Characterization of Peroxisomal Pex5p from Rat Liver

Abstract

Pex5p is the receptor for the vast majority of peroxisomal matrix proteins. Here, we show that about 15% of rat liver Pex5p is found in the peroxisomal fraction representing 0.06% of total peroxisomal protein. This population of Pex5p displays all the characteristics of an intrinsic membrane protein. Protease protection assays indicate that this pool of Pex5p has domains exposed on both sides of the peroxisomal membrane. The strong interaction of Pex5p with the membrane of the organelle is not affected by mild protease treatment of intact organelles, conditions that result in the partial degradation of Pex13p. Cytosolic Pex5p is a monomeric protein.In contrast, virtually all peroxisomal Pex5p was found to be part of a stable 250-kDa protein assembly. This complex was isolated and shown to comprise just two subunits, Pex5p and Pex14p.

The Import Competence of a Peroxisomal Membrane Protein Is Determined by Pex19p before the Docking Step

“…A cDNA encoding the 373 amino acids of human Pex3p (39) was amplified from human skin fibroblast total RNA by reverse transcription-PCR exactly as described before (38) using the primers 5Ј-CGCGTCGACATGCTGAGGTCTG-TATG-3Ј and 5Ј-CGCGTCGACTCATTTCTCCAGTTGCT-3Ј. The 1.1-kilobase pair cDNA fragment was cloned into the pGEM-T Easy vector according to the manufacturer's instructions (Promega).…”

“…In antibody inhibition experiments, purified IgGs (8 g) in SEM buffer were added to PNS fractions in import buffer and incubated for 20 min on ice before starting the import reaction with the 35 S-labeled reporter protein. Proteinase K treatment (400 g/ml final concentration) of import reactions in the absence or presence of detergents (1% (w/v) Triton X-100, 0.5% (w/v) sodium deoxycholate), sedimentation of organelles by centrifugation, treatment of organelle pellets with Na 2 CO 3 , pH 11.5 (37), or with solutions of high and low ionic strengths, protein precipitation with trichloroacetic acid, and SDS-PAGE analysis were done exactly as described before (38). Routinely, the gels were blotted onto a nitrocellulose membrane, exposed to an x-ray film, and afterward probed with several antibodies.…”

“…The nitrocellulose membrane was first exposed to an x-ray film to detect the 35 Slabeled protein (top pannel) and afterward probed with the following antisera: anti-KDEL (KDEL; recognizes GRP72 and GRP98, two endoplasmic reticulum proteins); anti-cytochrome c (Cyt c; a mitochondrial marker); anticatalase (CAT; a peroxisomal enzyme), and anti-Pex13p (an intrinsic protein of the peroxisomal membrane). This last serum recognizes on proteinase K-treated peroxisomes a 28-kDa fragment of Pex13p (Pex13pЈ) (38). Note that catalase remaining at the top of the gradients (lanes 11-14) derives from the leakage of peroxisomes during preparation of PNS fractions.…”

The N Terminus of the Peroxisomal Cycling Receptor, Pex5p, Is Required for Redirecting the Peroxisome-associated Peroxin Back to the Cytosol

“…It consists of incubating 35 S-labeled Pex5p with a post-nuclear supernatant from rat liver, and it explores the fact that Pex5p acquires a protease-resistant status when inserted into the peroxisomal membrane (38,39). By using this experimental strategy, it was shown that insertion of Pex5p into the peroxisomal membrane requires the presence of PTS1-containing proteins in the import medium and depends on available Pex14p, an intrinsic membrane component of the peroxisomal docking/translocation machinery (39,40).…”

Molecular Mechanisms and Physiological Significance of Organelle Interactions and Cooperation

“…overrepresentation of Pex14 in the membrane complex involving Pex5 (Gouveia et al, 2000). Interestingly, not only the Cterminal part of Pex5 that encodes the cargo-binding domain reaches into the peroxisomal matrix (Gouveia et al, 2003a), but also the N-terminal sequence (Dammai and Subramani, 2001).…”