Hanspeter Rottensteiner

The peroxin PEX14 of Neurospora crassa is essential for the biogenesis of both glyoxysomes and Woronin bodies

In the filamentous fungus Neurospora crassa, glyoxysomes and Woronin bodies coexist in the same cell. Because several glyoxysomal matrix proteins and also HEX1, the dominant protein of Woronin bodies, possess typical peroxisomal targeting signals, the question arises as to how protein targeting to these distinct yet related types of microbodies is achieved. Here we analyzed the function of the Neurospora ortholog of PEX14, an essential component of the peroxisomal import machinery. PEX14 interacted with both targeting signal receptors and was localized to glyoxysomes but was virtually absent from Woronin bodies. Nonetheless, a pex14Δ mutant not only failed to grow on fatty acids because of a defect in glyoxysomal β‐oxidation but also suffered from cytoplasmic bleeding, indicative of a defect in Woronin body‐dependent septal pore plugging. Inspection of pex14Δ mutant hyphae by fluorescence and electron microscopy indeed revealed the absence of Woronin bodies. When these cells were subjected to subcellular fractionation, HEX1 was completely mislocalized to the cytosol. Expression of GFP‐HEX1 in wild‐type mycelia caused the staining of Woronin bodies and also of glyoxysomes in a targeting signal‐dependent manner. Our data support the view that Woronin bodies emerge from glyoxysomes through import of HEX1 and subsequent fission.

Peroxisomes as Novel Players in Cell Calcium Homeostasis

Ca2+ concentration in peroxisomal matrix ([Ca2+]perox) has been monitored dynamically in mammalian cells expressing variants of Ca2+-sensitive aequorin specifically targeted to peroxisomes. Upon stimulation with agonists that induce Ca2+ release from intracellular stores, peroxisomes transiently take up Ca2+ reaching peak values in the lumen as high as 50–100 μm, depending on cell types. Also in resting cells, peroxisomes sustain a Ca2+ gradient, [Ca2+]perox being ∼20-fold higher than [Ca2+] in the cytosol ([Ca2+]cyt). The properties of Ca2+ traffic across the peroxisomal membrane are different from those reported for other subcellular organelles. The sensitivity of peroxisomal Ca2+ uptake to agents dissipating H+ and Na+ gradients unravels the existence of a complex bioenergetic framework including V-ATPase, Ca2+/H+, and Ca2+/Na+ activities whose components are yet to be identified at a molecular level. The different [Ca2+]perox of resting and stimulated cells suggest that Ca2+ could play an important role in the regulation of peroxisomal metabolism.

A Eukaryote without Catalase-Containing Microbodies : Neurospora crassa Exhibits a Unique Cellular Distribution of Its Four Catalases

ABSTRACT Microbodies usually house catalase to decompose hydrogen peroxide generated within the organelle by the action of various oxidases. Here we have analyzed whether peroxisomes (i.e., catalase-containing microbodies) exist in Neurospora crassa. Three distinct catalase isoforms were identified by native catalase activity gels under various peroxisome-inducing conditions. Subcellular fractionation by density gradient centrifugation revealed that most of the spectrophotometrically measured activity was present in the light upper fractions, with an additional small peak coinciding with the peak fractions of HEX-1, the marker protein for Woronin bodies, a compartment related to the microbody family. However, neither in-gel assays nor monospecific antibodies generated against the three purified catalases detected the enzymes in any dense organellar fraction. Furthermore, staining of an N. crassa wild-type strain with 3,3′-diaminobenzidine and H2O2 did not lead to catalase-dependent reaction products within microbodies. Nonetheless, N. crassa does possess a gene (cat-4) whose product is most similar to the peroxisomal type of monofunctional catalases. This novel protein indeed exhibited catalase activity, but was not localized to microbodies either. We conclude that N. crassa lacks catalase-containing peroxisomes, a characteristic that is probably restricted to a few filamentous fungi that produce little hydrogen peroxide within microbodies.

