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

Novel Peroxisome–Vacuole Contacts in Yeast

Peroxisomes are important organelles and present in almost all eukaryotic cells. Close associations between peroxisomes and other cell compartments are known for several decades. The first molecular details of physical contacts between peroxisomes and various other organelles are now beginning to emerge. We recently described a novel contact between peroxisomes and vacuoles in the yeast Hansenula polymorpha, which develops during conditions of strong peroxisome proliferation. At such conditions, Pex3-GFP forms focal patches at the peroxisome–vacuole contacts, while overproduction of Pex3 promotes their formation. These results reveal a novel function for Pex3 in the formation of these contacts, where it might act as a tethering protein. We speculate that the peroxisome–vacuole contact is important for membrane lipid transfer at conditions of strong organellar expansion.</p

Peroxisomes:surprisingly versatile organelles

AbstractPeroxisome development is a dynamic process that is not yet completely understood. We use the methylotrophic yeast Hansenula polymorpha as model in our studies on peroxisome homeostasis. Cells of this species may contain different types of peroxisomes that differ in protein composition and capacity to incorporate matrix proteins. This protein import machinery is highly flexible and can accommodate unfolded and complex folded proteins

Structure-function analysis of the ER-peroxisome contact site protein Pex32

In the yeast Hansenula polymorpha, the ER protein Pex32 is required for associating peroxisomes to the ER. Here, we report on a structure-function analysis of Pex32. Localization studies of various Pex32 truncations showed that the N-terminal transmembrane domain of Pex32 is responsible for sorting. Moreover, this part of the protein is sufficient for the function of Pex32 in peroxisome biogenesis. The C-terminal DysF domain is required for concentrating Pex32 at ER-peroxisome contact sites and has the ability to bind to peroxisomes. In order to better understand the role of Pex32 in peroxisome biogenesis, we analyzed various peroxisomal proteins in pex32 cells. This revealed that Pex11 levels are strongly reduced in pex32 cells. This may explain the strong reduction in peroxisome numbers in pex32 cells, which also occurs in cells lacking Pex11

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).

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.

Peroxisome Maintenance Depends on De Novo Peroxisome Formation in Yeast Mutants Defective in Peroxisome Fission and Inheritance

There is an ongoing debate on how peroxisomes form: by growth and fission of pre-existing peroxisomes or de novo from another membrane. It has been proposed that, in wild type yeast cells, peroxisome fission and careful segregation of the organelles over mother cells and buds is essential for organelle maintenance. Using live cell imaging we observed that cells of the yeast Hansenula polymorpha, lacking the peroxisome fission protein Pex11, still show peroxisome fission and inheritance. Also, in cells of mutants without the peroxisome inheritance protein Inp2 peroxisome segregation can still occur. In contrast, peroxisome fission and inheritance were not observed in cells of a pex11 inp2 double deletion strain. In buds of cells of this double mutant, new organelles likely appear de novo. Growth of pex11 inp2 cells on methanol, a growth substrate that requires functional peroxisomes, is retarded relative to the wild type control. Based on these observations we conclude that in H. polymorpha de novo peroxisome formation is a rescue mechanism, which is less efficient than organelle fission and inheritance to maintain functional peroxisomes

A critical reflection on the principles of peroxisome formation in yeast

We have evaluated the current knowledge on peroxisome proliferation in yeast. In wild-type cells, peroxisomes multiply predominantly by fission at conditions that require peroxisome function(s) for growth. In cells that lack peroxisomes, for instance in pex3 and pex19 mutants or in mutants that display inheritance defects, peroxisomes may form de novo. We propose a novel machinery for the de novo formation of peroxisomes in pex3 cells, in which new peroxisomes do not arise from the endoplasmic reticulum. This machinery is based on the recent observation that membrane vesicles are present in these cells that display peroxisomal characteristics in that they contain specific peroxisomal membrane and matrix proteins. These structures are the target for newly formed peroxisomes upon reintroduction of Pex3. Furthermore, we critically evaluate the principles of sorting of other peroxisomal membrane proteins to their target organelle and the function of the endoplasmic reticulum therein