Endoplasmic Reticulum Tubule Protein Reticulon 4 Associates withthe Legionella pneumophila Vacuole and with Translocated SubstrateCeg9

Eva Haenssler,a,b* Vinay Ramabhadran,a,b Connor S. Murphy,a,b Matthew I. Heidtman,b* Ralph R. Isberga,b

Howard Hughes Medical Institutea and Department of Molecular Biology and Microbiology,b Tufts University School of Medicine, Boston, Massachusetts, USA

Intracellular growth of Legionella pneumophila occurs in a replication vacuole constructed by host proteins that regulate vesicu-lar traffic from the host endoplasmic reticulum (ER). This process is promoted by a combination of approximately 300 Icm/Dottranslocated substrates (IDTS). One of these proteins, Ceg9, was previously identified in a screen for L. pneumophila IDTS thatmanipulate secretory traffic when overexpressed in yeast. Using ectopic expression of Ceg9 in mammalian cells, we demonstratethat Ceg9 interacts with isoforms of host reticulon 4 (Rtn4), a protein that regulates ER tubule formation. Binding occurs underconditions that prevent association with other known reticulon binding proteins, arguing that Ceg9 binding is stable. A tripar-tite complex was demonstrated among Rtn4, Ceg9, and atlastin 1, a previously characterized reticulon interacting partner. Thebinding of Ceg9 to Rtn4 was not due to bridging by atlastin 1 but resulted from the two interacting partners binding indepen-dently to reticulon. When Ceg9 is ectopically expressed in mammalian cells, it shows a localization pattern that is indistinguish-able from that of Rtn4, perhaps due to interactions between and similar structural features of the two proteins. Consistent withRtn4 playing a role in the formation of the Legionella-containing vacuole, it was recruited to almost 50% of the vacuoles within20 min postinfection. Our studies suggest that L. pneumophila proteins interact with ER tubules at an early stage of replicationvacuole formation.

Intravacuolar growth of intracellular pathogens involves inti-mate interaction with specific subsets of host cell membrane

compartments (1). In the case of Chlamydia trachomatis, the as-sociation of the replication vacuole with the trans-Golgi and theendoplasmic reticulum (ER) network is a key step during the es-tablishment of an intracellular niche (2–5). Similarly, vacuolesharboring Brucella, Salmonella, and Mycobacterium species alsointerface with defined membrane compartments that aid thegrowth of these bacteria in host cells (6–10). Failure to establishassociation with the appropriate organelles can have dire conse-quences, including loss of vacuolar membrane integrity, activa-tion of inflammatory host cell death, and trafficking of the micro-organism into degradative compartments (3, 6, 11, 12).

Legionella pneumophila is one such intravacuolar pathogenthat targets specific intracellular compartments during its replica-tion cycle (13–16). The bacterium is a pathogen of amoebae andhumans. In humans, L. pneumophila’s primary manifestation ofvirulence is Legionnaires’ disease, a potentially lethal pneumoniaresulting from growth of the bacteria within alveolar macrophages(17, 18). Biogenesis of the microbial replication vacuole is evolu-tionarily conserved from amoebae to eukaryotic cells (16, 19),with the bacterium growing in an ER-encompassed compartmentcalled the Legionella-containing vacuole (LCV) (13, 15, 20). Theconnection between the LCV and ER dynamics is well established,since the proper function of Sar1 and Arf1, host proteins involvedin budding and vesicular movement from the ER, is required foroptimal replication vacuole formation (13, 21, 22). It has beenargued that the LCV is localized near ER exit sites, ideally suited tohijack vesicles trafficking on the anterograde secretory pathway(13). Further tying intracellular growth to ER dynamics, many ofthe Icm/Dot translocated substrates (IDTS) have biochemical ac-tivities that control the function of the host Rab1 protein, which

controls membrane docking and fusion events of vesicles that exitfrom the ER (23–32).

Intracellular growth of L. pneumophila requires the function ofthe Icm/Dot type IV secretion system apparatus, which recognizestranslocation signals found on at least 300 different bacterial pro-teins (33–37). In the absence of this secretion system, bacteria failto localize in an ER-encompassed vacuole and are instead foundassociated with marker proteins derived from late endosomal orlysosomal compartments (14, 38). Deletions in only a few of thegenes for the 300 Icm/Dot translocated substrates cause partialdefects in intracellular growth, but loss of no single such generecapitulates the strong trafficking defects that result from loss ofthe Icm/Dot system (39). Strategies have been used to identifymultigene knockout strains that cause defective growth, but thesestrains seem to mimic single mutants that cause the breakdown of

Received 20 April 2015 Returned for modification 21 May 2015Accepted 12 June 2015

Accepted manuscript posted online 22 June 2015

Citation Haenssler E, Ramabhadran V, Murphy CS, Heidtman MI, Isberg RR. 2015.Endoplasmic reticulum tubule protein reticulon 4 associates with the Legionellapneumophila vacuole and with translocated substrate Ceg9. Infect Immun83:3479 –3489. doi:10.1128/IAI.00507-15.

