Towards the molecular architecture of the peroxisomal receptor docking complex.

Import of yeast peroxisomal matrix proteins is initiated by cytosolic receptors, which specifically recognize and bind the respective cargo proteins. At the peroxisomal membrane, the cargo-loaded receptor interacts with the docking protein Pex14p that is tightly associated with Pex17p. Previous data suggest that this interaction triggers the formation of an import pore for further translocation of the cargo.

Pex14p Phosphorylation Modulates Import of Citrate Synthase 2 Into Peroxisomes in .

The peroxisomal biogenesis factor Pex14p is an essential component of the peroxisomal matrix protein import machinery. Together with Pex13p and Pex17p, it is part of the membrane-associated peroxisomal docking complex in yeast, facilitating the binding of cargo-loaded receptor proteins for translocation of cargo proteins into the peroxisome. Furthermore, Pex14p is part of peroxisomal import pores.

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 , proteins exist that are efficiently imported into peroxisomes despite having neither PTS1 nor PTS2 and for which no other import signal has been determined.

The deubiquitination of the PTS1-import receptor Pex5p is required for peroxisomal matrix protein import.

Peroxisomal biogenesis depends on the correct import of matrix proteins into the lumen of the organelle. Most peroxisomal matrix proteins harbor the peroxisomal targeting-type 1 (PTS1), which is recognized by the soluble PTS1-receptor Pex5p in the cytosol. Pex5p ferries the PTS1-proteins to the peroxisomal membrane and releases them into the lumen.

Receptor recognition by the peroxisomal AAA complex depends on the presence of the ubiquitin moiety and is mediated by Pex1p.

The receptor cycle of type I peroxisomal matrix protein import is completed by ubiquitination of the membrane-bound peroxisome biogenesis factor 5 (Pex5p) and its subsequent export back to the cytosol. The receptor export is the only ATP-dependent step of the whole process and is facilitated by two members of the AAA family of proteins (ATPases associated with various cellular activities), namely Pex1p and Pex6p. To gain further insight into substrate recognition by the AAA complex, we generated an N-terminally linked ubiquitin-Pex5p fusion protein.

In Cellulo Approaches to Study Peroxisomal Protein Import - Yeast Immunofluorescence Microscopy.

Immunofluorescence microscopy is a powerful tool to analyze the localization of selected proteins in single cells. The technique allows the detection of endogenously expressed proteins as well as tags added to proteins of choice with specific antibodies. Originally evolved for human cell lines, protocols are now also available for yeast cells.

Pex17p-dependent assembly of Pex14p/Dyn2p-subcomplexes of the peroxisomal protein import machinery.

Peroxisomal matrix protein import is facilitated by cycling receptors that recognize their cargo proteins in the cytosol by peroxisomal targeting sequences (PTS). In the following, the assembled receptor-cargo complex is targeted to the peroxisomal membrane where it docks to the docking-complex as part of the peroxisomal translocation machinery. The docking-complex is composed of Pex13p, Pex14p and in yeast also Pex17p, whose function is still elusive.

Nucleotide-dependent assembly of the peroxisomal receptor export complex.

Pex1p and Pex6p are two AAA-ATPases required for biogenesis of peroxisomes. Both proteins form a hetero-hexameric complex in an ATP-dependent manner, which has a dual localization in the cytosol and at the peroxisomal membrane. At the peroxisomal membrane, the complex is responsible for the release of the import receptor Pex5p at the end of the matrix protein import cycle.

Peroxisomal Pex11 is a pore-forming protein homologous to TRPM channels.

More than 30 proteins (Pex proteins) are known to participate in the biogenesis of peroxisomes-ubiquitous oxidative organelles involved in lipid and ROS metabolism. The Pex11 family of homologous proteins is responsible for division and proliferation of peroxisomes. We show that yeast Pex11 is a pore-forming protein sharing sequence similarity with TRPM cation-selective channels.

Role of AAA(+)-proteins in peroxisome biogenesis and function.

Mutations in the PEX1 gene, which encodes a protein required for peroxisome biogenesis, are the most common cause of the Zellweger spectrum diseases. The recognition that Pex1p shares a conserved ATP-binding domain with p97 and NSF led to the discovery of the extended family of AAA+-type ATPases. So far, four AAA+-type ATPases are related to peroxisome function.

Structural insights into cargo recognition by the yeast PTS1 receptor.

