© 1999 Oxford University Press Human Molecular Genetics, 1999, Vol. 8, No. 71263–1269

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Presenilins interact with Rab11, a small GTPaseinvolved in the regulation of vesicular transportCécile Dumanchin 1, Christian Czech 2, Dominique Campion 1, Marie Hélène Cuif 3,Thomas Poyot 2, Cosette Martin 1, Françoise Charbonnier 1, Bruno Goud 3, Laurent Pradier 2

and Thierry Frebourg 1,+

1INSERM EPI 9906, Faculté de Médecine et de Pharmacie, 22 Boulevard de Gambetta, 76183 Rouen and IFRMP,Mont-Saint-Aignan, 76821 Rouen Cedex, France, 2Rhone Poulenc Rorer, Centre de Recherche de Vitry-Alfortville, 94403Vitry-sur-Seine Cedex, France and 3Unité Mixte de Recherche CNRS 144, Institut Curie, 75248 Paris Cedex 05, France

Received February 15, 1999; Revised and Accepted April 13, 1999

Presenilin 1 (PS1) mutations account for the majority ofearly-onset dominant cases of familial Alzheimer’sdisease. Presenilins (PSs) are located in many intra-cellular compartments such as the endoplasmic reticu-lum, Golgi apparatus, nuclear region and vesicularstructures. These proteins include from seven to nineputative transmembrane domains, with the N- andC-terminal ends and a large hydrophilic loop orientatedtowards the cytoplasm. We report an interactionbetween the human PS1 or PS2 hydrophilic loop andRab11, a small GTPase belonging to the Ras-relatedsuperfamily. Interaction domains were mapped tocodons 374–400 for PS1 and to codons 106–179 forRab11, a region including the fourth GTP-bindingdomain. Considering the implication of Rab proteins invesicular transport pathways, the PS–Rab11 inter-action suggests that PSs might be involved in amyloidprecursor protein vesicular routing.

INTRODUCTION

Alzheimer’s disease (AD), the first cause of neurodegenerativedementia, is characterized by two cerebral lesions, senileplaques resulting from the deposition of the extracellular Aβpeptide and intracellular neurofibrillary tangles of which themain component is the microtubule-associated protein tau.Mutations of the presenilin 1 gene (PS1), located on chromo-some 14q24.3, account for the majority of autosomal dominantearly-onset AD (EOAD) cases. More than 50PS1missensemutations (1–4), two in-frame deletions of exon 9 (5,6) andtwo splicing mutations affecting the donor site of intron 4 (7)have been described in EOAD families. In contrast, only twomissense mutations have been identified in the presenilin 2gene (PS2), the second member of the presenilin familylocated on chromosome 1q42.1 (8,9).

The PS1 and PS2 proteins, composed of 467 and 448 aminoacids, respectively, display 67% identity and are predicted tocontain between seven and nine transmembrane (TM)domains, with a large hydrophilic loop between the sixth and

the seventh TM domains (10–14). PS1 mutations are widdistributed within the coding region, with clusters at the Nterminal end of TM2 and between TM6 and the N-terminregion of the hydrophilic loop (4).

Presenilins (PSs) are ubiquitously expressed and, in braare expressed mostly in neurons (15–17). At the subcellulevel, PSs have been located predominantly in the endoplasreticulum (ER) and Golgi apparatus (18–23), in the nucleregion (kinetochore, centrosome and nuclear membrane) (2at the cell surface (22) and in vesicular structures within tsomatodendritic compartment (23). PS1 displays ~25 and 5identity with the Caenorhabditis elegansSPE4 and SEL12proteins, respectively (24,25). SPE4, during spermatogeneis involved in pre-packaging and delivery of macromoleculto spermatids in the fibrous body–membranous organelle coplexes. SEL12 facilitates signalling mediated by the lin-1Notch family of receptors involved in developmental cell fatspecification and lateral inhibition.

