Analysis of Mammalian Peroxin Interactions Using a Non-transcription-based Bacterial Two-hybrid Assay

Abstract

In recent years, substantial progress has been made in the identification of proteins involved in peroxisome biogenesis. However, with the exception of the peroxisome-targeting signal receptors and the receptor docking proteins, the function of most of these proteins, called peroxins, remains largely unknown. One step toward elucidating the function of a protein is to identify its interacting partners. We have used a non-transcription-based bacterial two-hybrid system to analyze the interactions among a set of 12 mammalian peroxins and a yeast protein three-hybrid system to investigate whether proteins that interact with the same peroxin and have overlapping binding sites cooperate or compete for this site. Here we report a detailed interaction map of these peroxins and demonstrate that (i) farnesylation, and not the CAAX motif, of Pex19p strongly enhances its affinity for Pex13p; (ii) the CAAXmotif, and not farnesylation, of Pex19p strongly enhances its affinity for Pex11pβ; and (iii) the C3HC4 RING (really interesting new gene) finger domain of Pex12p does not alter the binding properties of Pex5p for the C-terminal peroxisome-targeting signal PTS1. Finally, we show that the Pex5p-Pex13p interaction is bridged by Pex14p and that the latter molecule exists predominantly as a dimer in vivo. Collectively, as demonstrated in this work with peroxins, these results indicate that the bacterial two-hybrid system is an attractive complementary approach to the conventional transcription-based yeast two-hybrid system for studying protein-protein interactions. In recent years, substantial progress has been made in the identification of proteins involved in peroxisome biogenesis. However, with the exception of the peroxisome-targeting signal receptors and the receptor docking proteins, the function of most of these proteins, called peroxins, remains largely unknown. One step toward elucidating the function of a protein is to identify its interacting partners. We have used a non-transcription-based bacterial two-hybrid system to analyze the interactions among a set of 12 mammalian peroxins and a yeast protein three-hybrid system to investigate whether proteins that interact with the same peroxin and have overlapping binding sites cooperate or compete for this site. Here we report a detailed interaction map of these peroxins and demonstrate that (i) farnesylation, and not the CAAX motif, of Pex19p strongly enhances its affinity for Pex13p; (ii) the CAAXmotif, and not farnesylation, of Pex19p strongly enhances its affinity for Pex11pβ; and (iii) the C3HC4 RING (really interesting new gene) finger domain of Pex12p does not alter the binding properties of Pex5p for the C-terminal peroxisome-targeting signal PTS1. Finally, we show that the Pex5p-Pex13p interaction is bridged by Pex14p and that the latter molecule exists predominantly as a dimer in vivo. Collectively, as demonstrated in this work with peroxins, these results indicate that the bacterial two-hybrid system is an attractive complementary approach to the conventional transcription-based yeast two-hybrid system for studying protein-protein interactions. The biogenesis of peroxisomes requires the concerted action of at least 23 peroxins (1.Holroyd C. Erdmann R. Protein translocation machineries of peroxisomes.FEBS Lett. 2001; 501: 6-10Google Scholar) and one cytosolic, peroxisome-specific DnaJ-like protein (2.Hettema E.H. Ruigrok C.C.M. Koerkamp M.G. van den Berg M. Tabak H. Distel B. Braakman I. The cytosolic DnaJ-like protein Djp 1p is involved specifically in peroxisomal protein import.J. Cell Biol. 1998; 142: 421-434Google Scholar). The best characterized peroxin is Pex5p, which functions as a cycling signal recognition factor for newly synthesized peroxisomal matrix proteins containing a C-terminal peroxisome-targeting signal (PTS1) 1The abbreviations used are: PTS1, C-terminal peroxisomal targeting signal; PTS2, N-terminal peroxisomal targeting signal; AD, Gal4p activation domain; b-2HS, non-transcription-based bacterial two-hybrid system; BD, Gal4p DNA-binding domain; CHO, Chinese hamster ovary; DSP, dithiobis(succinimidyl propionate); HA, influenza virus hemagglutinin epitope tag; Pexp, peroxin, including a number corresponding to the order of discovery; SH3, Src homology 3; TBST, Tris-buffered saline containing 0.05% (v/v) Tween 20; T18, amino acids 225 to 399 of B. pertussis adenylate cyclase; T25, amino acids 1 to 224 of B. pertussis adenylate cyclase; y-2HS, transcription-based yeast two-hybrid system; y-3HS, transcription-based yeast protein three-hybrid system. (3.Dammai V. Subramani S. The human peroxisomal targeting signal receptor, Pex 5p, is translocated into the peroxisomal matrix and recycled to the cytosol.Cell. 2001; 105: 187-196Google Scholar, 4.Harano T. Nose S. Uezu R. Shimizu N. Fujiki Y. Hsp70 regulates the interaction between the peroxisome targeting signal type 1(PTS1)-receptor Pex5p and PTS1.Biochem. J. 2001; 357: 157-165Google Scholar). Peroxisomal matrix proteins containing an N-terminal peroxisome-targeting signal (PTS2) are recognized by Pex7p, another soluble PTS receptor (5.Subramani S. Koller A. Snyder W.B. Import of peroxisomal matrix and membrane proteins.Annu. Rev. Biochem. 2000; 69: 399-418Google Scholar). The PTS receptors dock at the peroxisome membrane via the peroxisomal membrane proteins Pex13p and Pex14p, which, together with Pex17p, constitute the receptor docking complex (6.Terlecky S.R. Fransen M. How peroxisomes arise.Traffic. 2000; 1: 465-473Google Scholar). In mammals, docking of Pex7p at the peroxisome membrane is mediated by the long isoform of Pex5p, Pex5pL (7.Matsumura T. Otera H. Fujiki Y. Disruption of the interaction of the longer isoform of Pex 5p, Pex5pL, with Pex7p abolishes peroxisome targeting signal type 2 protein import in mammals.J. Biol. Chem. 2000; 275: 21715-21721Google Scholar). In Saccharomyces cerevisiae, the targeting of Pex7p to the peroxisome membrane requires two functionally related proteins, Pex18p and Pex21p (8.Purdue P.E. Yang X. Lazarow P.B. Pex 18p and Pex21p, a novel pair of related peroxins essential for peroxisomal targeting by the PTS2 pathway.J. Cell Biol. 1998; 143: 1859-1869Google Scholar). In Yarrowia lipolytica, Pex20p is also required for PTS2 protein import (9.Titorenko V.I. Smith J.J. Szilard R.K. Rachubinski R.A. Pex20p of the yeast Yarrowia lipolytica is required for the oligomerization of thiolase in the cytosol and for its targeting to the peroxisome.J. Cell Biol. 1998; 142: 403-420Google Scholar). Interestingly, Einwächter et al. (10.Einwächter H. Sowinski S. Kunau W.H. Schliebs W. Yarrowia lipolytica Pex20p, Saccharomyces cerevisiae Pex18p/Pex21p and mammalian Pex5pL fulfil a common function in the early steps of the peroxisomal PTS2 import pathway.EMBO Rep. 2001; 2: 1035-1039Google Scholar) reported recently that this Y. lipolytica Pex20p can substitute functionally for S. cerevisiae Pex18p and Pex21p. Other peroxins directly implicated in the import pathway for peroxisomal matrix proteins are Pex1p, Pex2p, Pex4p, Pex6p, Pex8p, Pex9p, Pex10p, Pex12p, Pex15p, Pex22p, and Pex23p. Epistasis analyses suggest that Pex2p, Pex8p, Pex10p, and Pex12p act downstream of the receptor docking event and that Pex1p, Pex4p, Pex6p, and Pex22p function downstream of the matrix protein translocation process (11.Collins C.S. Kalish J.E. Morrell J.C. McCaffery J.M. Gould S.J. The peroxisome biogenesis factors Pex 4p, Pex22p, Pex1p, and Pex6p act in the terminal steps of peroxisomal matrix protein import.Mol. Cell. Biol. 2000; 20: 7516-7526Google Scholar, Gould S.J. The of peroxisome Rev. 2000; Scholar). that and Pex6p are involved in the of peroxisomal Subramani S. proteins, and interact with in an and are with Cell. Biol. 1998; Scholar, V.I. H. Rachubinski R.A. of peroxisomal in an early step in the in peroxisome