The peroxisomal matrix protein import is facilitated by cycling import receptors that shuttle between the cytosol and the peroxisomal membrane. The import receptor Pex5p mediates the import of proteins harboring a peroxisomal targeting signal of type I (PTS1). Purified recombinant Pex5p forms a dimeric complex with the PTS1-protein Pcs60p in vitro with a KD of 0.

Isolation of Peroxisomes from Yeast.

Peroxisomes are multifunctional, dynamic organelles present in nearly all eukaryotic cells. Determining their structural and functional characteristics often requires obtaining isolated and purified peroxisomes via subcellular fractionation. Subcellular fractionation techniques are generally based on a three-step procedure: preparation of a cell-free homogenate (postnuclear supernatant), generation of an organellar pellet by differential centrifugation, and density gradient centrifugation.

Large-Scale Purification of Peroxisomes for Preparative Applications.

This protocol is designed for large-scale isolation of highly purified peroxisomes from Saccharomyces cerevisiae using two consecutive density gradient centrifugations. Instructions are provided for harvesting up to 60 g of oleic acid-induced yeast cells for the preparation of spheroplasts and generation of organellar pellets (OPs) enriched in peroxisomes and mitochondria. The OPs are loaded onto eight continuous 36%-68% (w/v) sucrose gradients.

Small-Scale Purification of Peroxisomes for Analytical Applications.

This protocol describes the isolation of peroxisomes from Saccharomyces cerevisiae by density gradient centrifugation using a sucrose, OptiPrep, or OptiPrep/sucrose gradient. Oleic acid-induced cells are first converted to spheroplasts using lyticase for cell wall digestion. Spheroplasts are homogenized, and nuclei and cell debris are removed by low-speed centrifugation to produce a postnuclear supernatant (PNS).

Cysteine-specific ubiquitination protects the peroxisomal import receptor Pex5p against proteasomal degradation.

Peroxisomal matrix protein import is mediated by dynamic import receptors, which cycle between the peroxisomal membrane and the cytosol. Proteins with a type 1 peroxisomal targeting signal (PTS1) are bound by the import receptor Pex5p in the cytosol and guided to the peroxisomal membrane. After cargo translocation into the peroxisomal matrix, the receptor is released from the membrane back to the cytosol in an ATP-dependent manner by the AAA-type ATPases Pex1p and Pex6p.

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 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.

Distinct ubiquitination cascades act on the peroxisomal targeting signal type 2 co-receptor Pex18p.

Peroxisomal matrix protein import is facilitated by cycling receptors that recognize their cargo proteins in the cytosol by a peroxisomal targeting sequence (PTS) and ferry them to the peroxisomal membrane. Subsequently, the cargo is translocated into the peroxisomal lumen, whereas the receptor is released to the cytosol for further rounds of protein import. This cycle is controlled by the ubiquitination status of the receptor, which is best understood for the PTS1-receptor.

Give what you can and keep what you need!

EMBO J 32 18, 2439–2453 doi:; DOI: 10.1038/emboj.2013.

ATP-dependent assembly of the heteromeric Pex1p-Pex6p-complex of the peroxisomal matrix protein import machinery.

The peroxisomal matrix protein import is facilitated by soluble receptor molecules which cycle between cytosol and the peroxisomal membrane. At the end of the receptor cycle, the import receptors are exported back to the cytosol in an ATP-dependent manner catalyzed by Pex1p and Pex6p, two AAA (ATPases associated with various cellular activities) type ATPases. Pex1p and Pex6p interact and form a heteromeric complex.

The RING-type ubiquitin ligases Pex2p, Pex10p and Pex12p form a heteromeric complex that displays enhanced activity in an ubiquitin conjugating enzyme-selective manner.

The RING finger peroxins Pex2p, Pex10p and Pex12p are central components of the peroxisomal matrix protein import machinery. The RING domain enables each of these proteins to exhibit ubiquitin-protein ligase activity, which has been linked to ubiquitin-dependent regulation of the peroxisomal import receptor Pex5p. The RING peroxins are considered to form a heteromeric complex in vivo, although the elucidation of the structural assembly, as well as the functional interplay of the RING domains, has remained elusive.

Cysteine-dependent ubiquitination of Pex18p is linked to cargo translocation across the peroxisomal membrane.

The peroxisomal matrix protein import is facilitated by cycling receptor molecules that shuttle between the cytosol and the peroxisomal membrane. In the yeast Saccharomyces cerevisiae, the import of proteins harboring a peroxisomal targeting signal of type II (PTS2) is mediated by the receptor Pex7p and its co-receptor Pex18p. Here we demonstrate that Pex18p undergoes two kinds of ubiquitin modifications.