles, which contained typical peroxisomal matrix and mem brane proteins (alcohol oxidase, catalase, Pex3p, Pex10p and Pex14p), showed a relatively low density(1.18 g cm(-3)) after sucrose gradient centrifugation of WT::P-AOX.PEX3(7x) homogenates, compared to normal p eroxisomes (1.23 g cm(-3)). We furthermore demonstrated that these ear ly induced, small peroxisomes were protected against glucose-induced p roteolytic degradation and did not fuse to form larger organelles. Rem arkably, the induction of these small peroxisomes was paralleled by a partial defect in matrix protein import, reflected by the mislocalizat ion of minor amounts of alcohol oxidase protein in the cytosol. Howeve r, when the cells were subsequently placed under conditions in which t he synthesis of a new matrix enzyme (amine oxidase) was induced while simultaneously the excessive proliferation was repressed (by repressio n of the P-AOX), amine oxidase protein was selectively incorporated in to these organelles. This indicated that the small peroxisomes had reg ained a normal protein import capacity. Based on these results we argu e that peroxisome proliferation and matrix protein import are coupled processes in H. polymorpha. (C) 1997 John Wiley & Sons, Ltd.