The mechanisms through which mutations of PSs lead to Aare unknown. Plasma, fibroblasts and brains from patients wPS mutations have been shown to contain increased amoof the Aβ1-42/43 peptide, the amyloidergic form proteolytically released from the amyloid precursor protein (APP) byβ-and γ-secretases (26–27). Similar increases have beobserved in transfected cells lines and transgenic animalsexpress human mutant PSs (28,29). In neuronal cells derifrom PS1–/PS1– mouse embryos, there is an inhibition of APcleavage byγ-secretase (30). Human mutantPS1alleles, whichrescuePS1–/PS1– mouse embryos from lethality, also lead tan increase of Aβ1-42 peptide production (31,32). Furthermore, PS1 mutants down-regulate the production of noamyloidergicα-secretase-derived product of APP (33). Thnon-amyloidergic cleavage occurs in thetrans-Golgi network(TGN) and at the cell surface (34,35), whereas the amyloidgic cleavage mediated byγ-secretase normally occurs in theER for cleavage at position 42 and in the TGN for cleavageposition 40 (36). A direct interaction between APP and PSsthe ER compartment has been demonstrated recently (37–All these data suggest that mutations in PSs linked to familEOAD alter APP processing through a gain of function, whic

+To whom correspondence should be addressed. Tel: +33 2 32 88 81 82; Fax: +33 2 32 88 80 80; Email: frebourg@chu-rouen.fr

1264 Human Molecular Genetics, 1999, Vol. 8, No. 7

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remains to be elucidated. In order to characterize the functionof PSs, we have screened for proteins able to interact with PSs.

RESULTS

The small GTPase Rab11 interacts in yeast withhydrophilic loops of PSs

To identify proteins that interact with PSs, we have screenedan adult human brain cDNA expression library, using a two-hybrid system in yeast Y190 (40). The bait plasmid expresses,as a fusion protein, the mutant PS1 hydrophilic loop (codons263–407) carrying the Leu392Val substitution linked toEOAD in a large French family (2). The hydrophilic loop ofPS1, whose functional importance had been suggested by com-plementation performed inC.elegans, is orientated towards thecytoplasm, and is therefore more susceptible to interactingwith cytoplasmic proteins (41). We initially cloned the mutantPS1 loop into the pAS2-1 expression vector, but a toxic over-expression of the loop led us to subclone it into the pGBT9vector which contains anADH1 truncated promoter with alower level of expression. The pGBT9 (PS1LH392) construct,which did not lead by itself to the activation of the two reportergenesHIS3andLacZ, was used to screen ~600 000 transform-ants, and 23 clones were found to co-activate both reportergenes. cDNAs were isolated and retransformed into yeast toconfirm that these cDNAs encode proteins which interact withthe PS1 hydrophilic loop region. Sequencing analysis revealedthat one of these clones corresponds to theδ-catenin identified

by Zhou et al. (42). Two other clones correspond to the Cterminal half of Rab11, codons 100–216 and 106–216, resptively. These proteins did not interact with different irrelevanproteins including human lamin C, murine p53 (codons 7390) and GAL4 DNA-binding domain. Similarly, no interaction was detected between PS1 and the C-terminal half of Rand Rab6 (codons 106–216) (43). Partial Rab11 (codons 1216) was also found to interact, in the yeast two-hybrid systewith the wild-type PS1 and PS2 hydrophilic loops (Fig. 1).

Rab11 interacts with PSs in COS cells

To confirm the validity of the interaction of PSs with Rab11we performed immunoprecipitation–western blot experimenin COS cells. The partial Rab11 cDNA (codons 106–216derived from the library screening, and the full-length Rab1cDNA, derived from human immortalized lymphocytes, wecloned into the pCNW8 expression vector which confers anterminal myc tag, and the resulting plasmids were transfecwith vectors expressing full-length wild-type PS1 or PS2 (39Cell lysates were immunoprecipitated with a polyclonal anbody directed against either PS1 or PS2 (37), and immunopcipitates were analysed by western blot using an anti-mantibody (Fig. 2). Rab11 was clearly detected (Fig. 2B) in Pimmunoprecipitates only when both proteins were expresseCOS cells. No Rab11 was detected when immunoprecipitatwas performed with a pre-immune serum (data not shown)similar interaction, although weaker, was also observed in Pimmunoprecipitates with both partial and full-length Rab1(Fig. 2A).

