Analysis of Human Pex19p's Domain Structure by Pentapeptide Scanning Mutagenesis

doi:10.1016/j.jmb.2005.01.013 J. Mol. Biol. (2005) 346, 1275–1286

Analysis of Human Pex19p’s Domain Structure byPentapeptide Scanning Mutagenesis

Marc Fransen*, Ilse Vastiau, Chantal Brees, Vanessa BrysGuy P. Mannaerts and Paul P. Van Veldhoven

Katholieke Universiteit LeuvenFaculteit Geneeskunde, CampusGasthuisberg (O/N 6, box 601)Departement MoleculaireCelbiologie, AfdelingFarmacologie, Herestraat 493000 Leuven, Belgium

0022-2836/$ - see front matter q 2005 E

Abbreviations used: PMP, peroxiprotein; b-2HS, bacterial two-hybridtwo-hybrid system; GFP, green fluoE-mail address of the correspond

[email protected]

Pex19p, a primarily cytosolic protein, is essential for the biogenesis ofnumerous peroxisomal membrane proteins (PMPs); however, its precisefunction is unclear. Pex19p might function as a PMP-specific chaperone, acycling PMP-receptor protein, a PMP membrane insertion factor, or anassociation/dissociation factor of membrane-associated protein com-plexes. Alternatively, Pex19p might act as a multifunctional peroxin andparticipate in a number of these activities. Here, we have employedtransposon mutagenesis to generate a library of human pex19 alleles codingfor Pex19p variants containing random in-frame pentapeptide insertions.A total of 87 different variants were characterized to identify functionallyimportant regions. These studies revealed that Pex19p has a tripartitedomain structure consisting of: (i) an amino-terminal domain that binds toPex3p and is essential for docking at the peroxisome membrane; (ii) acentral domain that competes with Pex5p and Pex13p for binding toPex14p and may play a role in the assembly of PTS-receptor dockingcomplexes; and (iii) a carboxy-terminal domain that interacts withmultiple PMPs including Pex3p, Pex11pb, Pex12p, Pex13p, Pex16p, andPex26p. Whether the latter interactions constitute the chaperone ortransport functions (or both), remains to be determined. Finally, ourobservation that Pex19p contains two distinct binding sites for Pex3psuggests that the peroxin may bind PMPs in multiple places and formultiple purposes.

q 2005 Elsevier Ltd. All rights reserved.

Keywords: peroxisomes; biogenesis; protein import; Pex19p; transposonmutagenesis
*Corresponding author
Introduction

Peroxisomes play several vital roles in humanmetabolism.1 Mutations disrupting peroxisomeformation or function result in severe diseasephenotypes.2 The prototype of these is Zellwegersyndrome, an often fatal inherited disease causedby a peroxisome biogenesis deficiency.3 Peroxisomebiogenesis is a complex multi-step process thatdepends on the concerted action of a select group ofproteins, called peroxins (abbreviated Pexp andincluding a number corresponding to the order ofdiscovery; gene acronym: PEX).4 To date, 16 humanPEX-genes have been identified,5–9 and the

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somal membranesystem; y-2HS, yeastrescent protein.ing author:

corresponding gene products have been implicatedin the synthesis and assembly of peroxisomemembranes (e.g. Pex3p, Pex16p and Pex19p), theimport of peroxisomal matrix proteins (e.g. Pex1p,Pex2p, Pex5p, Pex6p, Pex7p, Pex10p, Pex12p,Pex13p, Pex14p and Pex26p), or the growth in sizeand number of the organelles (e.g. Pex11pa,Pex11pb and Pex11pg).6–9 However, the precisefunction of most of these peroxins from a mechan-istic standpoint remains largely unclear.An important aim of our recent studies was to

elucidate the role of human Pex19p10 in theperoxisome biogenesis process. Like its counterpartin Pichia pastoris,11 HsPex19p exhibits a broadbinding specificity for peroxisomal membraneproteins (PMPs).12–17 It has been found in thecytosol and, to a minor extent, on the outer surfaceof the peroxisome membrane.12,18 Human fibro-blasts, Chinese hamster ovary cells andSaccharomyces cerevisiae cells deficient in Pex19p

d.

1276 Dissection of Pex19p into Distinct Domains

appear to lack peroxisomal membrane ghosts.18–20

These observations and the finding that Pex19pinteracts with the targeting domain of a number ofPMPs have led to the hypothesis that Pex19p mightfunction as a cycling PMP-receptor protein.12,21–23

However, as (i) the Pex19p interaction domainand the targeting domain do not overlap for allPMPs,11,14,24 (ii) peroxisome remnants and struc-tures resembling wild-type peroxisomes have beenobserved in pex19D cells of P. pastoris and Yarrowialipolytica, respectively,11,25 (iii) overproduction ofPex3p restores peroxisome formation in pex19Dcells of Hansenula polymorpha26 and (iv) humanPex19p can alter the binding properties of thePex14p PTS-receptor docking complex in vitro,27

several non-transport functions have beenpostulated. Among the functions suggested arechaperoning of newly synthesized PMPs21 andregulating membrane-associated protein com-plexes.11 Alternatively, Pex19p might be acting asa multifunctional peroxin.

Many multifunctional proteins are composed ofmultiple domains, each having a specific anddistinct function. The identification and character-ization of these domains is essential for gaininginsight into the protein’s functional activities. Here,we have employed pentapeptide scanning muta-genesis to pinpoint protein segments of humanPex19p involved in interactions with a select set ofPMPs. Pentapeptide scanning mutagenesis is afacile transposon-based procedure for the randominsertion of a variable five amino acid residuecassette into a target protein.28 This study providesevidence that human Pex19p has a tripartitedomain structure. How these domains appear tofunction is discussed.

