Pex12p of Saccharomyces cerevisiae is a component of a multi-protein complex essential for peroxisomal matrix protein import

We have isolated the Saccharomyces cerevisiae pex12-1 mutant from a screen to identify mutants defective in peroxisome biogenesis, The pex12 Delta deletion strain fails to import peroxisomal matrix proteins through both the PTS1 and PTS2 pathway. The PEX12 gene was cloned by functional complementation of the pex12-1 mutant strain and encodes a polypeptide of 399 amino acids. ScPex12p is orthologous to Pex12 proteins from other species and like its orthologues, S, cerevisiae Pex12p contains a degenerate RING finger domain of the C3HC4 type in its essential carboxy-terminus. Localization studies demonstrate that Pex12p is an integral peroxisomal membrane protein, with its NH2-terminus facing the peroxisomal lumen and with its COOH-terminus facing the cytosol, Pex12p - deficient cells retain particular structures that contain peroxisomal membrane proteins consistent with the existence of peroxisomal membrane remnants ("ghosts") in pex12 Delta null mutant cells. This finding indicates that pex12 Delta cells are not impaired in peroxisomal membrane biogenesis. In immunoisolation experiments Pex12p was co-purified with the RING finger protein Pex10p, the PTS1 receptor Pex5p and the docking proteins for the PTS1 and the PTS2 receptor at the peroxisomal membrane, Pex13p and Pex14p, Furthermore, two-hybrid experiments suggest that the two RING finger domains are sufficient for the Pex1op-Pex12p interaction. Our results suggest that Pex12p is a component of the peroxisomal translocation machinery for matrix proteins.</p

Molecular snapshots of the Pex1/6 AAA + complex in action

The peroxisomal proteins Pex1 and Pex6 form a heterohexameric type II AAA+ ATPase complex, which fuels essential protein transport across peroxisomal membranes. Mutations in either ATPase in humans can lead to severe peroxisomal disorders and early death. We present an extensive structural and biochemical analysis of the yeast Pex1/6 complex. The heterohexamer forms a trimer of Pex1/6 dimers with a triangular geometry that is atypical for AAA+ complexes. While the C-terminal nucleotide-binding domains (D2) of Pex6 constitute the main ATPase activity of the complex, both D2 harbour essential sub-strate-binding motifs. ATP hydrolysis results in a pumping motion of the complex, suggesting that Pex1/6 function involves substrate translocation through its central channel. Mutation of the Walker B motif in one D2 domain leads to ATP hydrolysis in the neighbouring domain, giving structural insights into inter-domain communication of these unique heterohexameric AAA + assemblies

The Peroxisomal Targeting Signal 3 (PTS3) of the Budding Yeast Acyl-CoA Oxidase Is a Signal Patch

The specificity of import of peroxisomal matrix proteins is dependent on the targeting signals encoded within their amino acid sequences. Two known import signals, peroxisomal targeting signal 1 (PTS1), positioned at the C-termini and PTS2 located close to N-termini of these proteins are recognized by the Pex5p and Pex7p receptors, respectively. However, in several yeast species, including Saccharomyces cerevisiae, proteins exist that are efficiently imported into peroxisomes despite having neither PTS1 nor PTS2 and for which no other import signal has been determined. An example of such a protein is S. cerevisiae acyl-CoA oxidase (AOx) encoded by the POX1 gene. While it is known that its import is driven by its interaction with the N-terminal segment of Pex5p, which is separate from its C-terminal PTS1-recognizing tetratricopeptide domain, to date, no AOx polypeptide region has been implicated as critical for this interaction, and thus would constitute the long-sought PTS3 signal. Using random mutagenesis combined with a two-hybrid screen, we identified single amino acid residues within the AOx polypeptide that are crucial for this interaction and for the peroxisomal import of this protein. Interestingly, while scattered throughout the primary sequence, these amino acids come close to each other within two domains of the folded AOx. Although the role of one or both of these regions as the PTS3 signal is not finally proven, our data indicate that the signal guiding AOx into peroxisomal matrix is not a linear sequence but a signal patch

The cytosolic domain of Pex22p stimulates the Pex4p-dependent ubiquitination of the PTS1-receptor