Identification of a Novel, Intraperoxisomal Pex14-Binding Site in Pex13: Association of Pex13 with the Docking Complex Is Essential for Peroxisomal Matrix Protein Import

ABSTRACT The peroxisomal docking complex is a key component of the import machinery for matrix proteins. The core protein of this complex, Pex14, is thought to represent the initial docking site for the import receptors Pex5 and Pex7. Associated with this complex is a fraction of Pex13, another essential component of the import machinery. Here we demonstrate that Pex13 directly binds Pex14 not only via its SH3 domain but also via a novel intraperoxisomal site. Furthermore, we demonstrate that Pex5 also contributes to the association of Pex13 with Pex14. Peroxisome function was affected only mildly by mutations within the novel Pex14 interaction site of Pex13 or by the non-Pex13-interacting mutant Pex5W204A. However, when these constructs were tested in combination, PTS1-dependent import and growth on oleic acid were severely compromised. When the SH3 domain-mediated interaction of Pex13 with Pex14 was blocked on top of that, PTS2-dependent matrix protein import was completely compromised and Pex13 was no longer copurified with the docking complex. We conclude that the association of Pex13 with Pex14 is an essential step in peroxisomal protein import that is enabled by two direct interactions and by one that is mediated by Pex5, a result which indicates a novel, receptor-independent function of Pex5.

Farnesylation of Pex19p Is Required for Its Structural Integrity and Function in Peroxisome Biogenesis

The conserved CaaX box peroxin Pex19p is known to be modified by farnesylation. The possible involvement of this lipid modification in peroxisome biogenesis, the degree to which Pex19p is farnesylated, and its molecular function are unknown or controversial. We resolve these issues by first showing that the complete pool of Pex19p is processed by farnesyltransferase in vivo and that this modification is independent of peroxisome induction or the Pex19p membrane anchor Pex3p. Furthermore, genomic mutations of PEX19 prove that farnesylation is essential for proper matrix protein import into peroxisomes, which is supposed to be caused indirectly by a defect in peroxisomal membrane protein (PMP) targeting or stability. This assumption is corroborated by the observation that mutants defective in Pex19p farnesylation are characterized by a significantly reduced steady-state concentration of prominent PMPs (Pex11p, Ant1p) but also of essential components of the peroxisomal import machinery, especially the RING peroxins, which were almost depleted from the importomer. In vivo and in vitro, PMP recognition is only efficient when Pex19p is farnesylated with affinities differing by a factor of 10 between the non-modified and wild-type forms of Pex19p. Farnesylation is likely to induce a conformational change in Pex19p. Thus, isoprenylation of Pex19p contributes to substrate membrane protein recognition for the topogenesis of PMPs, and our results highlight the importance of lipid modifications in protein-protein interactions.

The N-domain of Pex22p can functionally replace the Pex3p N-domain in targeting and peroxisome formation.

Pex3p is a central component of the import machinery for peroxisomal membrane proteins (PMPs) that can reach peroxisomes via the endoplasmic reticulum (ER) and even trigger de novo peroxisome formation from the ER. Pex19p is the import receptor for type I PMPs, whereas targeting of type II PMPs, of which Pex3p so far represents the only species, does not require Pex19p. Pex3p possesses two domains with distinct function: a short N-terminal domain, which harbors the information for peroxisomal (and ER) targeting, and a C-terminal domain, which faces the cytosol and serves as a docking site for Pex19p, thereby delivering newly synthesized PMPs to the peroxisome. Here we show that the N-terminal domain of Pex3p can be functionally replaced by the N-terminal peroxisomal membrane targeting signal (mPTS) of Pex22p, a supposedly unrelated component of the import machinery for peroxisomal matrix proteins. An exchange of the mPTS of Pex22p by that of Pex3p likewise fully preserved the function of Pex22p. Neither of the two mPTS interacted with Pex19p, and in the absence of Pex19p, colocalization of Pex3p and Pex22p was observed, indicating that also Pex22p is targeted to peroxisomes by a type II mPTS. When a type I mPTS was hooked to the C-terminal domains of Pex22p and Pex3p, function was retained in the case of Pex22p and in part even for Pex3p. The C-terminal domain of Pex3p thus contains the relevant information required for de novo peroxisome formation, thereby challenging the concept of the N terminus of Pex3p being key in that process.