Figure 1. An overview of the peroxin-binding proper-ties of Pex19p variants containing random pentapeptideinsertions. The protein sequence of human Pex19p isshown. The pentapeptide insertions are indicated by avertical arrow and named by the amino acid residue tothe N-terminal side of the insertion followed by thesequence of the insertion. The peroxin-binding propertiesof the Pex19p variants were studied by employing theyeast (Pex3p, Pex12p, Pex13p, Pex16p, and Pex26p) andbacterial (Pex3p, Pex11pb, Pex14p, Pex16p, Pex26p) two-hybrid systems. Pentapeptide insertions retaining thesame peroxin-binding properties as wild-type Pex19p aremarked in black. Insertions resulting in a stronglyreduced binding affinity for Pex3p or Pex14p areindicated in blue and green, respectively. Insertionsresulting in a strongly diminished binding affinity forPex11pb, Pex12p, Pex13p, Pex16p, and Pex26p aremarked in red. Insertions yielding unstable Pex19pmolecules in the yeast two-hybrid system are indicatedin purple. Insertions analysed in the bacterial two-hybridsystem after removal of the N-terminal Pex3p-bindingsite (see Table 2) are shaded in blue. Grey-shadedregions indicate a-helices as predicted by the PSA-server(probability R0.85).

Results

Generation of an in-frame, five amino acidresidue insertion library of human Pex19p

To gain insight into the domain organization ofhuman Pex19p, a plasmid encoding Gal4pAD-Pex19p was mutated using the bacteriophageMuA transposase (MGS Mutation GenerationSystem, Finnzymes). This system inserts artificialtransposons, designated as entranceposons, largelyat random, into any target plasmid in vitro. Aftertransforming Escherichia coli with the transpositionreaction mixture, 1224 clones were obtained, andthe insertion position of M1-KanR, the entrance-poson employed, was determined by colony PCR.This analysis yielded 103 clones in which theentranceposon insertions occurred in the PEX19cDNA. The corresponding plasmids were digestedwith NotI and closed by self-ligation to remove thebody of the entranceposon. Sequence analysis ofthese plasmids revealed the presence of 87 unique15 base-pair insertions that, after translation, resultin five (variable) extra amino acid residues(Figure 1). Note that the pentapeptide insertions

are distributed fairly evenly, with the largestinterval between adjacent insertions being 14amino acid residues.

Dissection of Pex19p into Distinct Domains 1277

Analysis of the peroxin-binding properties ofPex19p variants

In order to identify and map different functionaldomains of Pex19p, the variant Pex19p proteinswere analysed for binding to Pex3p, Pex11pb,Pex12p, Pex13p, Pex14p, Pex16p, and Pex26p inthe bacterial two-hybrid system (b-2HS) or in theyeast two-hybrid system (y-2HS). These Pex19p-interaction partners were chosen for the followingreasons: previously, we reported that Pex3p,Pex12p, Pex13p and Pex16p interact with Pex19pin the y-2HS;14 we showed also that Pex3p,Pex11pb, Pex14p and Pex16p bind to Pex19p in anon-transcription-based b-2HS;15 and recently wehave identified Pex26p5 as a novel interactionpartner of Pex19p (unpublished results; thiswork). The peroxin-binding properties of the87 variant Pex19p proteins were evaluatedinitially by employing indicator plates (b-2HS)and colony lift b-galactosidase filter assays(y-2HS). In follow-up experiments, we performedliquid culture b-galactosidase assays for a select

Table 1. Effect of pentapeptide insertions on the peroxin bind

Normalised absorbance

AD-Pex19p BD-Pex3p BD-Pex12p

– 0.30G0.02 0.54G0.21

WT 302G37 2.33G1.17

16VRPHR 79.2G8.9 0.67G0.0719DAAAE 14.8G10.2 0.47G0.05

22DAAAL 3.02G2.16 1.76G0.9425LRPHA 8.16G11.69 1.28G0.25

67DAAAQ 213G77 1.11G0.5567VRPHQ 216G70 1.21G0.1267CGRTQ 144G16 1.40G0.6271HAAAF 188G91 1.48G0.28

219VRPHQ 541G78 0.43G0.09223CGRNS 664G130 0.27G0.01


T18-Pex19p T25-Pex3p T25-Pex11pb

– 87G61 43G34

WT 5354G276 424G162

16VRPHR 4721G268 356G7419DAAAE 4024G1079 342G144

22DAAAL 1306G447 422G8625LRPHA 510G232 500G187

67DAAAQ 4548G484 160G1867VRPHQ 2323G109 120G7567CGRTQ 4511G422 119G4471HAAAF 4601G538 209G72

219VRPHQ 3461G310 14G17223CGRNS 4790G1241 39G29

A select set of Pex19p variants, fused to Gal4pAD (y-2HS) or T18 (bPex13p, Pex14p, Pex16p, and Pex26p, fused to Gal4pBD (y-2HS) or Ttransformants expressing one of the bait (BD and T25, respectively) anselected and assayed for b-galactosidase activity using o-nitrophenyl-measured at 420 nm, normalized for culture densities (optical densitcorrected for the blank (“empty” plasmids only). The values givemeasurements performed on cultures derived from independent coactivity of the wild-type protein after subtracting the average backgrouare underlined. – Z“empty” T18 (b-2HS) or Gal4pAD (y-2HS) plasm

group of mutants. Summaries of these results areshown in Figure 1 and Table 1. Two variants,Pex19p22DAAAL and Pex19p25LRPHA, displayed asignificantly reduced binding affinity for Pex3p.Four pentapeptide insertions, situated betweenamino acid residues 67 and 72, resulted in a severelyreduced binding affinity for Pex14p, and 28mutants, located in the region spanning aminoacid residues 173 and 264, displayed no, or astrongly diminished, binding affinity for Pex11pb,Pex12p, Pex13p, Pex16p and Pex26p. Note thatnearly all pentapeptide insertions affecting thebinding properties of Pex19p are located withinpredicted a-helices (see Discussion). The obser-vation that each of these mutants displayed at leastone affinity similar to that of the wild-type proteinindicates that these variants are probably expressedequally. The remaining 53 mutated proteinsbehaved like the wild-type molecule, at least inthe b-2HS. In the y-2HS, two of these mutantsdisplayed a reduced binding affinity for all inter-action partners. Immunoblot analysis studies pointto protein instability as the cause for this lack of