Figure 1. Specific interaction of the hydrophilic loops of PSs with Rab11.Saccharomyces cerevisiaestrains harbouring the bait plasmids pGBT9 (PS1LH392pGBT9 (PS1LHWT) or pGBT9 (PS2LHWT) (see Materials and Methods) were transformed with pACT2-1 (Rab11106–216). (A) Individual Trp+Leu+ clones werestreaked onto SD-His-Trp-Leu plates and incubated for 8–10 days at 30°C. Positive control was pVA3-1/pTD1-1, and negative controls were pGBT9 + pACT2(Rab11106–216), pLAM 5'-1 + pACT2-1 (Rab11106–216), pGBT9 (PS1LH392) + pACT2-1 and pGBT9 (PS1LHWT) + pACT2-1. (B) His+Trp+Leu+ clones were filterassayed forβ-galactosidase activity.

Human Molecular Genetics, 1999, Vol. 8, No. 71265

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Identification of the binding domains on PS1 and Rab11

We generated a set of wild-type and mutant PS1 deletionmutants fused to the GAL4 DNA-binding domain and a set ofRab11 deletion mutants fused to the GAL4 activation domainin order to identify the binding domains on both proteins.Yeasts were co-transformed with different combinations ofplasmids and assayed for their ability to grow on medium with-out histidine and forβ-galactosidase expression. As shown inFigure 3, we found that the essential interaction domain of PS1was located between amino acids 374 and 400, corresponding

to the C-terminal end of the large hydrophilic loop. The essetial interaction domain of Rab11 was located between amiacids 106 and 179, which includes the fourth GTP-bindindomain.

DISCUSSION

Our results indicate that Rab11 interacts with presenilins as:two distinct cDNAs corresponding to the C-terminal half oRab11 were identified in a two-hybrid screen using mutaLeu392Val PS1 hydrophilic loop as a bait; (ii) Rab11 washown to interact not only with the hydrophilic loop of PS1but also with that of PS2, the second member of the PS famand (iii) the interaction was confirmed in cells transfected wiplasmids expressing the full-length PSs and Rab11 as welRab11106–216. The hypothesis of a gain of function led us to peform the screening with a mutant form of PS1. In yeast, wwere unable to detect any difference in the binding to Rabbetween the wild-type and mutant form of PS1, which does nexclude a subtle difference in the affinity of both proteins.transfected COS cells, the PS1–Rab11 interaction was mdifficult to detect than the PS2–Rab11 interaction (Fig. 2which might be explained by a difference in the quality of PSand PS2 antibodies. The alternative hypothesis is that the inaction between Rab11 and PS1 is either transient or of laffinity. It should be noted that the Rab11-binding domainPS1 (amino acids 374–400) corresponds to a region of the lohighly conserved between PS1 and PS2 (89% identity), whsupports the functional importance of this domain.

Rab11, which is composed of 216 amino acids with a prdicted molecular weight of ~25 kDa, belongs to the Rab sugroup of the Ras-related superfamily of small GTPases, whcomprises >30 members. Rab proteins have emerged as imtant regulatory components of the vesicular transport aorganelle dynamics in eucaryotic cells, each step of cellutrafficking involving a different set of Rab proteins (44,45)Rab proteins contain four consensus regions (G1–4) knownbe involved in GTP/GDP binding and, as is the case for othmembers of the Ras superfamily, Rab proteins undergo a futional cycle between a GDP-bound inactive and a GTP-bouactive conformation. Interestingly, the domain of Rab1involved in PS1 binding involves the G4 region (46), whicmight suggest that such an interaction might interfere with tGTP/GDP binding status of Rab11. Rab11 is ubiquitousexpressed and, at the subcellular level, has been localizedifferent cell types within different compartments includinthe TGN and TGN-derived vesicles (47), the pericentriolrecycling endosome (48) and apical tubulovesicle membra(49). At the present time, the function of Rab11 has not beelucitated fully. The association of Rab11 with transferrin-cotaining recycling compartments (50) suggests a role in the trficking and recycling of internalized proteins. Rab11 has albeen implicated in protein transport from the TGN througpost-Golgi vesicles to the plasma membrane (51,52). Thefore, considering that PSs have also been localized in the Gapparatus and perinuclear vesicles, the interaction thatreport between PSs and Rab11 might be relevantin vivo. Fur-thermore, recent studies have implicated several Rab protein trafficking and recycling of APP in specific cell compartments. It has been proposed that Rab1b protein might reguearly steps in exocytic transport and processing of APP (5

Figure 2. Western blot analysis ofin vitro interaction between PSs and Rab11in COS cells. Cells were transfected with PSs and/or myc-tagged Rab11expression plasmids and cell lysates were immunoprecipitated with PS1 (A) orPS2 (B) antibodies. Lysates (25µg) or immunoprecipitated lysates (500µg)were subjected to immunoblotting with anti-myc antibody. The position of themolecular weight marker (pre-stained SDS–PAGE standard; Bio-Rad,Hercules, CA) is indicated. Arrows indicate Rab11 and Rab11106–216positions.