pathway of Yarrowia Cell Biol. 2000; Scholar). Pex9p, Pex15p, and have not been in step of the matrix protein import pathway Gould S.J. The of peroxisome Rev. 2000; Scholar). and Pex19p are implicated in peroxisome membrane biogenesis. Cell in these peroxins peroxisomal membrane proteins and have peroxisomal Gould S.J. The of peroxisome Rev. 2000; Scholar). peroxin, peroxisomal V.I. Rachubinski R.A. The of the Rev. 2001; 2: Scholar). However, van et al. Tabak van den Berg M. E.H. a in a process that peroxisome number and in Saccharomyces Cell Biol. 2000; Scholar) reported recently that in S. cerevisiae a in the pathway and the that this pathway regulates the of a molecule that peroxisome number and the interactions between peroxins is an essential step toward the of these proteins function and proteins are translocated the peroxisomal the best to between peroxins is the transcription-based yeast two-hybrid system S. novel system to protein-protein Scholar). used to identify or to interactions between peroxins are and a transcription-based mammalian two-hybrid and the of (5.Subramani S. Koller A. Snyder W.B. Import of peroxisomal matrix and membrane proteins.Annu. Rev. Biochem. 2000; 69: 399-418Google Scholar, M. S.R. Subramani S. of a human receptor docking protein directly required for peroxisomal protein S. A. 1998; C. C. J.E. of the mammalian peroxisomal import Pex2p, Pex5p, Pex12p, and Pex14p are of the same protein Biol. Chem. 2001; Scholar). Here we report a detailed interaction map of 12 mammalian peroxins that are in peroxin map by the y-2HS, as as a non-transcription-based bacterial two-hybrid system The latter by and J. A. bacterial two-hybrid system a signal S. A. 1998; the of the domain of pertussis adenylate proteins of are to and T25, two complementary that are essential for adenylate the corresponding proteins In an in adenylate this can as as the or are and to a We show that the non-transcription-based and the transcription-based are complementary and with interactions. In results the that of Pex19p enhances strongly its affinity for Pex13p M. T. C. Pex19p peroxisomal membrane proteins at Cell. Biol. 2001; Scholar) and the of human Pex14p to in and to the Pex5p-Pex13p interaction in Finally, we have the protein interaction of Pex5p and Pex14p and show that Pex5p has overlapping binding sites for the C3HC4 RING finger domain of Pex12p and The used for as as for the of The adenylate used for the bacterial two-hybrid two-hybrid and protein three-hybrid the S. cerevisiae Chinese hamster in with (v/v) 2 and and in a The the proteins by with the and into the yeast two-hybrid or the yeast protein three-hybrid or the bacterial two-hybrid or J. T. Scholar). the of into the bacterial two-hybrid the sites of and the which sites for and by a pair containing sites for and the which sites for and by a pair containing sites for and The corresponding of and and The detailed the of of the number of can the corresponding The of essential by The of in yeast and the as by the The interactions as or the and The with as also as by the However, for this the yeast for in and or in and in the or of 1 two-hybrid as A. bacterial two-hybrid system that a in 2000; Scholar) the bacterial with or with and containing and The interactions as or the and of the mediated by interaction between two proteins by in A. bacterial two-hybrid system that a in 2000; Scholar). in with of One of with of containing of dithiobis(succinimidyl to to for at the by with a The to a and the proteins by the of of and of (v/v) the with 1 of in of (v/v) and in the or of (v/v) The for to to and with the the 1 of human to the (PTS1) M. B. The cytosolic protein that the C-terminal of is not a peroxisomal import receptor a Scholar) a the with TBST, the with in for 1 with of bacterial as M. C. The of peroxisomal proteins containing a C-terminal targeting by the PTS Biochem. containing or M. S.R. Subramani S. of a human receptor docking protein directly required for peroxisomal