ing properties of Pex19p

BD-Pex13p BD-Pex16p BD-Pex26p

0.11G0.02 0.43G0.38 0.33G0.17

0.54G0.06 22.0G0.90 5.94G1.50

0.27G0.10 5.03G1.15 2.12G0.640.21G0.03 4.83G0.88 1.18G1.16

0.56G0.03 19.05G8.53 4.33G1.340.52G0.12 20.08G8.74 4.54G0.97

0.59G0.11 13.52G6.04 3.14G1.440.44G0.15 31.72G0.99 6.48G2.690.54G0.13 27.60G10.50 7.08G3.180.32G0.09 17.47G3.80 5.21G1.16

0.13G0.02 0.29G0.06 0.40G0.100.18G0.09 0.39G0.13 0.68G0.36

T25-Pex14p T25-Pex16p T25-Pex26p

5G8 36G36 15G7

477G41 1310G233 298G100

281G47 422G46 221G51264G106 815G169 302G84

644G75 1135G448 355G161424G58 1093G350 267G57

101G46 797G189 235G7028G32 1103G74 148G1817G22 1033G89 165G4710G23 576G56 236G45

205G64 62G19 8G6230G149 76G24 26G28

-2HS), were tested for interaction with Pex3p, Pex11pb, Pex12p,25 (b-2HS), in the yeast or bacterial two-hybrid system. Doubled one of the prey (AD and T18, respectively) fusion proteins wereb-D-galactopyranoside as the substrate. The optical densities werey at 600 nmZ10) and time (b-2HS: 1 hour; y-2HS: 24 hours), andn are the mean (G the standard deviation) of at least threelonies. Strongly diminished interactions (!25% of the bindingnd activity of the correspondingGal4pBD- or T25-fusion proteins)id; WTZwild-type Pex19p.


binding activity (data not shown). Summarized, ourresults suggest that Pex19p consists of three distinctdomains: one that is involved in Pex3p binding; asecond that is involved in Pex14p binding; and athird that is involved in binding to Pex11pb,Pex12p, Pex13p, Pex16p and Pex26p.

Pex19p has a tripartite domain structure

By employing transposon-delivered peptide tags,we have “deconstructed” Pex19p into a multi-domain peroxin (Figure 1). To investigate whetherthese domains could be physically separated, weperformed deletion analysis studies in the b-2HS(Figure 2). These studies: (i) confirmed our previousy-2HS results that the N-terminal 51 amino acidresidues of Pex19p contain a binding site forPex3p;14 (ii) revealed that amino acid residues60–91 were required and sufficient for binding toPex14p; and (iii) showed that amino acid residues124–299 determine specific binding to Pex11pb,Pex16p and Pex26p. Unexpectedly, the latterdomain did retain a significant binding affinity forPex3p (Figure 2). The observation that Pex19p hastwo distinct binding sites for Pex3p, combinedwith the fact that Pex19p molecules lacking theN-terminal 30 amino acid residues do not interactwith Pex3p in the y-2HS,14 may explain why thepentapeptide insertions 22DAAAL and 25LRPHAdecrease the affinity of Pex19p for Pex3p to 2% ofwild-type in the y-2HS and to some 20% in theb-2HS (Table 1). Aside from this apparentdiscrepancy, the transposon mutagenesis results(Figure 1; Table 1) show consistent agreement withthe results of the deletion analysis studies (Figure 2):variants displaying a severely reduced bindingaffinity for Pex14p cluster between amino acidresidues 67 and 72; and all pentapeptide insertionsresulting in a strongly diminished binding for

Figure 2.Mapping of the protein–protein interaction domaitested for interaction with Pex3p, Pex11pb, Pex14p, Pex16psystem. Double transformants were selected, and b-galactosiThe binding activities of the tested protein pairs are indicated48 hours, (K) the staining intensity of the colonies was not ableft indicate the amino acid residues present in the correpentapeptide VRPHQ at position 219. The delineated interactilength Pex19p molecule: 3A, 3B, Pex3p; 11b, Pex11pb; 14, Pex

Pex11pb, Pex16p and Pex26p are situated betweenamino acid residues 173 and 264.

Further characterization of the C-terminalPex3p-binding site of Pex19p

To investigate whether Pex3p binds to the sameregion within the C-terminal domain of Pex19p asPex11pb, Pex16p and Pex26p, we generated aconstruct coding for Pex19p(61–299,219VRPHQ), aprotein lacking the N-terminal Pex3p-bindingdomain. In the indicator plate assay, this proteindid interact with Pex14p, but not with Pex11pb,Pex16p or Pex26p (Figure 2). In addition,Pex19p(61–299,219VRPHQ) binds to Pex3p (Figure 2).These results suggest that Pex3p and Pex11pb/Pex16p/Pex26p do not share the same binding sitewithin the C-terminal domain of Pex19p. Todocument this point more carefully, we generatedadditional constructs including pentapeptide inser-tions within the region spanning amino acidresidues 124–49 and performed quantitative (liquidculture) b-galactosidase assays (Table 2). Theseexperiments showed no direct correlation betweenbinding of Pex19p to Pex3p and binding of thisperoxin to Pex11pb, Pex16p and Pex26p (Table 2).For example: (i) Pex19p(61–299,134GAAAS) interactedwith Pex11pb, Pex16p, and Pex26p, but not withPex3p; (ii) Pex19p(61–299,176MRPQQ) bound weaklyto Pex3p, but not at all to Pex11pb, Pex16p orPex26p (although the significance of theseextremely low values is difficult to assess);(iii) Pex19p(61–299,223CGRNS) bound to Pex3p, butnot to Pex11pb, Pex16p or Pex26p; and (iv)Pex19p(61–299,147CGRTS) interacted with Pex3p,Pex11pb, Pex16p and Pex26p. These experimentsrevealed that four out of six pentapeptide insertionspresent in the region spanning amino acid residues124–149 have a strong negative impact on thebinding affinity of Pex19p(61–299) for Pex3p

