P. Edward Purdue

Pex7p translocates in and out of peroxisomes in Saccharomyces cerevisiae

Pex7p is the soluble receptor responsible for importing into peroxisomes newly synthesized proteins bearing a type 2 peroxisomal targeting sequence. We observe that appending GFP to Pex7ps COOH terminus shifts Pex7ps intracellular distribution from predominantly cytosolic to predominantly peroxisomal in Saccharomyces cerevisiae. Cleavage of the link between Pex7p and GFP within peroxisomes liberates GFP, which remains inside the organelle, and Pex7p, which exits to the cytosol. The reexported Pex7p is functional, resulting in import of thiolase into peroxisomes and improved growth of the yeast on oleic acid. These results support the “extended shuttle” model of peroxisome import receptor function and open the way to future studies of receptor export.

Eci1p uses a PTS1 to enter peroxisomes: either its own or that of a partner, Dci1p.

Saccharomyces cerevisiae delta3,delta2-enoyl-CoA isomerase (Eci1p), encoded by ECI1, is an essential enzyme for the betaoxidation of unsaturated fatty acids. It has been reported, as well as confirmed in this study, to be a peroxisomal protein. Unlike many other peroxisomal proteins, Ecilp possesses both a peroxisome targeting signal type 1 (PTS1)-like signal at its carboxy-terminus (-HRL) and a PTS2-like signal at its amino-terminus (RIEGPFFIIHL). We have found that peroxisomal targeting of a fusion protein consisting of Eci1p in front of green fluorescent protein (GFP) is not dependent on Pex7p (the PTS2 receptor), ruling out a PTS2 mechanism, but is dependent on Pex5p (the PTS1 receptor). This Pex5p-dependence was unexpected, since the putative PTS1 of Ecilp is not at the C-terminus of the fusion protein; indeed, deletion of this signal (-HRL-) from the fusion did not affect the Pex5p-dependent targeting. Consistent with this, Pex5p interacted in two-hybrid assays with both Eci1p and Eci1PdeltaHRL. Ecilp-GFP targeting and Eci1pdeltaHRL interaction were abolished by replacement of Pex5p with Pex5p(N495K), a point-mutated Pex5p that specifically abolishes the PTS1 protein import pathway. Thus, Eci1p peroxisomal targeting does require the Pex5p-dependent PTS1 pathway, but does not require a PTS1 of its own. By disruption of ECI1 and DCI1, we found that Dci1p, a peroxisomal PTS1 protein that shares 50% identity with Eci1p, is necessary for Eci1p-GFP targeting. This suggests that the Pex5p-dependent import of Eci1p-GFP is due to interaction and co-import with Dci1p. Despite the dispensability of the C-terminal HRL for import in wild-type cells, we have also shown that this tripeptide can function as a PTS1, albeit rather weakly, and is essential for targeting in the absence of Dci1p. Thus, Eci1p can be targeted to peroxisomes by its own PTS1 or as a hetero-oligomer with Dcilp. These data demonstrate a novel, redundant targeting pathway for Eci1p.

An intronic duplication in the alanine: glyoxylate aminotransferase gene facilitates identification of mutations in compound heterozygote patients with primary hyperoxaluria type 1

SummaryWe report here the identification of a duplication within the first intron of the gene encoding human alanine:glyoxylate aminotransferase (AGT); this duplication is closely linked to two point mutations associated with peroxisome-to-mitochondrion mistargeting of AGT in primary hyperoxaluria type 1 (PH1) patients. Polymerase chain reaction amplification of regions of the AGT gene including the insertion site from individuals heterozygous for this duplication, produces allele-specific fragments of different sizes. We have taken advantage of this to identify a nonsense mutation within a non-expressed allele of a compound heterozygote PH1 patient with mitochondrial AGT.

Rhizomelic Chondrodysplasia Punctata, a Peroxisomal Biogenesis Disorder Caused by Defects in Pex7p, a Peroxisomal Protein Import Receptor: A Minireview

Rhizomelic chondrodysplasia punctata (RCDP) is a lethal autosomal recessive disease correspondingto complementation group 11 (CG 11), the second most common of the thirteen CGs of peroxisomalbiogenesis disorders (PBDs). RCDP is characterized by proximal limb shortening, severely disturbedendochondrial bone formation, and mental retardation, but there is an absence of the neuronal migrationdefect found in the other PBDs. Plasmalogen biosynthesis and phytanic acid oxidation are deficient, butvery long chain fatty acid (VLCFA) oxidation is normal. At the cellular level, RCDP is unique in thatthe biogenesis of most peroxisomal proteins is normal, but a specific subset of at least four, and maybemore, peroxisomal matrix proteins fail to be imported from the cytosol. In this review, we discuss recentadvances in understanding RCDP, most prominently the cloning of the affected gene, PEX7,and identification of PEX7 mutations in RCDP patients. Human PEX7 wasidentified by virtue of its sequence similarity to its Saccharomyces cerevisiae ortholog, whichhad previously been shown to encode Pex7p, an import receptor for type 2 peroxisomal targetingsequences (PTS2). Normal human PEX7 expression rescues the cellular defects in culturedRCDP cells, and cDNA sequence analysis has identified a variety of PEX7 mutations in RCDP patients,including a deletion of 100 nucleotides, probably due to a splice site mutation, and a prevalent nonsensemutation which results in loss of the carboxyterminal 32 amino acids. Identification of RCDP as a PTS2import disorder explains the observation that several, but not all, peroxisomal matrix proteins aremistargeted in this disease; three of the four proteins deficient in RCDP have now been shown to bePTS2-targeted.