Identification of PEX33, a novel component of the peroxisomal docking complex in the filamentous fungus Neurospora crassa

The docking complex of peroxisomal matrix protein import is composed of PEX13 and PEX14 in all species analyzed so far, whereas only yeast appears to possess an additional component, PEX17. In this report we isolated PEX14 complexes of Neurospora crassa. Among the complex constituents, one protein designated as PEX33 possessed homology to PEX14 but only in a short N-terminal domain. The PEX14/PEX33 interaction was verified by means of two-hybrid analysis. Moreover, PEX33 was shown to interact with itself and the PTS1-receptor PEX5. Localization studies demonstrated that PEX33 constitutes a glyoxysomal protein. Growth tests of the pex33 deletion strain revealed a defect of this strain in the biogenesis of glyoxysomes and Woronin bodies. As the function of PEX33 was not redundant to that of PEX14, it is a genuine novel peroxin. Based on our experimental data, the function of PEX33 seems to resemble that of yeast PEX17 despite clear structural differences.

De novo synthesis of peroxisomes upon mitochondrial targeting of Pex3p.

Peroxisomes can form either by growth and division of pre-existing peroxisomes or by de novo synthesis from the endoplasmic reticulum. Pex3p is the key component for both pathways and its targeting to the ER is thought to initiate the de novo formation of peroxisomes. Here, we addressed the question whether Pex3p also can induce peroxisome formation from mitochondrial membranes. Pex3p was targeted to mitochondria by fusion with the mitochondrial targeting signal of Tom20p. The Tom20p-Pex3p-fusion protein was expressed in Pex3p-deficient cells, which are characterized by the lack of peroxisomal membranes. De novo formation of import-competent peroxisomes was observed upon expression of the mitochondrial Pex3p in the mutant cells. This de novo synthesis is independent of the GTPases Vps1p and Dnm1p, two proteins required for peroxisome fission. We conclude that natural or artificial targeting of Pex3p to any endomembrane may initiate peroxisome formation and that also Pex3p-containing mitochondria can serve as source for the de novo synthesis of peroxisomes.

A Eukaryote without Catalase-Containing Microbodies: Neurospora crassa Exhibits a Unique Cellular Distribution of Its Four Catalases

ABSTRACT Microbodies usually house catalase to decompose hydrogen peroxide generated within the organelle by the action of various oxidases. Here we have analyzed whether peroxisomes (i.e., catalase-containing microbodies) exist in Neurospora crassa. Three distinct catalase isoforms were identified by native catalase activity gels under various peroxisome-inducing conditions. Subcellular fractionation by density gradient centrifugation revealed that most of the spectrophotometrically measured activity was present in the light upper fractions, with an additional small peak coinciding with the peak fractions of HEX-1, the marker protein for Woronin bodies, a compartment related to the microbody family. However, neither in-gel assays nor monospecific antibodies generated against the three purified catalases detected the enzymes in any dense organellar fraction. Furthermore, staining of an N. crassa wild-type strain with 3,3′-diaminobenzidine and H2O2 did not lead to catalase-dependent reaction products within microbodies. Nonetheless, N. crassa does possess a gene (cat-4) whose product is most similar to the peroxisomal type of monofunctional catalases. This novel protein indeed exhibited catalase activity, but was not localized to microbodies either. We conclude that N. crassa lacks catalase-containing peroxisomes, a characteristic that is probably restricted to a few filamentous fungi that produce little hydrogen peroxide within microbodies.

Channel-forming activities of peroxisomal membrane proteins from the yeast Saccharomyces cerevisiae.

Highly‐purified peroxisomes from the yeast Saccharomyces cerevisiae grown on oleic acid were investigated for the presence of channel (pore)‐forming proteins in the membrane of these organelles. Solubilized membrane proteins were reconstituted in planar lipid bilayers and their pore‐forming activity was studied by means of multiple‐channel monitoring or single‐channel analysis. Two abundant pore‐forming activities were detected with an average conductance of 0.2 and 0.6 nS in 1.0 m KCl, respectively. The high‐conductance pore (0.6 nS in 1.0 m KCl) is slightly selective to cations (PK+/PCl− ∼ 1.3) and showed an unusual flickering at elevated (> ±40 mV) holding potentials directed upward relative to the open state of the channel. The data obtained for the properties of the low‐conductance pore (0.2 nS in 1.0 m KCl) support the notion that the high‐conductance channel represents a cluster of two low‐conductance pores. The results lead to conclusion that the yeast peroxisomes contain membrane pore‐forming proteins that may aid the transfer of small solutes between the peroxisomal lumen and cytoplasm.