Deviant Pex3p levels affect normal peroxisome formation in Hansenula polymorpha: high steady-state levels of the protein fully abolish matrix protein import.

Abstract

PEX3 encodes at 52 kDa peroxisomal membrane protein (PMP), essential for peroxisome biogenesis in the yeast Hansenula polymorpha. The relation between Pex3p levels and peroxisome formation was studied in wild type (WT) and delta pex3 strains expressing additional copies of PEX3 under control of a substrate-inducible promoter, namely the strong alcohol oxidase (PAOX) or the weaker amine oxidase (PAMO) promoter. In glucose-grown delta pex3 cells, containing PAOX.PEX3, Pex3p was undetectable and peroxisomes were absent. After induction of these cells on methanol, peroxisomes were rapidly formed. At Pex3p levels up to 7-10 times the values observed in WT controls normal peroxisomes were present. However, at further enhanced Pex3p levels a general matrix protein import defect was observed. This phenomenon was paralleled by aberrant peroxisome assembly and the formation of numerous small vesicles. These vesicles contained Pex3p, together with other H. polymorpha PMPs, but lacked the major matrix proteins which has accumulated in the cytosol. The implications of our results on PEX3 gene regulation and functioning of the peroxisomal matrix protein import machinery in H. polymorpha are discussed.