ns of Pex19p. Pex19p deletion mutants (fused to T18) were, and Pex26p (fused to T25) in the bacterial two-hybriddase activities monitored by employing indicator plates.as follows: (C) the colony staining times were less than

ove background level after 72 hours. The numbers on thesponding Pex19p deletion mutants. X, insertion of theon domains are indicated on the representation of the full-14p; 16, Pex16p; and 26, Pex26p.

Table 2. Effect of deletions on the peroxin binding properties of Pex19p variants


T18-Pex19p T25-Pex3p T25-Pex11pb T25-Pex14p T25-Pex16p T25-Pex26p

– 87G61 43G34 5G8 36G36 15G7

1–299 (WT) 5354G276 424G162 477G41 1310G233 298G1001–299 (133SAAAL) 2371G772 586G103 653G45 664G198 492G881–299 (133CGRTL) 2693G854 243G43 733G73 924G187 335G4921–299 (134GAAAS) 2130G549 167G60 378G132 813G212 246G531–299 (135CGRSG) 2069G483 242G57 552G96 743G220 188G431–299 (147CGRTS) 3065G607 431G43 518G104 943G217 355G941–299 (147MRPHS) 2970G430 408G119 453G180 1033G246 298G1241–299 (176MRPQQ) 3510G460 33G7 279G81 64G25 42G301–299 (219VRPHQ) 3461G310 14G17 205G94 62G19 8G61–299 (223CGRNS) 4790G1241 39G29 230G149 76G24 26G28

61–299 (WT) 2767G455 1344G297 1377G124 2744G365 892G11661–299 (133SAAAL) 728G329 571G104 1257G128 2646G282 530G13561–299 (133CGRTL) 148G82 221G10 889G55 1447G246 113G4761–299 (134GAAAS) 48G38 410G128 997G240 2408G267 183G6261–299 (135CGRSG) 219G92 447G79 1035G287 1753G172 218G6761–299 (147CGRTS) 1450G684 474G187 1250G220 2018G539 495G1361–299 (147MRPHS) 1638G691 785G104 1313G147 2549G239 332G11761–299 (176MRPQQ) 164G40 57G12 540G90 23G18 39G1661–299 (219VRPHQ) 697G89 66G12 660G53 215G58 11G661–299 (223CGRNS) 1339G117 59G20 591G99 18G16 11G4

150–299 64G9 10G7 16G9 22G14 10G931–299 610G175 693G216 490G84 688G56 141G4194–176 43G6 43G28 23G7 20G15 30G131–125 1460G302 10G8 865G344 8G5 10G91–51 1219G400 4G3 58G391 13G5 19G360–93 53G18 9G7 1606G219 12G11 16G11

A select set of Pex19p variants, fused to T18, were tested for interaction with Pex3p, Pex11pb, Pex14p, Pex16p, and Pex26p, fused to T25,in the bacterial two-hybrid system. Double transformants expressing one of the bait and one of the prey fusion proteins were selectedand assayed for b-galactosidase activity using o-nitrophenyl-b-D-galactopyranoside as the substrate. The optical densities weremeasured at 420 nm, normalized for culture densities (optical density at 600 nmZ10) and time (1 hour), and corrected for the blank(“empty” plasmids only). The values given are the mean (G the standard deviation) of at least three measurements performed oncultures derived from independent colonies. Strongly diminished interactions (!25% of the binding activity of the full-length wild-typeprotein after subtracting the average background activity of the corresponding T25-fusion proteins) are underlined. – Z“empty” T18plasmid; WTZwild-type protein.


(Table 2). However, note that at least compared tothe corresponding truncated wild-type protein,these pentapeptide insertions do also (slightly)decrease the affinity of Pex19p(61–299) for the otherinteraction partners. Whether this is a direct effector the result of local conformational changesremains to be investigated. As the region of Pex19pflanked by amino acid residues 124 and 149 is, onthe one hand, not sufficient for binding to Pex3p(Table 2, see results obtained with T18-Pex19p(94–176))and, on the other hand, essential for bindingto Pex11pb, Pex16p and Pex26p (Figure 2), it ishighly unlikely that the binding sites for Pex3pand Pex11pb/Pex16p/Pex26p present in theC-terminal domain of Pex19p can be physicallyseparated.

The N-terminal Pex3p-binding site of Pex19p isessential for docking at the peroxisomemembrane

The integral peroxisomal membrane proteinPex3p is required for recruitment of Pex19p toperoxisomes.17,29–31 As our data show that Pex19phas two distinct binding sites for Pex3p (Figure 2;Table 2), we investigated whether the docking

process is mediated by its N-terminal or C-terminalbinding site. Mouse fibroblasts were transfectedwith plasmids encoding the peroxisomal markerprotein DsRed-SKL and green fluorescent protein(GFP), GFP-Pex19pWT, GFP-Pex19p25LRPHA, GFP-Pex19p67CGRTQ, GFP-Pex19p134GAAAS or GFP-Pex19p219VRPHQ. Fluorescence microscopy analysisof cells expressing GFP-Pex19p67CGRTQ, GFP-Pex19p134GAAAS, or Pex19p219VRPHQ revealed aprominent cytosolic staining and some punctatestaining (Figure 3). Double labelling with theperoxisomal marker protein DsRed-SKL identifiedthe punctate structures as peroxisomes (Figure 3).A virtually identical staining pattern was observedin cells expressing the wild-type GFP-fusion protein(Figure 3). Interestingly, the punctate stainingpattern was completely absent from cells expres-sing GFP-Pex19p25LRPHA (Figure 3). Combining theobservations that (i) the expression levels of thePex19p-fusion proteins are comparable (Figure 4),and (ii) the pentapeptide insertions do not drasti-cally alter the stability of the various Pex19pproteins (Figure 4; and see Discussion), these datashow that the N-terminal, and not the C-terminal,Pex3p-binding site of Pex19p is required for itsdocking at the peroxisome membrane.