Peroxisomal biogenesis is an ubiquitin-dependent process because the receptors required for the import of peroxisomal matrix proteins are controlled via their ubiquitination status. A key step is the monoubiquitination of the import receptor Pex5p by the ubiquitin-conjugating enzyme (E2) Pex4p. This monoubiquitination is supposed to take place after Pex5p has released the cargo into the peroxisomal matrix and primes Pex5p for the extraction from the membrane by the mechano-enzymes Pex1p/Pex6p. These two AAA-type ATPases export Pex5p back to the cytosol for further rounds of matrix protein import. Recently, it has been reported that the soluble Pex4p requires the interaction to its peroxisomal membrane-anchor Pex22p to display full activity. Here we demonstrate that the soluble C-terminal domain of Pex22p harbours its biological activity and that this activity is independent from its function as membrane-anchor of Pex4p. We show that Pex4p can be functionally fused to the trans-membrane segment of the membrane protein Pex3p, which is not directly involved in Pex5p-ubiquitination and matrix protein import. However, this Pex3(N)-Pex4p chimera can only complement the double-deletion strain pex4Δ/pex22Δ and ensure optimal Pex5p-ubiquitination when the C-terminal part of Pex22p is additionally expressed in the cell. Thus, while the membrane-bound portion Pex22(N)p is not required when Pex4p is fused to Pex3(N)p, the soluble Pex22(C)p is essential for peroxisomal biogenesis and efficient monoubiquitination of the import receptor Pex5p by the E3-ligase Pex12p in vivo and in vitro. The results merge into a picture of an ubiquitin-conjugating complex at the peroxisomal membrane consisting of three domains: the ubiquitin-conjugating domain (Pex4p), a membrane-anchor domain (Pex22(N)p) and an enhancing domain (Pex22(C)p), with the membrane-anchor domain being mutually exchangeable, while the Ubc- and enhancer-domains are essential

Fluidity and lipid composition of membranes of peroxisomes, mitochondria and the ER from oleic acid-induced saccharomyces cerevisiae

The maintenance of a fluid lipid bilayer is key for organelle function and cell viability. Given the critical role of lipid compositions in determining membrane properties and organelle identity, it is clear that cells must have elaborate mechanism for membrane maintenance during adaptive responses to environmental conditions. Emphasis of the presented study is on peroxisomes, oleic acid-inducible organelles that are essential for the growth of yeast under conditions of oleic acid as single carbon source. Here, we isolated peroxisomes, mitochondria and ER from oleic acid-induced Saccharomyces cerevisiae and determined the lipid composition of their membranes using shotgun lipidomics and compared it to lipid ordering using fluorescence microscopy. In comparison to mitochondrial and ER membranes, the peroxisomal membranes were slightly more disordered and characterized by a distinct enrichment of phosphaditylinositol, indicating an important role of this phospholipid in peroxisomal membrane associated processes

Autosomal recessive cerebellar ataxia caused by mutations in the PEX2 gene

<p>Abstract</p> <p>Objective</p> <p>To expand the spectrum of genetic causes of autosomal recessive cerebellar ataxia (ARCA).</p> <p>Case report</p> <p>Two brothers are described who developed progressive cerebellar ataxia at 3 1/2 and 18 years, respectively. After ruling out known common genetic causes of ARCA, analysis of blood peroxisomal markers strongly suggested a peroxisomal biogenesis disorder. Sequencing of candidate <it>PEX </it>genes revealed a homozygous c.865_866insA mutation in the <it>PEX2 </it>gene leading to a frameshift 17 codons upstream of the stop codon. <it>PEX </it>gene mutations usually result in a severe neurological phenotype (Zellweger spectrum disorders).</p> <p>Conclusions</p> <p>Genetic screening of PEX2 and other PEX genes involved in peroxisomal biogenesis is warranted in children and adults with ARCA.</p

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 pex14 Delta mutant not only failed to grow on fatty acids because of a defect in glyoxysomal beta-oxidation but also suffered from cytoplasmic bleeding, indicative of a defect in Woronin body-dependent septal pore plugging. Inspection of pex14 Delta 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