protein S. A. 1998; as in with TBST, Pex5p, Pex14p, and Pex13p to and The at by 1 of human to the M. B. The cytosolic protein that the C-terminal of is not a peroxisomal import receptor a Scholar) as of a peroxisomal protein import in a new The and are M. T. C. Pex19p peroxisomal membrane proteins at Cell. Biol. 2001; Scholar). The The Pex5p by M. by the a step toward the of a peroxin we a matrix R. X. N. M. Protein interaction in C. proteins involved in 2000; Scholar) in which 12 mammalian peroxins for interactions in the yeast two-hybrid system the and proteins the not the protein to the receptor, Pex5p, with its docking Pex14p, a predominantly cytosolic protein that a CAAX the peroxisomal membrane proteins Pex10p, Pex12p, and results that these protein interact in the M. T. C. Pex19p peroxisomal membrane proteins at Cell. Biol. 2001; Scholar, Gould S.J. with and and downstream of receptor docking in peroxisomal matrix protein import.J. Cell Biol. Scholar, S. Y. Fujiki Y. The peroxin membrane in peroxisome Biol. Cell. 2000; Scholar, H. J. J. Fransen M. of novel peroxisome-targeting signal 1 proteins in J. 2001; 357: Scholar). However, interactions and not peroxin demonstrated to interact including and (7.Matsumura T. Otera H. Fujiki Y. Disruption of the interaction of the longer isoform of Pex 5p, Pex5pL, with Pex7p abolishes peroxisome targeting signal type 2 protein import in mammals.J. Biol. Chem. 2000; 275: 21715-21721Google Scholar, W. J. B. Kunau W.H. human peroxisomal targeting signal receptor for interaction of with Biol. Chem. Scholar, Gould S.J. with and and downstream of receptor docking in peroxisomal matrix protein import.J. Cell Biol. Scholar, N. R. Y. Otera H. S. T. S. Fujiki Y. The peroxin Pex14p, by a Chinese hamster and Biol. Chem. Scholar, J.M. X. Y. Gould S.J. peroxisomal membrane proteins, is predominantly and is required for peroxisome membrane Cell Biol. 2000; Scholar) two peroxins the and of the reported interactions between peroxins not in the y-2HS, we and a new matrix a non-transcription-based J. A. bacterial two-hybrid system a signal S. A. 1998; Scholar). In this which a in in adenylate of the 12 peroxins the or and peroxin and specifically in of the two-hybrid of these interactions and also in the The two and not in the The that Pex14p and Pex19p peroxins in the largely the that these peroxins are or to to the yeast However, of a of human Pex14p are in S. cerevisiae interactions and in the not in the this related to a or to as we to T18, T25, Pex10p, and Pex12p, we to the of and we have whether the and the interactions in the However, an M. T. C. Pex19p peroxisomal membrane proteins at Cell. Biol. 2001; we to demonstrate that Pex13p in with the that the of Pex19p strongly enhances its affinity for Pex13p M. T. C. Pex19p peroxisomal membrane proteins at Cell. Biol. 2001; we that the of interaction between these two peroxins is by the that Pex19p is not in Interestingly, a a affinity for Pex19p the molecule in the M. T. C. Pex19p peroxisomal membrane proteins at Cell. Biol. 2001; does a binding to Pex19p in the the we that the CAAX motif, and not is an in the affinity of Pex19p for Pex19p and to interact in the and in the b-2HS, and the Pex19p in in a strongly binding affinity for M. T. C. Pex19p peroxisomal membrane proteins at Cell. Biol. 2001; and In this is to that Pex19p and are and for Pex14p, and and have binding properties in the in a new the two-hybrid of 12 mammalian peroxins of the interactions a substantial number of reported interactions between peroxins and not However, as the of mammalian peroxins in yeast and in we whether interactions peroxin of this we to that the C3HC4 domain of Pex12p, not the with Pex5p and in the Gould S.J. with and and downstream of receptor docking in peroxisomal matrix protein import.J. Cell Biol. Scholar, H. J. J. Fransen M. of novel peroxisome-targeting signal 1 proteins in J. 2001; 357: Scholar, I. Fujiki Y. of the peroxin Pex12p, finger domain is