We have cloned and characterized the Hansenula polymorpha PER9 gene by functional complementation of the per9-1 mutant of H. polymorpha, which is defective in peroxisome biogenesis. The predicted product, Per9p, is a polypeptide of 52 kDa with sequence similarity to Pas3p, a protein involved in peroxisome biogenesis in Saccharomyces cerevisiae. In a per9 disruption strain (⌬per9), peroxisomal matrix and membrane proteins are present at wild-type levels. The matrix proteins accumulated in the cytoplasm. However, the location of the membrane proteins remained obscure; fully induced ⌬per9 cells lacked residual peroxisomal vesicles ("ghosts"). Analysis of the activity of the PER9 promoter revealed that PER9 expression was low in cells grown on glucose, but was enhanced during growth of cells on peroxisome-inducing substrates. The highest expression levels were observed in cells grown on methanol. Localization studies revealed that Per9p is an integral membrane protein of the peroxisome. Targeting studies suggested that Per9p may be sorted to the peroxisome via the endoplasmic reticulum. Overexpression of PER9 induced a significant increase in the number of peroxisomes per cell, a result that suggests that Per9p may be involved in peroxisome proliferation and/or membrane biosynthesis. When PER9 expression was placed under the control of a strongly regulatable promoter and switched off, peroxisomes were observed to disintegrate over time in a manner that suggested that Per9p may be required for maintenance of the peroxisomal membrane.Peroxisomes are cell organelles that are present in virtually all eukaryotic cells. They perform specific metabolic functions that are often related to the developmental stage and/or the organism in which they occur (1). The metabolic importance of peroxisomes in humans is demonstrated by the fact that the absence of the organelles leads to severe abnormalities, followed by an early death (e.g. Zellweger syndrome (2)). Consequently, many studies are now devoted to unravel the molecular mechanisms of peroxisome biogenesis and function. Yeasts are excellent model systems for such studies having the advantages that (i) the induction and protein composition of peroxisomes can readily be manipulated by varying growth conditions and (ii) in the absence of peroxisomes, yeasts are viable (3, 4). Hence, peroxisome-deficient mutants have been isolated from different yeast species (4), and the corresponding genes are being cloned and characterized.In yeast, peroxisomes normally develop by growth and fission from pre-existing ones. Peroxisomal matrix proteins are nuclear-encoded, synthesized in the cytoplasm, and directed to the organelle by topogenic signals (PTSs).1 Two PTSs have been identified and are located either at the extreme C terminus (PTS1) or the N terminus of the protein (PTS2) (4). Our knowledge on the sorting of peroxisomal membrane proteins is still limited, and consensus topogenic sequences have yet to be identified (5).In our laboratory, we use the methylotrophic yeast Hansenula po...

PER genes are essential for the assembly of peroxisomes in Hansenula polymorpha. Here we describe the PER3 gene which was cloned by functional complementation of a H. polymorpha per3 mutant. The complementing PER3 gene encodes a protein of 569 amino acids (Per3p) with a calculated mass of 63.9 kDa; Per3p belongs to the tetratricopeptide repeat protein family and is located in both the cytosol and the peroxisomal matrix. Remarkably, Per3p does not contain a known targeting signal (PTS1 or PTS2). The PER3 gene product shows similarity to the Saccharomyces cerevisiae Pas10p (40% identity) and the Pichia pastoris Pas8p (55% identity). However, their function apparently cannot be interchanged since the P. pastoris PAS8 gene failed to functionally complement a H. polymorpha per3 disruption mutant. The per3 disruption mutant contained normal but small peroxisomes in which PTS2 proteins (both homologous and heterologous) were imported. Other matrix proteins (in particular PTS1 proteins) resided in the cytosol where they were normally assembled and active. We argue that Per3p is a component of the peroxisomal import machinery and most probably shuttles matrix proteins from the cytosol to the organellar matrix.

We have cloned the Hansenula polymorpha PEX4 gene by functional complementation of a peroxisomedeficient mutant. The PEX4 translation product, 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 (Δpex4) showed a specific defect in import of peroxisomal matrix proteins containing a C-terminal targeting signal (PTS1) and of malate synthase, whose targeting signal is not yet known. Import of the PTS2 protein amine oxidase and the insertion of the peroxisomal membrane proteins Pex3p and Pex14p was not disturbed in Δpex4 cells. The PTS1 protein import defect in Δpex4 cells could be suppressed by overproduction of the PTS1 receptor, Pex5p, in a dose-response related manner. In such cells, Pex5p is localized in the cytosol and in peroxisomes. The peroxisome-bound Pex5p specifically accumulated at the inner surface of the peroxisomal membrane and thus differed from Pex5p in wild-type peroxisomes, which is localized throughout the matrix. We hypothesize that in H.polymorpha Pex4p plays an essential role for normal functioning of Pex5p, possibly in mediating recycling of Pex5p from the peroxisome to the cytosol.

We have cloned the Hansenula polymorpha PEX1 and PEX6 genes by functional complementation of the corresponding peroxisome‐deficient (pex) mutants. The gene products, HpPex1p and HpPex6p, are ATPases which both belong to the AAA protein family. Cells deleted for either gene (Δpex1 or Δpex6) were characterized by the presence of small peroxisomal remnants which contained peroxisomal membrane proteins and minor amounts of matrix proteins. The bulk of the matrix proteins, however, resided in the cytosol. In cell fractionation studies HpPex1p and HpPex6p co‐sedimented with the peroxisomal membrane protein HpPex3p in both wild‐type cells and in Δpex4, Δpex8 or Δpex14 cells. Both proteins are loosely membrane‐bound and face the cytosol. Furthermore, HpPex1p and HpPex6p physically and functionally interact in vivo. Overexpression of PEX6 resulted in defects in peroxisomal matrix protein import. By contrast, overexpression of PEX1 was not detrimental to the cells. Interestingly, co‐overproduction of HpPex1p rescued the protein import defect caused by HpPex6p overproduction. Overproduced HpPex1p 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 © 1999 John Wiley & Sons, Ltd.

We have analyzed the presence of peroxisomal remnants ('ghosts') in three peroxisome-deficient (per) mutants of the yeast Hansenula polymorpha, namely Aper4, Aper5 and AperlO. Under peroxisome-inducing growth conditions peroxisomal membrane proteins (PMPs) were normally synthesized in cells of these mutants. In addition, these cells contained clusters of small membranous vesicles, which were absent in cells grown under peroxisome-repressing growth conditions. These structures displayed typical peroxisomal properties in that they proliferated upon overproduction of Per8p, the H. polymorpha peroxisome proliferation factor. Moreover, in Aper4 and Aper5 these vesicles were susceptible to glucose-induced proteolytic degradation.