Figure 3. Intracellular localization of variant Pex19p proteins in mouse fibroblasts. Mouse fibroblasts transfectedtransiently with plasmids expressing the peroxisomal marker protein DsRed-SKL and GFP (K), Pex19pWT (WT),Pex19p25LRPHA (25LRPHA), Pex19p67CGRTQ (67CGRTQ), Pex19p134GAAAS (134GAAAS), or Pex19p219VRPHQ (216VRPHQ)N-terminally fused to GFPwere examined for direct fluorescence 48 hours after transfection. The subcellular localizationof the GFP-fusion proteins was determined by the staining pattern: cytosol (25LRPHA), cytosol-nucleus (K), cytosol-peroxisome (WT, 67CGRTQ, 134GAAAS, 219VRPHQ). The punctate structures observed (WT, 67CGRTQ, 134GAAAS,219VRPHQ) are peroxisomes, as illustrated by their colocalization with DsRed-SKL. The scale bar represents 20 mm.


Discussion

The analysis of proteins harbouring randomartificial-peptide insertions can provide valuableinsight into protein structure–function relation-ships, domain organization, regions that are insen-sitive or hypersensitive to the effects of insertions,and the different parts of multifunctional proteinsthat are implicated in various activities.32 Here,we have employed pentapeptide scanning

mutagenesis to pinpoint domains of humanPex19p involved in the interactions with a selectset of PMPs. Of the 87 mutants analysed, 51pentapeptide insertions did not alter any of thePMP-binding properties of full-length Pex19p, andtwo mutants were poorly expressed or unstable inyeast. Among the remaining 34 mutants, twovariants displayed a reduced affinity for Pex3p,four insertions resulted in a strongly reducedaffinity for Pex14p, and 28 variants failed to bind

Figure 4. Variant Pex19p proteins are expressed equallyin mouse fibroblasts. Equal amounts of whole cell lysatesfrom mouse fibroblasts transfected transiently withplasmids expressing (1) GFP, (2) Pex19pWT, (3)Pex19p25LRPHA, (4) Pex19p67CGRTQ, (50 Pex19p134GAAAS,and (6) Pex19p219VRPHQ were separated by SDS-PAGE,transferred to nitrocellulose, and probed with an anti-serum raised against human Pex19p. The GFP-Pex19pfusion proteins are indicated by an arrow (full-lengthprotein) and arrowheads (degradation products), respect-ively. Note that the titer of the anti-Pex19p antiserum isnot high enough to detect endogenous Pex19p. Molecularmass markers (expressed in kDa) are indicated on the left.


to Pex11pb, Pex12p, Pex13p, Pex16p and Pex26p.Interestingly, pentapeptide insertions resulting inPex19p mutants with equal binding propertiesclustered within three restricted regions of themolecule: insertions reducing the affinity forPex3p were located between amino acid residues16 and 20; insertions affecting Pex14p-binding werepresent between amino acid residues 67 and 72; andinsertions abolishing the affinity for Pex11pb,Pex12p, Pex13p, Pex16p and Pex26p were locatedin the region spanning amino acid residues 173 and264. Note that, on the basis of previous observationssuggesting that farnesylation of Pex19p is animportant determinant in the affinity of Pex19p forPex12p and Pex13p,14,15 it might seem surprisingthat Pex19pWT and Pex19p295CAAAQ display equiv-alent affinities for these peroxins. However, as far aswe can tell, there is no reason why a pentapeptideinsertion adjacent to the CaaX box would interferewith farnesylation. Whether this pentapeptideinsertion will alter the function of Pex19p in vivoremains to be investigated.

Interestingly, the regions of Pex19p containingpentapeptide insertions affecting the PMP-bindingproperties of the protein are separated from eachother by protein segments that are highly tolerant ofinsertions. In general, amino acid stretches at whichthe protein is tolerant of mutations function as inter-domain regions.32,33 Pentapeptide insertions insuch disordered regions are less deleterious toprotein function than insertions in a-helices orb-sheets.28 At first glance, this appears to be the casefor human Pex19p. That is, according to secondarystructure predictions performed at the ProteinSequence Analysis (PSA) server, 30 out of 34

pentapeptide insertions affecting the bindingaffinity of Pex19p are situated in a-helices, while37 out of 41 insertions located between thepredicted a-helices do not alter the bindingproperties of Pex19p (Figure 1). In this context, itis important to point out that circular dichroismstudies of (His)10-Pex19p(1–156) and (His)10-Pex19p(156–296) report a 6% and 55% content ofa-helix, respectively.34 Note that the value obtainedfor (His)10-Pex19p(156–296) is in good agreement withthe value predicted from the amino acid sequence(PSA server 43%; data not shown). However, thevalue obtained for (His)10-Pex19p(1–156) is notconsistent with that obtained from the theoreticalprediction (PSA server 49%; data not shown).Therefore, the number, length and location of thepredicted a-helices in human Pex19p should beinterpreted with care, especially within theN-terminal part of the protein.By studying the peroxin-binding properties of