Editor: C. R. Roy

Address correspondence to Ralph R. Isberg, ralph.isberg@tufts.edu.

* Present address: Eva Haenssler, Qiagen GmbH, Hilden, Germany; Matthew I.Heidtman, Glycosyn LLC, Medford, Massachusetts, USA.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00507-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00507-15

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an established replication vacuole, indicating that they have de-fects in the maintenance of vacuole integrity (40).

The ER is subdivided broadly into two distinct domains: peri-nuclear sheets that are rich in ribosome-studded rough ER andperipherally localized tubular structures that are involved inmaintaining direct contact of the ER with the host cell plasmamembrane (41–43). The morphology of these regions is main-tained by the microtubule network, as well as by ER-associatedproteins such as Climp-63, Dp1, reticulon family members, andreticulon-like proteins, which are evolutionarily conserved fromyeast to humans (44–48). The sheet structure is collaborativelymaintained by Climp-63 and the reticulons, while the peripheraltubules require tubulin with support from the reticulons (49). Thereticulon (Rtn) family members include a number of genes andsplice variants, with most mammalian cells having some combi-nation of the splice variants of Rtn4/Nogo (referred to as Rtn4a/Nogo-A, Rtn4b/Nogo-B1, and Rtn 4d/Nogo-B2) (48). The factthat tubules are found in the peripheral areas of cells, which are theprimary regions of phagocytosis, indicates that the reticulon-richtubular ER is likely to be the initial site of contact with pathogens.In fact, reticulon has been shown to be recruited about the repli-cation vacuole during replication of the plant brome mosaic virus(50).

In a previous study, we screened a yeast expression bank har-boring 130 known and putative L. pneumophila IDTS for proteinsthat delay the export of well-characterized secreted proteins car-boxypeptidase Y and alkaline phosphatase in Saccharomycescerevisiae (51). Such a delay in secretion is presumably a conse-quence of direct misregulation of host proteins involved in eventsassociated with the early secretory traffic and ER biogenesis (52),the likely site of membrane material that is trafficked to the Legio-nella replication vacuole during intracellular growth (13). Amongthe IDTS identified that interfere with yeast protein secretion isCeg9, a 241-amino-acid protein that colocalizes with the ER whenexpressed in HeLa cells (51). In the present study, we show thatCeg9 associates with mammalian Rtn4 isoforms that are recruitedto the L. pneumophila vacuole shortly after bacterial contact withhost cells, consistent with Rtn4 playing a key role during the earlystages of bacterial infection.

MATERIALS AND METHODSBacterial strains and media. The L. pneumophila strains used in the pres-ent study were derived from the clinical isolate Philadelphia-1 (38). TheLp02 [thyA �(hsdR-lvh) rpsL], Lp03 (dotA3), and Lp02�ceg9 strains werecultivated on charcoal-N-(2-acetamido)-2-aminoethanesulfonic acid(ACES)–yeast extract–thymidine (CYET) plates and in ACES-yeast ex-tract-thymidine (AYET) liquid medium (51, 53, 54). For the constructionof the ceg9 deletion strain Lp02�ceg9, a derivative of the suicide vectorpSR47S (55) was introduced into Lp02 by electroporation (54), and thedeletion strain was constructed as described previously (56, 57). The in-frame deletion of ceg9 was confirmed using the primers Ceg9ups andCeg9downs (see below). For the infection of mammalian cells, L. pneu-mophila strains were grown overnight to post-exponential phase to a mo-tile state as described previously (53, 58).

Primers and plasmids. The plasmid encoding hemagglutinin (HA)-tagged human atlastin-1 (ATL1) pGW1-ATL1 was kindly provided by C.Blackstone (46), and the plasmid Rtn4/NogoA-Myc encoding humanRtn4 with a C-terminal myc tag was kindly provided by S. Strittmatter(59) and T. Rapoport (Harvard Medical School, Boston, MA).

