Location of catalase in crystalline peroxisomes of methanol-grown Hansenula polymorpha

We have studied the intraperoxisomal location of catalase in peroxisomes of methanol-grown Hansenula polymorpha by (immuno)cytochemical means. In completely crystalline peroxisomes, in which the crystalline matrix is composed of octameric alcohol oxidase (AO) molecules, most of the catalase protein is located in a narrow zone between the crystalloid and the peroxisomal membrane. In non-crystalline organelles the enzyme was present throughout the peroxisomal matrix. Other peroxisomal matrix enzymes studied for comparison, namely dihydroxyacetone synthase, amine oxidase and malate synthase, all were present throughout the AO crystalloid. The advantage of location of catalase at the edges of the AO crystalloids for growth of the organism on methanol is discussed.

Pay32p of the Yeast Yarrowia lipolytica Is an Intraperoxisomal Component of the Matrix Protein Translocation Machinery

Pay mutants of the yeast Yarrowia lipolytica fail to assemble functional peroxisomes. One mutant strain, pay32-1, has abnormally small peroxisomes that are often found in clusters surrounded by membranous material. The functionally complementing gene PAY32 encodes a protein, Pay32p, of 598 amino acids (66,733 D) that is a member of the tetratricopeptide repeat family. Pay32p is intraperoxisomal. In wild-type peroxisomes, Pay32p is associated primarily with the inner surface of the peroxisomal membrane, but ~30% of Pay32p is localized to the peroxisomal matrix. The majority of Pay32p in the matrix is complexed with two polypeptides of 62 and 64 kD recognized by antibodies to SKL (peroxisomal targeting signal-1). In contrast, in peroxisomes of the pay32-1 mutant, Pay32p is localized exclusively to the matrix and forms no complex. Biochemical characterization of the mutants pay32-1 and pay32-KO (a PAY32 gene disruption strain) showed that Pay32p is a component of the peroxisomal translocation machinery. Mutations in the PAY32 gene prevent the translocation of most peroxisome-bound proteins into the peroxisomal matrix. These proteins, including the 62-kD anti-SKL-reactive polypeptide, are trapped in the peroxisomal membrane at an intermediate stage of translocation in pay32 mutants. Our results suggest that there are at least two distinct translocation machineries involved in the import of proteins into peroxisomes.

The Hansenula polymorpha PER8 Gene Encodes a Novel Peroxisomal Integral Membrane Protein Involved in Proliferation

We previously described the isolation of mutants of the methylotrophic yeast Hansenula polymorpha that are defective in peroxisome biogenesis. Here, we describe the characterization of one of these mutants, per8, and the cloning of the PER8 gene. In either methanol or methylamine medium, conditions that normally induce the organdies, per8 cells contain no peroxisome-like structures and peroxisomal enzymes are located in the cytosol. The sequence of PER8 predicts that its product (Per8p) is a novel polypeptide of 34 kD, and antibodies against Per8p recognize a protein of 31 kD. Analysis of the primary sequence of Per8p revealed a 39-amino-acid cysteine-rich segment with similarity to the C3HC4 family of zinc-finger motifs. Overexpression of PER8 results in a markedly enhanced increase in peroxisome numbers. We show that Per8p is an integral membrane protein of the peroxisome and that it is concentrated in the membranes of newly formed organdies. We propose that Per8p is a component of the molecular machinery that controls the proliferation of this organelle.

A comparative study of peroxisomal structures in Hansenula polymorpha pex mutants

In a recent study, we performed a systematic genome analysis for the conservation of genes involved in peroxisome biogenesis (PEX genes) in various fungi. We have now performed a systematic study of the morphology of peroxisome remnants (‘ghosts’) in Hansenula polymorpha pex mutants (pex1–pex20) and the level of peroxins and matrix proteins in these strains. To this end, all available H. polymorpha pex strains were grown under identical cultivation conditions in glucose-limited chemostat cultures and analyzed in detail. The H. polymorpha pex mutants could be categorized into four distinct groups, namely pex mutants containing: (1) virtually normal peroxisomal structures (pex7, pex17, pex20); (2) small peroxisomal membrane structures with a distinct lumen (pex2, pex4, pex5, pex10, pex12, pex14); (3) multilayered membrane structures lacking apparent matrix protein content (pex1, pex6, pex8, pex13); and (4) no peroxisomal structures (pex3, pex19).

PTS1-independent sorting of peroxisomal matrix proteins by Pex5p

Most peroxisomal matrix proteins contain a peroxisomal targeting signal 1 (PTS1) for sorting to the correct organelle. This signal is located at the extreme C-terminus and generally consists of only three amino acids. The PTS1 is recognized by the receptor protein Pex5p. Several examples have been reported of peroxisomal matrix proteins that are sorted to peroxisomes via Pex5p, but lack a typical PTS1 tripeptide. In this contribution we present an overview of these so-called non-PTS1 proteins and discuss the current knowledge of the molecular mechanisms involved in their sorting. (C) 2006 Elsevier B.V. All rights reserved

Peroxisome biogenesis in Hansenula polymorpha: different mutations in genes, essential for peroxisome biogenesis, cause different peroxisomal mutant phenotypes

In Hansenula polymorpha, different monogenic recessive mutations mapped in either of two previously identified genes, PER1 and PER3, produced different peroxisomal mutant phenotypes. Among five per1 mutants, four showed a Pim- phenotype: the cells contained few small peroxisomes while the bulk of the matrix enzymes resided in the cytosol. One of these mutants, per1-124 had an enhanced rate of peroxisome proliferation. The fifth mutant completely lacked peroxisomes (Per- phenotype). Of seven per3 mutants, four displayed a Pim- phenotype, two others a Per- phenotype, while one mutant showed pH-dependent growth on methanol and was affected in oligomerization of peroxisomal matrix protein. Thus, the protein products of both PER1 and PER3 genes appear to be essential in different aspects of peroxisome assembly/proliferation.

Reprogramming Hansenula polymorpha for penicillin production: expression of the Penicillium chrysogenum pcl gene

We aim to introduce the penicillin biosynthetic pathway into the methylotrophic yeast Hansenula polymorpha. To allow simultaneous expression of the multiple genes of the penicillin biosynthetic pathway, additional markers were required. To this end, we constructed a novel host–vector system based on methionine auxotrophy and the H. polymorpha MET6 gene, which encodes a putative cystathionine β-lyase. With this new host–vector system, the Penicillium chrysogenum pcl gene, encoding peroxisomal phenylacetyl-CoA ligase (PCL), was expressed in H. polymorpha. PCL has a potential C-terminal peroxisomal targeting signal type 1 (PTS1). Our data demonstrate that a green fluorescent protein–PCL fusion protein has a dual location in the heterologous host in the cytosol and in peroxisomes. Mutation of the PTS1 of PCL (SKI-COOH) to SKL-COOH restored sorting of the fusion protein to peroxisomes only. Additionally, we demonstrate that peroxisomal PCL–SKL produced in H. polymorpha displays normal enzymatic activities.