Pex19p-deletion proteins, we were able to show thatthe three distinct domains identified by penta-peptide scanning mutagenesis could be physicallyseparated. In this context, we cite the work ofMuntau and colleagues, who have reported that, onthe basis of computational database studies, humanPex19p consists of three domains: D1 (amino acidresidues 1–87), D2 (amino acid residues 88–272),and D3 (amino acid residues 273–299).16 Theseauthors demonstrated that: (i) a splice variantlacking D1 did bind to peroxisomal ABC-transporters and full-length, but not N-terminallytruncated, Pex3p; (ii) a splice variant lacking D3displayed binding properties for peroxisomal ABC-transporters and (truncated) Pex3p similar to thoseof full-length Pex19p; and (iii) under conditions ofoverexpression, the D3 splice variant could comple-ment peroxisome biogenesis in a Pex19p-deficientcell line. However, our experimental studies showthat the D1 domain can be separated further, bothfunctionally and physically, into a Pex3p-bindingand a Pex14p-binding domain. With respect to theD2 domain, our results extend the observationsreported by Muntau and co-workers, by demon-strating that this domain binds to Pex11pb, Pex12p,Pex13p, Pex16p and Pex26p. With respect to the D3domain, our observation that this region of Pex19pis not required directly for binding to the investi-gated Pex19p-binding partners is in agreement withthe fact that, at least under certain experimentalconditions, D3 is dispensable for function. Morerecently, Kato and co-workers investigated thedomain structure of Pex19p.34 By employinglimited proteolysis studies, these authors showedthat Pex19p consists of a rigid C-terminal domainand a flexible, disordered N-terminal region. Thus,it may not be surprising that pentapeptide inser-tions in the N-terminal half of Pex19p (e.g.25LRPHA and 67CGRTQ) have a greater effect onprotein stability than insertions in the C-terminalhalf of the protein (e.g. 219VRPHQ) (Figure 4). Notealso that the region between positions 173 and 263in human Pex19p, in which 28 out of the 35


pentapeptide insertions abolished detectable bind-ing to Pex11pb, Pex12p, Pex13p, Pex16p andPex26p, is the mostly conserved part of themolecule among all Pex19p species (data notshown).

Our Pex19p deletion analysis studies in thebacterial two-hybrid system confirm and extendthe earlier observations of Muntau and co-workers,who reported that Pex19p might contain distinctbinding sites for Pex3p.16 Here, we report that thePex3p-binding site in the N-terminal, but not in theC-terminal, domain of Pex19p is required for itsdocking at the peroxisome membrane. Note thatthis result is in good agreement with the very recentobservation that the N-terminal 56 amino acidresidues of Pex19p are sufficient for docking toperoxisomes.31 The functional significance of theinteraction between the C-terminal domain ofPex19p and Pex3p is unclear. As this domain isnot involved in the Pex19p-docking event, it isunlikely that the separate Pex3p-binding domainsfunction cooperatively. In addition, as our resultssuggest that Pex3p and Pex11pb/Pex16p/Pex26pdo not share the same binding site within theC-terminal domain of Pex19p, the functional sig-nificance of these interactions is likely to bedifferent. Moreover, as these binding sites are atleast partially overlapping, it is tempting to specu-late that, after docking of Pex19p at the peroxisomemembrane, the interaction between the C-terminaldomain and Pex3p serves to dissociate other PMPinteractions occurring through this domain. Such amodel would fit perfectly within the hypothesis ofPex19p being a cycling PMP-receptor protein.12

With respect to the Pex19p–Pex14p interaction, itis important to point out that we have shownpreviously that for Pex14p, Pex19p-binding andperoxisomal sorting are not functionally linked.27

Therefore, it is unlikely that the Pex14p-bindingdomain of Pex19p is involved directly in the sortingprocess of Pex14p. In addition, we provided in vitroevidence that Pex19p can alter the binding proper-ties of the Pex14p docking complex, suggesting a

Table 3. Synthetic oligonucleotide primers used in this study

Name Nucleotide sequence

PGAD424SalIA 5 0-GGGGTCGACACCAAACCCpGBT9NotIRv 5 0-GGGCGCGCGGCCGCCATApGBT9XhoIB 5 0-GGGGCTCGAGGTTGACTGM1-KAN.RV FITC-50-CGAGCAAGACGTTTCpUT18C.RV2 5 0-GCGTCAGCGGGTGTTGGC19BTHAA94.fw 5 0-AGGGGATCCGTTGGCTGA19.BTH1 5 0-AGGGGATCCGATGGCCGC19.BTH2 5 0-GAGGGGTACCTCACATGAPex19.1 5 0-AGGGATCCAGATGGCCGCPex19.2 5 0-TGTCTCGAGTCACATGATCPex19.5 5 0-ATGGGATCCATGGCCGCCGPex19.8 5 0-TGTGTCGACGTGATGGGTAPex19.61fw 5 0-GAGGATCCGGATGCCCTCPex19.BTH2 5 0-GAGGGGTACCTCACATGAPex19.EELTfw 5 0-AGGGAATTCTGCAGGGAAHsPex26.3 5 0-CCGGAATTCATGAAGAGCHsPex26.4 5 0-GCCGGTCGACTCAGTCAC

potential role for this peroxin in assembly of PTS-receptor docking complexes.27 Note that theresidues around which the Pex14p interaction-impaired mutants cluster is a relatively small anddiscrete region within a predicted a-helix. In thiscontext, we note that the PTS1 receptor, Pex5p,interacts with Pex14p through WXXXF/Y penta-peptide motifs,35–37 and the tendency of such motifsto form a helical conformation may be important forPex14p-binding.38 Whether the Pex14p-bindingdomain of Pex19p consists of such a small linearepitope remains to be investigated. However,secondary structure predictions of Pex19p67DAAAQ,Pex19p67VRPHQ, Pex19p67CGRTQ, Pex19p68CGRKE andPex19p71HAAAF suggest that there is no directcorrelation between the a-helical content of thisregion of Pex19p and Pex14p-binding activity (datanot shown).