1266 Human Molecular Genetics, 1999, Vol. 8, No. 7

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APP has been localized in Rab5-covered vesicles and, in spo-radic AD cases, an increased neuronal endocytosis and pro-tease delivery to Rab5-positive early endosomes, which mightbe a mechanism of increasedβ amyloidogenesis, has beenreported (54,55).

PS mutations linked to familial AD lead to an increased pro-duction of Aβ1-42/43 peptide. Although the biological rele-vance of the PS–Rab11 interaction that we describe in thisstudy remains to be confirmed, our results led us to speculatethat this altered processing of APP might be tightly linked to anabnormal vesicular routing since amyloidergic and non-amyloidergic cleavages occur in different cells compartments.

MATERIALS AND METHODS

Strains and plasmids

Saccharomyces cerevisiaestrain Y190, pAS2-1 and pGBT9expression vectors, containing the GAL4 DNA-bindingdomain, pACT2-1 containing the GAL4 activation domain andcontrol plasmids pVA3-1, pTD1-1 and pLAM 5'-1 were pur-

chased from Clontech (Palo Alto, CA). The coding regionPS1, corresponding to the hydrophilic loop (codons 263–40was PCR-amplified from lymphocyte cDNAs of an FAD ROfamily affected member harbouring the heterozygoLeu392Val mutation (2), using primers containing additionrestriction sites to facilitate cloning into pAS2-1. The resultinbait plasmids, containing the wild-type and mutant hydrophiloop sequence, were named pAS2-1 (PS1LHWT) and pAS(PS1LH392), respectively. The PS1 cDNAs derived frothese plasmids subsequently were cloned into pGBT9 to genate pGBT9 (PS1LHWT) and pGBT9 (PS1LH392). PS1 deltion mutant cDNAs were generated by PCR from theplasmids and subcloned into pGBT9. The region of the PcDNA corresponding to the hydrophilic loop (codons 269388) was PCR-amplified from normal lymphocyte cDNAs ancloned into pGBT9 to generate pGBT9 (PS2LHWT). Baplasmids were transformed into yeast Y190 by the lithiuacetate method (56) and plated onto SD-Trp plates. The flength Rab11 cDNA was PCR-amplified from lymphocytcDNAs and cloned into pBluescriptSK (+) to generate thpBSK (Rab11fl) plasmid. Rab11 deletion mutant cDNAs wer

Figure 3. (A) Identification of the PS1- and Rab11-binding domains. PS1 deletion mutants cloned into pGBT9 and Rab11 deletion mutants cloned into pwere transformed intoS.cerevisiaestrains harbouring the plasmids pACT2-1 (Rab11106–216) and pGBT9 (PS1LHWT), respectively. Y190 transformants weselected on SD-His-Trp-Leu plates, and His+ colonies were assayed forβ-galactosidase activity. The relative blue colour development was scored visually: +dark blue colonies; –, white colonies; +/–, appearance of faint blue. The shaded box corresponds to the fourth GTP-binding domain of Rab11 (G4). Identical resultswere obtained with pGBT9 (PS1LH392). (B) Amino acid sequence of human PS1 (GenBank accession no. 4050086) and Rab11 (GenBank accession no.The PS1 hydrophilic loop (amino acids 263–407) and the region corresponding to amino acids 106–216 of Rab11 are underlined. Dotted lines indicate thbinding domains of Rab11. Open boxes correspond to the PS1- and Rab11-binding domains (G1–4).

