The ubiquitin-conjugating enzyme Pex4p of Hansenula polymorpha is required for efficient functioning of the PTS1 import machinery

Hansenula polymorpha Pex3p plays an essential role in the biogenesis and maintenance of the peroxisomal membrane. In the initial report, bakers' yeast Pex3p was suggested to represent an integral component of the peroxisomal membrane, containing one membranespanning region that exposes the N terminus of the protein into the organellar matrix. Biochemically, HpPex3p behaved like an integral membrane protein as it was resistant toward high salt and carbonate treatment. However, urea fully removed Pex3p from the membrane under conditions in which the integral membrane protein Pex10p was resistant to this treatment. Additional experiments, including protease protection assays and pre-embedding labeling experiments on purified organellar fractions from cells that produced Pex3ps carrying Myc epitopes at various selected locations in the protein, revealed that invariably all Myc tags were accessible for externally added proteases and antibodies, independent of the presence of detergents. Also, overproduction of Pex3p failed to demonstrate the typical integral membrane protein structures in fracture faces of freeze-fractured peroxisomes. Taken together, our data suggest that HpPex3p does not span the peroxisomal membrane but instead is tightly associated to the cytosolic face of the organelle where it may be present in focal protein clusters.

Section: Methodsmentioning

confidence: 99%

Hansenula polymorpha Pex3p Is a Peripheral Component of the Peroxisomal Membrane

Haan¹,

Faber²,

Baerends³

et al. 2002

“…Pex4p, which is attached to the membrane by the integral membrane protein Pex22p, has been identified as the ubiquitin-conjugating enzyme Ubc10p (22,23). Pex4p and Pex22p together with the AAA ATPases Pex1p and Pex6p may act late in peroxisomal matrix protein import, possibly in the recycling of the receptor to the cytoplasm (24,25).…”

mentioning

confidence: 99%

The Saccharomyces cerevisiae Peroxisomal Import Receptor Pex5p Is Monoubiquitinated in Wild Type Cells

Kragt

Voorn-Brouwer

Berg

et al. 2005

121

113

Pex5p is a mobile receptor for peroxisomal targeting signal type I-containing proteins that cycles between the cytoplasm and the peroxisome. Here we show that Pex5p is a stable protein that is monoubiquitinated in wild type cells. By making use of mutants defective in vacuolar or proteasomal degradation we demonstrate that monoubiquitinated Pex5p is not a breakdown intermediate of either system. Monoubiquitinated Pex5p is localized to peroxisomes, and ubiquitination requires the presence of functional docking and RING finger complexes, which suggests that it is a late event in peroxisomal matrix protein import. In pex1, pex4, pex6, pex15, and pex22 mutants, all of which are blocked in the terminal steps of peroxisomal matrix protein import, polyubiquitinated forms of Pex5p accumulate, ubiquitination being dependent on the ubiquitin-conjugating enzyme Ubc4p. However, Ubc4p is not required for Pex5p ubiquitination in wild type cells, and cells lacking Ubc4p are not affected in peroxisome biogenesis. These results indicate that Pex5p monoubiquitination in wild type cells serves to regulate rather than to degrade Pex5p, which is supported by the observed stability of Pex5p. We propose that Pex5p monoubiquitination in wild type cells is required for the recycling of Pex5p from the peroxisome, whereas Ubc4p-mediated polyubiquitination of Pex5p in mutants blocked in the terminal steps of peroxisomal matrix protein import may function as a disposal mechanism for Pex5p when it gets stuck in the import pathway.

“…Receptors for PTS1 and PTS2 have been identified, as have docking proteins for the PTS receptors on the peroxisomal surface (2,3). Receptor and cargo may translocate through the membrane before receptors are recycled to the cytosol (4,5).…”

mentioning

confidence: 99%

Discrete Targeting Signals Direct Pmp47 to Oleate-induced Peroxisomes in Saccharomyces cerevisiae

Wang

Unruh

Goodman

2001

Pmp47 is a peroxisomal membrane protein consisting of six transmembrane domains (TMDs). We previously showed that the second matrix loop containing a basic cluster of amino acids is important for peroxisomal targeting, and similar basic targeting motifs have been found in other peroxisomal membrane proteins. However, this basic cluster by itself targets to peroxisomes very poorly. We have developed a sensitive quantitative localization assay based on the targeting of Pmp47-GFP fusion proteins to identify the important elements of the basic cluster and to search for other targeting information on Pmp47. Our data suggest that side-chain structure and position as well as charge are important for targeting by the basic cluster. Analysis of other regions of Pmp47 indicates that all TMDs except TMD2 can be eliminated or substituted without significant loss of targeting. TMD2 plus an adjacent cytoplasmic-oriented sequence is crucial for targeting. Cytoplasmic-oriented sequences from two other peroxisomal membrane proteins, ScPex15p and ScPmp22, could partially substitute for the analogous sequence in Pmp47. Targeting with high fidelity to oleate-induced peroxisomes required the following elements: the cytoplasmic-oriented sequence and TMD2, a short matrix loop containing a basic cluster, and a membrane-anchoring TMD.Peroxisomes consist of a dense matrix and surrounding membrane. Proteins are targeted to the peroxisomal matrix from the cytosol by virtue of peroxisomal targeting signals (PTSs) 1 (1). Most matrix proteins contain a PTS1 sequence at their extreme carboxyl terminus consisting of three amino acids identical or related to the tripeptide SKL. A few matrix proteins contain a PTS2 sequence close to the amino terminus, which is often cleaved upon entering the peroxisomal assembly pathway. Receptors for PTS1 and PTS2 have been identified, as have docking proteins for the PTS receptors on the peroxisomal surface (2, 3). Receptor and cargo may translocate through the membrane before receptors are recycled to the cytosol (4, 5).Integral membrane proteins lack these PTS sequences and must be targeted by alternative sequences. We have used Pmp47 from the methylotrophic yeast Candida boidinii as a model protein to understand targeting to the peroxisome membrane. Pmp47 shares homology with members of the mitochondrial carrier protein family (6). These proteins span the membrane six times, and sequence similarity within them suggests that the family was derived from an ancient gene triplication. Pmp47 is important in the metabolism of medium-chain fatty acids and may transport ATP (7), and it is also critical for the import of dihydroxyacetone synthase, an abundant matrix protein (8). Pmp47 contains two loops of amino acids on the matrix side and three on the cytoplasmic side of the membrane. We previously identified a targeting motif contained within the second matrix loop that we termed the mPTS (9). We showed that the most important attribute of the targeting loop is a basic cluster of amino acids of the sequence KIKKR...