Summarized, the data presented above suggestthat Pex19p may bind PMPs in multiple places andfor multiple purposes. Interestingly, Pex19p hasbeen implicated recently in processes other thanperoxisome biogenesis. Indeed, it has been reportedthat this peroxin dampens the p19ARF-p53-p21tumour suppressor pathway in mice,39,40 andmay be actively involved in controlling theinternalization and trafficking of the Na/Pi IIaco-transporter.41 Future molecular modelling andstructural studies of Pex19p will provide the nextlevel of insight into its biological function and itsrole in health and disease. It will be interesting tosee how the PMP-binding properties of the penta-peptide insertion mutants reported here correlatewith the three-dimensional structure ultimatelydetermined.

Materials and Methods

Plasmids

The plasmids encoding Gal4pBD-HsPex3p (pMF158),14

T18-HsPex3p (pMF369),15 T25-HsPex3p (pMF392),15

(restriction sites are underlined)

AAAAAAAGAGATC-3 0

AGAAATTCGCCCGGAAT-30

TATCGCCGGAATTC-30

CCGTTG-30

GGG-3 0

GGAAGAACCCCAC-3 0

CGCTGAGGAA-3 0

TCAGACACTG-3 0

CGCTGAGGAAGG-30

AGACACTGTTC-3 0

CTGAGGAAGG-3 0

CAGCACATC-3 0

TTCGCTTCCC-30

TCAGACACTG-3 0

GAAGAGCTGACC-3 0

GATTCTTCGACC-3 0

GGATGCGGAGCTG-30

Table 4. Plasmids constructed for this study

Name Protein Cloning vector Insert

pMF123 GFP-HsPex19p pEGFP-C1 (BglII/SalI) BamHI/XhoI-digest of PCR product: template pMF133 (Pex19.2, Pex19.5)pMF763 GFP-HsPex19p25LRPHA pEGFP-C1 BglII/SalI BamHI/XhoI-digest of PCR product: template pMF827 (cl.10) (Pex19.2, Pex19.5)pMF894 (cl.11) GFP-HsPex19p67CGRTQ pEGFP-C1 BglII/SalI BamHI/XhoI-digest of PCR product: template pMF827 (cl.38) (Pex19.2, Pex19.5)pMF759 GFP-HsPex19p219VRPHQ pEGFP-C1 BglII/SalI BamHI/XhoI-digest of PCR product: template pMF827 (cl.12) (Pex19.2, Pex19.5)pMF154 BD-HsPex19p(124–299) pGBT9 Eco RI-digestion of pMF132 and religation of plasmidpMF788 T18-HsPex19p(150–299) pMF424 Pst I/SalI XhoI/Pst I-digest of PCR product: template pMF133 (Pex19p.EELTfw/Pex19.2)pMF818 T18-HsPex19p(124–299) pMF424 SalI/Not I XhoI/Not I-digest of PCR product: template pMF154 (pGBT9XhoIB/pGBT9NotIRv)pMF907 T18-HsPex19p(94–299) pMF424 BamHI/KpnI BamHI/KpnI-digest of PCR product: template pMF368 (19.BTHAA94.fw, Pex19.BTH2)pMF975 T18-HsPex19p(94–176) pMF424 BamHI/Not I BamHI/Not I-digest of PCR product: template pMF815 (19.BTHAA94.fw, pUT18C.RV2)pMF817 T18-HsPex19p(61–299,WT) pMF424 BamHI/KpnI BamHI/KpnI-digest of PCR product: template pMF133 (Pex19.61fw, Pex19.BTH2)pMF929 T18-HsPex19p(61–299,133SAAAL) pMF424 BamHI/KpnI BamHI/KpnI-digest of PCR product: template pMF827 (cl.83) (Pex19.61fw, Pex19.BTH2)pMF928 T18-HsPex19p(61–299,133CGRTL) pMF424 BamHI/KpnI BamHI/KpnI-digest of PCR product: template pMF827 (cl.41) (Pex19.61fw, Pex19.BTH2)pMF932 T18-HsPex19p(61–299,134GAAAS) pMF424 BamHI/KpnI BamHI/KpnI-digest of PCR product: template pMF827 (cl.69) (Pex19.61fw, Pex19.BTH2)pMF936 T18-HsPex19p(61–299,135CGRSG) pMF424 BamHI/KpnI BamHI/KpnI-digest of PCR product: template pMF827 (cl.19) (Pex19.61fw, Pex19.BTH2)pMF939 T18-HsPex19p(61–299,147CGRTS) pMF424 BamHI/KpnI BamHI/KpnI-digest of PCR product: template pMF827 (cl.84) (Pex19.61fw, Pex19.BTH2)pMF938 T18-HsPex19p(61–299,147MRPHS) pMF424 BamHI/KpnI BamHI/KpnI-digest of PCR product: template pMF827 (cl.49) (Pex19.61fw, Pex19.BTH2)pMF949 T18-HsPex19p(61–299,176MRPQQ) pMF424 BamHI/KpnI BamHI/KpnI-digest of PCR product: template pMF827 (cl.44) (Pex19.61fw, Pex19.BTH2)pMF909 T18-HsPex19p(61–299,219VRPHQ) pMF424 BamHI/KpnI BamHI/KpnI-digest of PCR product: template pMF827 (cl.12) (Pex19.61fw, Pex19.BTH2)pMF940 T18-HsPex19p(61–299,223CGRNS) pMF424 BamHI/KpnI BamHI/KpnI-digest of PCR product: template pMF827 (cl.9) (Pex19.61fw, Pex19.BTH2)pMF816 T18-HsPex19p(31–299) pMF424 SalI SalI/XhoI-digest of PCR product: template pMF202 (pGAD424SalIA/Pex19.2)pMF815 T18-HsPex19p(1–176) pMF424 SalI SalI/XhoI-digest of PCR product: template pMF133 (pGAD424SalIA/Pex19.8)pMF897 (cl. 1/103) T18-HsPex19p variants pUTC18C BamHI/KpnI BamHI/KpnI-digest of PCR product: template pMF827 (cl. 1/103) (19.BTH1/19.BTH2)pMF898 (cl. 1/103) T25-HsPex19p variants pKT25 BamHI/KpnI BamHI/KpnI-digest of PCR product: template pMF827 (cl. 1/103) (19.BTH1/19.BTH2)pMF808 T18-HsPex26p pMF424 SalI/Not I XhoI/Not I-digest of PCR product: template pMF937 (pGBT9XhoIB/pGBT9NotIRv)pMF807 T25-HsPex26p pMF413 XhoI/Not I XhoI/Not I-digest of PCR product: template pMF937 (pGBT9XhoIB/pGBT9NotIRv)pMF937 BD-HsPex26p pGBT9 Eco RI/SalI Eco RI/SalI-digest of PCR product: template human liver cDNA (HsPex26.3/HsPex26.4)