The full-length open reading frame of lpg0246 (ceg9) was cloned intothe mammalian expression vector 3�FLAG-CMV7.1 (Sigma-Aldrich)using the primers MH325 (AAA CGC GGA TCC ATG TAT AAA AAT

ATA CTG TTT) and MH326 (AAA CGC GGA TCC TTA AAT GGA CATACG AAT TAG) (restriction sites are underlined). For the plasmid3�FLAG-Ceg9121–241 encoding Ceg9 lacking the transmembrane do-main, the primers Ceg9141f (GCG CGC GAA TTC AAC GGT TAA ATGCCT GGT TGA G) and Ceg9rnew (GCG CGC GGA TCC TTA AAT GGACAT ACG AAT TAG) were used. The EGFP-Ceg9 fusion has been de-scribed previously (51), and for the construction of enhanced green fluo-rescent protein (EGFP) fusions with Ceg9 truncations, i.e., pEGFP-Ceg961–241, pEGFP-Ceg9121–241, and pEGFP-Ceg91–120, the open readingframes were subcloned with EcoRI and BamHI from 3�FLAG constructsinto the pEGFP-C1 backbone (Clontech). 3�FLAG-Ceg961–241 was con-structed with Ceg91hf (GCG CGC GAA TTC ATT TTC GAT GCT AGTCGC GGG) and Ceg9rnew (GCG CGC GGA TCC TTA AAT GGA CATACG AAT TAG). For 3�FLAG-Ceg91–120, the primers Ceg9N_fwd (GCGCGC GAA TTC ATA TAA AAA TAT ACT GTT TTT TTC AAA TTTTAG) and Ceg9N_rev141 (GCG CGC GGA TCC TTA TGC AAT ACATCG GCC TAC AGT ACT) were used. ceg19 (lpg1121) was cloned into3�FLAG-CMV7.1 (Sigma-Aldrich) using the primers MH323 (AAACGC GGA TCC CTG TTT AAA GAA ATT ATC ACT) and MH324 (AAACGC GGA TCC TTA AAT TAT ATT CAC ATT ATC). Ceg9 1-60 wascloned into eGFP-C1 using the primers RI35 (GAT CCT CGA GAT ATGTAT AAA AAT ATA CTG TTT TTT TCA AAT TTT AG) and RI36 (GATCGA ATT CAG AAG CAG TAA CTG GCT GGA AT). Human Rtn4b wascloned into eGFP-N1 using the primers MinisogF (GCG GCG GAA TTCATG GAA GAC CTG GAC CAG TCT C) and MinisogR (GCG GCG GGTACC CAT TCA GCT TTG CGC TTC AAT C).

The suicide vector for constructing the Lp02�ceg9 strain was gener-ated with the primers MH417 (GGA CGC GTC GAC TCG CAT CAA TGTTTA TTT GAA TTT), MH424 (TGA TTT TTA AAC TAA TCT AAA TTAAAC AGG TGC TTC GTC TTT TGA AAG), MH420 (AAG AAG GAGCTC AGA GCA TCA GCA AAG TAT TTT TTA), GC-MH424 (CTT TCAAAA GAC GAA GCA CCT GTT TAA TTT AGA TTA GTT TAA AAATCA), Ceg9ups (GCG CGC GGA TCC TCG CAT CAA TGT TTA TTTGAA TTT), and Ceg9downs (GCG CGC GGA TCC AGA GCA TCA GCAAAG TAT TTT TTA) using a pCR Blunt II TOPO kit (Invitrogen).

Mammalian cell culture, transfection, and subcellular localizationstudies. Bone marrow-derived macrophages were isolated from the fe-murs of A/J mice as previously described (15, 60, 61) and frozen in fetalbovine serum (FBS) with 10% dimethyl sulfoxide. Cultivation was per-formed in RPMI 1640 (Gibco) supplemented with 10% FBS and 2 mMglutamine at 37°C with 5% CO2. HEK293T and Cos7 cells were cultivatedin Dulbecco modified Eagle medium (Gibco) supplemented with 10%FBS (Gibco) at 37°C with 5% CO2.

For subcellular localization studies using immunofluorescence, 2 �104 Cos7 cells were seeded on glass coverslips in 24-well dishes, and on thenext day 0.5 �g of plasmids encoding EGFP-Ceg9 fusions and 0.2 �g ofRtn4a-myc (or Rtn4b-GFP) were transfected using 0.5 �l of Lipo-fectamine 2000 (Invitrogen). After 24 h, the cells were fixed with 4%paraformaldehyde for 10 min at room temperature and permeabilizedwith 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 10 min atroom temperature. After blocking in 4% goat serum (Gibco) in PBS for atleast 15 min, the following antibodies were used for detection: mouseanti-myc antibody (1:500; Santa Cruz Biotechnology, Inc.) and TexasRed-conjugated anti-mouse antibody (1:500; Invitrogen).

