Identification of PEX5 Homologue in Rat by RT-PCR and Sequencing




Matthew B Cascio

Introduction

Newly synthesized peroxisomal proteins are targeted for transportation to a peroxisome by the presence of one of two known peroxisomal targeting sequences (PTS1, PTS2). PTS1 is a short, three amino acid sequence of Ser-Lys-Leu (SKL) at the immediate C-terminal end of a peroxisomal protein, whereas PTS2 is a much longer N-terminal signal. PTS1 is the signal present on the great majority of peroxisomal matrix proteins, including catalase, and is recognized by the cytosolic receptor Pex5p (Lodish). The mobile Pex5p is responsible for transportation of proteins presenting PTS1 to the peroxisome via interaction with the peroxisome membrane bound Pex14p. Recent evidence implicates Pex5p in transport of proteins containing PTS2 as well, however the exact mechanism is still unclear (Fujiki et al., 2001 and Legakis & Terlecky 2001). Certain mutations that can occur in Pex5p result in a condition known as Zellweger syndrome. Patients suffering from this condition are deficient in the import of most peroxisomal proteins into the peroxisome matrix (Singh et al., 1997).
The sequence of the gene that encodes Pex5p (PEX5) has been determined for several organisms including humans, mice, and a variety of yeast, but has yet to be elucidated in rats. Mouse PEX5 is approximately 1.9kb and encodes a 639 amino acid protein. This report presents the sequence of a 1.2kb segment of the PEX5 homologue present in rat tissue and a comparison of homology between this experimental sequence and the published mouse PEX5 sequence, available from GenBank.

Figures


Figure 1-Extraction and DNAse treatment of RNA. Total RNA extracted from rat liver homogenate as described in materials and methods is shown here on an agarose gel(A). RNA was DNAse treated after extraction and run on an agarose gel(B).


Figure 2-PCR products from reactions using primers designed to mouse PEX5 sequence. Primers used: MmPx1,4 (lane1); MmPx3,2 (lane2); MmPx3,4 (lane3); MmPx3,6 (lane4); MmPx5,2 (lane 5); MmPx5,6 (lane6).


Figure 3-Alignment of published mouse PEX5 sequence (top) with PEX5 homologue from rat (bottom). This alignment figure was made using MacVector.


Rat RNA was extracted from liver tissue and DNAse treated, as described in materials and methods, then run on an agarose gel (Figure 1A and B). The pattern seen on the gels in figure 1 is consistent with samples containing 28S RNA and 18S RNA.
PCR reactions yielded products of sizes that would be expected based on the locations of the primers used to amplify these segments (Figure 2). More than one band is visible in lane 1 of figure 2. The upper band is approximately 1.5kb, the expected length of product for that reaction. This band was isolated as described in materials and methods. These PCR products were used as templates for sequencing reactions. Sequencing reactions were carried out using various combinations of primers PCR product templates.
The PCR products shown in figure 2 represent overlapping DNA segments that span from primer MmPx1 (at the start codon of M.mus PEX5) to MmPx2 (past the stop codon). A 1252bp portion of rat cDNA was sequenced that has about 85% homology to the published sequence of mouse PEX5 (Figure 3). This sequence was determined by matching overlapping sequences obtained from reactions run with primers MmPx2,3, and 5, in addition to RnPx7,8,and 9. Primers RnPx7, 8, and 9 were designed from rat sequences obtained via reactions run using primers to mouse sequences. RnPx primers were used for primer walking.
The cDNA sequence reported in this paper shows high homology to previously reported sequences of the PEX5 gene. Although the 1.2kb sequence reported does not represent the entire PEX5 gene, it provides evidence that such a homologue does exist in rats, and that the techniques reported herein are sufficient to isolate and identify this gene.
The tetratrico peptide repeat (TPR) motif is a five amino acid residue present in many proteins and is involved in crucial protein-protein interactions from cell-cycle regulation to chaperone function (Kumar et al., 2001). The TPR motif consists of the amino acid sequence W-X-X-X-Y/F, and is necessary in Pex5p for proper interaction with PTS1 (deWalque et al., 1999), (Szilard and Rachubinski 2000), (Jardim et al., 2000), (Klein et al., 2001). The mouse PEX5 gene encodes 7 such regions, 4 of which fall within the 1.2kb sequence reported in this paper. Three of the four regions coding for TPR motifs show perfect homology to the mouse PEX5 sequence, while the fourth, although not perfectly homologous, still encodes a W-X-X-X-Y amino acid residue. Two of the other segments of the PEX5 gene that encode TPR motifs are fewer than 100bp upstream from the beginning of the 1.2kb sequence reported here. The third is coded by a sequence between bases 840 and 1040 (fig. 3) and all three could very likely be sequenced by primer walking. The presence of these regions coding for TPR motifs provides further evidence that the sequence reported is a PEX5 homologue.

Full Paper

Acknowledgments

I would like to thank Rocco Coli, Ira Daly, and Sabrina Volpi for their infinite patience and support, without which this report would not have been possible. In addition I would like to thank Dr. Berish Rubin for the opportunity to perform this experiment, and Ms. Amy Kozak and Dr. Sylvia Anderson for help and tolerance that was not overlooked. Finally, I would like to thank Dr. Levente Kapas for graciously donating materials.

References

Fujiki Y, Okumoto K, Otera H, Tamura S.(2000) Peroxisome biogenesis and molecular defects in peroxisome assembly disorders. Cell Biochem Biophys;32 Spring:155-64

Jardim A, Liu W, Zheleznova E, Ullman B.(2000) Peroxisomal targeting signal-1 receptor protein PEX5 from Leishmania donovani: molecular, biochemical, and immunocytochemical characterization. J Biol Chem. 2000 May 5;275(18):13637-44.

Klein AT, Barnett P, Bottger G, Konings D,Tabak HF, Distel B. (2001) Recognition of peroxisomal targeting signal type 1 by the import receptor pex5p. J Biol Chem 4;276(18):15034-41

Kumar A, Roach C, Hirsh IS, Turley S, deWalque S, Michels PA, Hol WG. (2001) An unexpected extended conformation for the third TPR motif of the peroxin PEX5 from Trypanosoma brucei. J Mol Biol 307(1):271-82.

Legakis JE, Terlecky SR.(2001) PTS2 import into mammalian peroxisomes.Traffic 4: 252-60.

Lodish H, Berk A, Zipursky SA, Matsudaira P, Baltimore D, and Darnell J. (2000)Molecular Cell Biology 4th Edition. W.H. Freeman and Co. New York.

Szilard RK, Rachubinski RA.(2000) Tetratricopeptide repeat domain of Yarrowia lipolytica Pex5p is essential for recognition of the type 1 peroxisomal targeting signal but does not confer full biological activity on Pex5p. Biochem J 346 Pt 1:177-84

Singh I, Voigt RG, Sheikh FG, Kremser K, Brown FR 3rd.(1997) Biochemical features of a patient with Zellweger-like syndrome with normal PTS-1 and PTS-2 peroxisomal protein import systems: a new peroxisomal disease. Biochem Mol Med 61(2):198-207

de Walque S, Kiel JA, Veenhuis M, Opperdoes FR, Michels PA. 1999. Cloning and analysis of the PTS-1 receptor in Trypanosoma brucei. Mol Biochem Parasitol 104: 106-19.



This document was last modified 01/31/2006.
This site is powered by the versatile Zope platform.
This is a project of the Biology Department of Fordham University
Biotechniques.org Home