Human Molecular Genetics, 1999, Vol. 8, No. 71267

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generated by PCR from pBSK (Rab11fl) and cloned intopACT2-1. Canine Rab5 and human Rab6 cDNAs (codons106–216) were PCR-amplified from plex (Rab5) and plex(Rab6) expression vectors (43) and cloned into pACT2-1. Full-length and partial Rab11 cDNAs (codons 106–216) were PCR-amplified from pBSK (Rab11fl) and cloned into a modifiedversion of the mammalian pCDNA3 expression vector(Invitrogen, CH-Groningen, The Netherlands), pCNW8, con-taining an N-terminal tag corresponding to the myc epitope(codons 409–418). Full-length wild-type PS1 and PS2 cDNAswere cloned into pCDNA3 (39). All constructs were validatedby DNA sequencing using the PRISM AmpliTaqFS ReadyReaction Dye Terminators sequencing kit (Applied Biosys-tems, Perkin Elmer-Cetus, Foster City, CA) and an AppliedBiosystems model 373A automated sequencer.

Library screening

A single colony of Y190 harbouring the bait plasmid pGBT9(PS1LH392) was grown overnight in SD-Trp and transformedwith a human adult brain Matchmaker expression librarycloned in pACT2-1 (Clontech). A total of 600 000 independenttransformants were plated on 22.5× 22.5 cm plates containingSD-His-Trp-Leu medium with 40 mM 3-amino-1,2,4-triazole(Sigma, St Louis, MO) to overcome leakyHIS3 reporter geneexpression. After incubation at 30°C for 8–10 days, His+

clones were assayed forβ-galactosidase activity by filter assay.Replica filters were frozen in liquid nitrogen for 20 s andthawed to room temperature to permeabilize the cells. Filterswere then incubated in an X-gal solution at 30°C for a maxi-mum of 8 h. LacZ+ clones were streaked onto SD-His-Trp-Leumedium and re-assayed to confirm the His+LacZ+ phenotype.Plasmid DNA was extracted, as previously described (56), frompositive clones grown overnight in 2 ml of SD-Leu, and was usedto transform C600 competent bacteria (Clontech), which werethen plated on M9-Leu medium containing 50µg/ml ampicillin.Single colonies were incubated in 2 ml of LB medium, andplasmid DNA was extracted using an RPM kit (BIO101, LaJolla, CA). Inserts were sequenced as indicated above andDNA searches were performed using the BLAST 2.0 programat the NCBI server (http://www.ncbi.nlm.nih.gov/cgi-bin/BLAST/nph-newblast?Jform=1 ).

Cell culture, transfection and immunoprecipitation

Rab11 expression vectors (4µg) were transfected with PSexpression vectors or empty pCDNA3 (4µg) using lipo-fectamine (Boehringer Mannheim, Mannheim, Germany),according to the manufacturer’s instructions, into COS cells at70% confluence in 10 cm plates. Two days after transfection,cells were lysed in 800µl of 50 mM Tris, pH 7.5, 150 mMNaCl, 2 mM EDTA, 1% NP-40, 10% glycerol, in the presenceof a protease inhibitor cocktail (Complete; Boehringer Man-nheim). To solubilize membrane proteins, cell lysates weresonicated and gently agitated overnight at 4°C. Aftercentrifugation for 20 min at 15 000g, supernatants were recov-ered as detergent-soluble fractions. The detergent-solublelysates (150–500µg) were incubated for 1 h at room tempera-ture in 400µl of 150 mM NaCl, 50 mM Tris, pH 7.5, 0.5%deoxycholic acid and 1% NP-40 with protein A–Sepharose(Amersham Pharmacia Biotech, Little Chalfont, UK), andimmunoprecipitations were performed using polyclonal

antibodies B1-03 or 95-041 (37) raised against PS1 and Ppeptides, respectively. Proteins were separated on a 2.8 abis-acrylamide 8–16% Tris–glycine gel (Novex, San DiegCA) and transferred to poly(vinylidene difluoride) membraneWestern blot analysis was performed with anti-myc antibousing an ECL detection kit (Amersham Pharmacia Biotecaccording to the manufacturer’s instructions.

ACKNOWLEDGEMENTS

B1-03 antibody was a generous gift from Dr L. Bué(INSERM U422, Vieillissement Cérébral et DégénerescenNeuronale, Lille, France). This work was supported by a grafrom Le Conseil Régional de Haute Normandie (to C.D.).

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