† http://bmerc-www.bu.edu/psa/


T18-HsPex11pb (pMF422),15 T25-HsPex11pb (pMF423),15

Gal4pBD-HsPex12p (pMF304),42 Gal4p-HsPex13p(pMF103),14 T18-HsPex14p (pMF365),15 T25-HsPex14p(pMF366),15 Gal4p-HsPex16p (pMF187),14 T18-HsPex16p(pMF428),15 T25-HsPex16p (pMF429),15 Gal4pBD-HsPex19p (pMF132),15 Gal4pAD-HsPex19p (pMF133),14

Gal4pAD-HsPex19p(31–299) (pMF202),14 Gal4pAD-HsPex19p(61–299) (pMF203),14 T18-HsPex19p (pMF367),15

T25-HsPex19p (pMF368)15 and DsRed-SKL (pMF578)14

are described elsewhere. The oligonucleotides (Invitro-gen) and plasmids constructed for this study arecompiled in Tables 3 and 4, respectively. Cloning vectorswere obtained from Clontech (pEGFP-N1, pGBT9, andpGAD424) and Hybrigenics (pUT18C and pKT25). Themodified bacterial two-hybrid vectors (pMF413 andpMF424) are reported elsewhere.15 The human livercDNA library was obtained from TaKaRa. PCR appli-cations were performed routinely using Pfx DNA poly-merase (InVitrogen). The E. coli strain Top10F 0

(Invitrogen) was used for all DNA manipulations. Theidentities of essential constructs were confirmed by DNAsequencing.

MGS mutagenesis procedure

The in vitro transposition reaction, employing the targetplasmid pMF133 and the M1-KanR entranceposon(Finnzymes), was done according to the manufacturer’sinstructions. The reaction mixture was used to transform(heat-shock method) E. coli Top10F 0 (InVitrogen) cellsyielding 1224 independent clones. The insertion positionof the M1-KanR entranceposon in the correspondingplasmids (pMF796/clones 1–1224) was determined bycolony PCR43 (oligonucleotides Pex19.1 and Pex19.2; TaqDNA polymerase (MBI Fermentas)). Plasmids in whichthe entranceposon insertion occurred in the open readingframe of Pex19p were (i) subjected to DNA sequencing(oligonucleotide M1-KAN.RV) to determine the exactinsertion position, and (ii) digested with NotI, re-ligatedand transformed in competent Top10F 0 E. coli cells toobtain plasmids (pMF827/clones 1–103) coding forGal4pAD-HsPex19p fusion proteins containing randomin-frame pentapeptide insertions. The construction of theplasmids coding for the corresponding T18- and T25-HsPex19p fusion proteins is described in Table 4.

Cell culture, transfections, fluorescence microscopy,and immunoblot analysis of Pex5C/K mousefibroblasts

Pex5C/K mouse fibroblasts were cultured asdescribed.42,44 After transfer to coverslips, the cells weretransiently transfected by employing the Magnetofectiontransfection technology (OZ Biosciences), and processedfor direct fluorescence as described.43 The peroxisomallocalization of the GFP-fusion proteins was confirmed byco-localization studies with the peroxisome-targetedDsRed-KSKL reporter protein.14 Fluorescence wasobserved under a Leica DMR microscope equippedwith FITC/RSGFP/Bodipy/Fluo3/DIO and Texas redfilters. For immunoblot analysis, transfected cells, grownin culture dishes to 90% confluency, were freed from thesedishes by scraping, resuspended in ice-cold phosphate-buffered saline (pH 7.2), transferred to a microfuge tube,and precipitated and processed as described.15

Other

Bacterial (E. coli strain BTH101) and yeast (S. cerevisiae

strain SFY526) two-hybrid analyses were performed asdescribed.14,15 Secondary structure predictions wereobtained from the BioMolecular Engineering ResearchCenter (BMERC) Protein Sequence Analysis (PSA)server†.45 The rabbit polyclonal antiserum raised against(His)6-HsPex19p is described elsewhere.14

Acknowledgements

We thank Dr Stanley Terlecky (Wayne StateUniversity School of Medicine, MI) for criticallyreading the manuscript, Dr Myriam Baes(Katholieke Universiteit Leuven, Belgium) for theimmortalized Pex5C/K-fibroblasts, and MarijkeBrams (Katholieke Universiteit Leuven, Belgium)for excellent technical support. This work wassupported by grants from the Flemishgovernment (Geconcerteerde OnderzoeksactiesGOA/99/09 and GOA/2004/08) and the Fondsvoor Wetenschappelijk Onderzoek-Vlaanderen(Onderzoeksproject G.0237.04).

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Edited by J. Karn

(Received 18 October 2004; received in revised form 4 January 2005; accepted 5 January 2005)

Analysis of Human Pex19p's Domain Structure by Pentapeptide Scanning Mutagenesis

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