essential for Pex12p function and with the peroxisome-targeting signal type Pex5p and a peroxin, Biol. Chem. 2000; 275: Scholar) for the W. J. B. Kunau W.H. human peroxisomal targeting signal receptor for interaction of with Biol. Chem. Scholar, H. T. M. S. A. A. Shimizu N. Fujiki Y. The mammalian peroxin Pex the longer isoform of the peroxisome targeting signal type 1 the protein complex into peroxisomes via its docking Biol. Chem. 2000; 275: Scholar) and (7.Matsumura T. Otera H. Fujiki Y. Disruption of the interaction of the longer isoform of Pex 5p, Pex5pL, with Pex7p abolishes peroxisome targeting signal type 2 protein import in mammals.J. Biol. Chem. 2000; 275: 21715-21721Google Scholar) interactions not However, the domain we that the domain of Pex13p to Pex14p an interaction in a M. S.R. Subramani S. of a human receptor docking protein directly required for peroxisomal protein S. A. 1998; Scholar). Interestingly, this domain of Pex13p also to interact with at least in the Finally, the to the of Pex14p for and Pex19p binding we show that the amino acids and not the Pex14p also interact with Pex14p and in the (i) two-hybrid results that suggest that Pex14p, not Pex5p, in and (ii) that Pex5p functions as a C. C. J.E. of peroxisomal Pex5p Biol. Chem. 2000; 275: a H. T. M. S. A. A. Shimizu N. Fujiki Y. The mammalian peroxin Pex the longer isoform of the peroxisome targeting signal type 1 the protein complex into peroxisomes via its docking Biol. Chem. 2000; 275: Scholar) or a protein W. J. B. Kunau W.H. human peroxisomal targeting signal receptor for interaction of with Biol. Chem. we the of these peroxins in mammalian protein-protein we the and of the of the that Pex14p, not Pex5p, exists as a complex in of suggest that this complex is in a of (i) Pex14p can is in (ii) the complex of to a as the of not and (iii) the complex is not recognized by the peroxins Pex5p, and Pex19p not Interestingly, in the of a of Pex14p as a dimer However, that the Pex14p is not by between the by its The of and its the of a interaction in Erdmann R. C. and of in the J. 1998; Scholar). of Pex14p (i) have an affinity for the interacting peroxins, and (ii) are to peroxisome biogenesis in a in Pex14p remains to In of that the domain of yeast Pex13p functions as a peroxisomal membrane receptor for Pex5p Y. A. T. van den Berg M. B. T. Tabak Distel B. The domain of the Saccharomyces cerevisiae peroxisomal membrane protein Pex13p functions as a docking for Pex5p, a receptor for the import of Cell Biol. Scholar, R. of a peroxisomal membrane receptor for the recognition Cell Biol. Scholar, S.J. Kalish J.E. Morrell J.C. J. Pex13p is an protein of the peroxisome membrane and a docking for the predominantly PTS Cell Biol. to an interaction between the corresponding human in the bacterial and yeast two-hybrid However, in of a peroxisomal protein import complex of Pex5p, Pex14p, and the domain of Pex13p acids to demonstrated that Pex14p, not the domain of with Pex5p with In these show that Pex14p can to Pex5p and these results suggest that in (i) Pex14p, and not functions as the docking protein for Pex5p, and (ii) the Pex5p-Pex13p interaction is bridged by Pex14p Pex5p and Pex14p interact with and a number of We the yeast and bacterial two-hybrid to the involved in the binding of these peroxins to partners. Pex5p, we the required for and Pex12p interaction to the and In we that Pex5p has binding sites for Pex14p, and these binding sites are in the N-terminal of the molecule by Schliebs et al. W. J. B. Kunau W.H. human peroxisomal targeting signal receptor for interaction of with Biol. Chem. Scholar). In these that the in the N-terminal of Pex5p are the for the interaction with this we in the which with Pex14p containing one motif, the amino to at The that the binding affinity of for Pex14p, not for and to that the are for Pex14p to Pex14p, we have a of to map the the and the domain that is of the sites for Pex5p and Pex19p not and the amino acids to The and required for of Pex5p are for Pex14p in a new of the interaction of Pex14p to the Gal4p or for interaction with Pex5p, Pex14p, and to the Gal4p or in the yeast or bacterial two-hybrid system. one of the and T25, and one of the and T18, proteins and as The binding of the protein are as interaction interaction or interaction The the indicate the amino in the corresponding Pex14p The and the domain are in and in The in and is with The interaction are the of the Pex14p The that (i) the domain and the domain of Pex5p are overlapping and (ii) Pex12p downstream of the docking event (11.Collins C.S. Kalish J.E. Morrell J.C. McCaffery J.M. Gould S.J. The peroxisome biogenesis factors Pex 4p, Pex22p, Pex1p, and Pex6p act in the terminal steps of peroxisomal matrix protein import.Mol. Cell. Biol. 2000; 20: 7516-7526Google that Pex12p have a in the binding properties of investigate whether this is the we In this which can as an of the y-2HS, the of a protein or the binding properties of two proteins the Gal4p protein and the Gal4p activation domain can that in the we the of the protein by the C. R. J. J.M. or the of a in a three-hybrid yeast Biol. Chem. Scholar). in the binding properties of Pex5p not the molecule at the compete with for Pex5p binding with the that with Pex5p in the Gould S.J. with and and downstream of receptor docking in peroxisomal matrix protein import.J. Cell Biol. and I. Fujiki Y. of the peroxin Pex12p, finger domain is essential for Pex12p function and with the peroxisome-targeting signal type Pex5p and a peroxin, Biol. Chem. 2000; 275: Scholar, and suggest that Pex5p has binding sites for the C3HC4 RING finger domain of Pex12p and proteins and that Pex5p and have binding has binding sites for the C3HC4 RING finger domain of Pex12p and in a new the peroxisomal protein import is essential to identify the interactions between the is the most used to these interactions. However, as this results R. S. of that in the two-hybrid Scholar, and A. to in yeast two-hybrid Scholar, protein interaction two-hybrid Lett. 2000; Scholar) and results protein interaction two-hybrid Lett. 2000; Scholar, T. T. R. M. M. Y. two-hybrid to the yeast protein S. A. 2001; is that interactions are by at least one used to yeast two-hybrid interactions and However, these in are and proteins, proteins, or of an to these in the J. A. bacterial two-hybrid system a signal S. A. 1998; Scholar, A. interactions between by a bacterial two-hybrid 2001; Scholar). in interaction system the in that (i) a bacterial and not a yeast is used as and (ii) is the of a signal pathway and not the of a is for the a step to the b-2HS, we a matrix in which 12 mammalian peroxins for interactions and these results with yeast two-hybrid interacting peroxin and to interact in two-hybrid and in the y-2HS, and two and in the peroxin interactions in two-hybrid are to have a has been reported that Pex14p functions as a protein M. S.R. Subramani S. of a human receptor docking protein directly required for peroxisomal protein S. A. 1998; Scholar, M. X. Erdmann Kunau W.H. Erdmann R. and of the human of yeast Cell. Biol. Scholar, S. Y. Fujiki Y. The peroxin membrane in peroxisome Biol. Cell. 2000; Scholar) and that a molecule required for peroxisome membrane to a of peroxisomal membrane proteins Gould S.J. The of peroxisome Rev. 2000; Scholar). to the interactions that in the y-2HS, is essential to that has been reported that the CAAX of Pex19p is an in the affinity of Pex19p for Pex10p, Pex12p, and Pex13p M. T. C. Pex19p peroxisomal membrane proteins at Cell. Biol. 2001; Scholar). In with the that protein is a process of a suggest that and not the CAAX is an in the affinity of Pex19p for Pex10p, Pex12p, and However, with to the we that the CAAX motif, and not of Pex19p to the CAAX is required directly for binding to or whether the corresponding the of Pex19p in a that binding to sites is is not to the interactions that in the b-2HS, has to the proteins the identification of interactions of T. T. R. M. M. Y. two-hybrid to the yeast protein S. A. 2001; Scholar, and two-hybrid 2000; Scholar). the that the not the of Pex14p with in the this Protein by by interactions with in the these newly interactions not with the an of this we and to interactions. this approach one novel and and peroxin M. S.R. Subramani S. of a human receptor docking protein directly required for peroxisomal protein S. A. 1998; Scholar, Gould S.J. with and and downstream of receptor docking in peroxisomal matrix protein import.J. Cell Biol. Scholar, I. Fujiki Y. of the peroxin Pex12p, finger domain is essential for Pex12p function and with the peroxisome-targeting signal type Pex5p and a peroxin, Biol. Chem. 2000; 275: Scholar) reported peroxin W. J. B. Kunau W.H. human peroxisomal targeting signal receptor for interaction of with Biol. Chem. Scholar, H. T. M. S. A. A. Shimizu N. Fujiki Y. The mammalian peroxin Pex the longer isoform of the peroxisome targeting signal type 1 the protein complex into peroxisomes via its docking Biol. Chem. 2000; 275: Scholar) and H. T. M. S. A. A. Shimizu N. Fujiki Y. The mammalian peroxin Pex the longer isoform of the peroxisome targeting signal type 1 the protein complex into peroxisomes via its docking Biol. Chem. 2000; 275: not in the yeast and bacterial two-hybrid However, we have that human Pex7p is not is in yeast or M. and for in a mammalian Pex7p does interact with Pex5pL Gould S.J. of human and to S. cerevisiae Pex18p and Biol. Chem. 2001; Scholar) In another of we the bacterial and yeast two-hybrid to the interaction of Pex5p and Pex14p that are involved in the binding to partners. to Pex5p, we for the that the of this molecule are for Pex14p binding W. J. B. Kunau W.H. human peroxisomal targeting signal receptor for interaction of with Biol. Chem. and that the domain and the domain this work in by et al. Gould S.J. of human and to S. cerevisiae Pex18p and Biol. Chem. 2001; Scholar) and et al. J. A. W. Kunau W.H. Schliebs W. The of the human peroxisome import receptor are affinity binding sites for the peroxisomal membrane protein Biol. Chem. 2001; Scholar). However, by a y-3HS, we also show that and not compete for the same binding to Pex14p, we also that the sites for Pex5p and Pex19p not as Pex19p not interact with Pex14p in the y-2HS, we not to a yeast protein three-hybrid to investigate whether Pex19p is to alter the Pex5p binding properties of Pex14p The demonstrate that the bacterial and yeast two-hybrid are complementary for the of protein interactions. In the with the y-2HS, a proteins with Pex5p and which in the act as are to the Gal4p DNA-binding domain M. in yeast by a to a binding not the and in the one of the of the non-transcription-based two-hybrid the of the between the proteins and the activation investigate the of Pex5p and Pex14p, we an in approach that Pex14p exists predominantly as a in its In results also suggest that Pex5p exists predominantly as a in mammalian these results are in with two-hybrid not the that Pex5p can In this is interesting to that Pex5p does W. J. B. Kunau W.H. human peroxisomal targeting signal receptor for interaction of with Biol. Chem. Scholar). However, the that the same molecule to in W. J. B. Kunau W.H. human peroxisomal targeting signal receptor for interaction of with Biol. Chem. Scholar) that these in have to with Finally, as we not an interaction between Pex5p and Pex13p in two-hybrid we the of the domain of Pex13p in the docking we an in binding in which a peroxisomal protein import complex of Pex5p, Pex14p, and that Pex14p, and not functions as the docking for are by the that Pex5p the peroxisomal membrane in mammalian that are in Pex13p or Pex14p and remains cytosolic in in Pex14p H. T. M. S. A. A. Shimizu N. Fujiki Y. The mammalian peroxin Pex the longer isoform of the peroxisome targeting signal type 1 the protein complex into peroxisomes via its docking Biol. Chem. 2000; 275: Scholar). We are to V. and J. for to M. for the and to S. for