Immunoprecipitation and Western blotting. For transfection pre-ceding immunoprecipitation, HEK293T cells were plated at a density of5 � 105 to 6 � 105 cells per well in a tissue culture-treated six-well dish.For single transfections 2 �g of plasmid DNA and for double transfections1 �g of each plasmid were used with 2.5 �l of Lipofectamine 2000.HEK293T cells were harvested 24 h after transfection by removing mediaand washing, followed by resuspension in lysis buffer and centrifugation.Cell lysis in 1% Triton X-100 (20 mM Tris [pH 7], 150 mM NaCl, 5 mMMgCl2) and Complete protease inhibitor cocktail (Roche) was carried outfor 20 min at 4°C, and cell debris was removed by centrifugation at�14,000 � g for 10 min at 4°C. When using the combination of HKM lysis

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buffer, which contained 1% digitonin in 25 mM HEPES, 150 mM potas-sium acetate, and 2 mM magnesium acetate (pH 7.4), and Complete pro-tease inhibitor cocktail (Roche), the incubation time was extended to 1 hat 4°C as described previously (62). For lysis using 0.5% CHAPS {3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate} in PBS, in-cubation was carried out for 30 min. Cleared lysates were transferred to afresh microcentrifuge tube, diluted with an equal volume of detergent-free buffer, and mixed with 50 to 70 �l per sample of washed resin to allowbinding at 4°C under end over end rotation 4 h to overnight. For immu-noprecipitations, anti-FLAG M2 affinity gel (Sigma-Aldrich) and EZviewRed Anti-HA affinity gel (Sigma-Aldrich) were used. Washes of boundresin were performed four or five times in cold HKM buffer, phosphatebuffer, or buffer containing 0.1% Triton X-100 in 20 mM Tris (pH 7), 150mM NaCl, and 5 mM MgCl2. For elution, samples were boiled in samplebuffer without reducing reagents and loaded onto sodium dodecyl sulfate(SDS)-polyacrylamide gels for Western blot analyses.

For immunoprecipitations using an anti-Rtn4 antibody coupled toprotein A-Sepharose beads, transfected HEK293T cells were harvestedafter 16 to 24 h, solubilized for 10 min at 4°C in buffer containing 1%Triton X-100, and centrifuged at �14,000 � g for 10 min at 4°C. Thesupernatant was transferred to a fresh microcentrifuge tube, diluted withan equal volume of detergent-free buffer, and incubated with 1 �g of goatanti-Rtn4 antibody (Santa Cruz) and 35 �l of 20% protein A-Sepharosebeads for 3 h. Bound resin was washed five times with buffer containing0.1% Triton X-100, and samples were eluted by boiling in sample buffer.

The following antibodies were used in subsequent immunoblot exper-iments: rabbit anti-Reticulon 4 antibody (1:5,000; Life Span Biosciences),mouse anti-FLAG antibody (1:5,000; Sigma-Aldrich), rabbit anti-FLAGantibody (1:1,000 –1:2,500; Sigma-Aldrich), rabbit anti-RhoGDI anti-body (1:2,000 –1:5,000; Santa Cruz Biotechnology, Inc.), anti-S6 antibody(1:1,000; Cell Signaling), and rabbit anti-HA antibody (1:1,000; SantaCruz Biotechnology, Inc.). ECL Plus Western blotting reagent (Pierce)was used for detection.

Mass spectrometry and protein identification. 3�FLAG-Ceg9 andvector control immunoprecipitates were subjected to SDS–10% PAGEand silver staining (Invitrogen). Proteins of interest were excised from thegel and analyzed at Boston Children’s Hospital by H. Steen.

Intracellular growth and Rtn4 localization assays. To determine theintracellular growth of L. pneumophila strains and Rtn4 localization dur-ing infection, 105 bone marrow-derived macrophages isolated from fe-murs of A/J mice were seeded per well on glass coverslips in a 24-well dishin RPMI 1640 supplemented with 10% FBS, 2 mM glutamine, and 200 �gof thymidine/ml. Infections were carried out at a multiplicity of infection(MOI) of 1 with L. pneumophila cultures in post-exponential phase. At 1and 8 h postinfection the cells were washed and fixed in 4% paraformal-dehyde in PBS for 10 min at room temperature. The cells were permeab-ilized using 0.1% Triton X-100 and stained with rat anti-Legionella serum(1:10,000) and Hoechst DNA dye (1:10,000; Invitrogen) after blocking forat least 30 min. Bacterial replication was quantified by determining thenumber of host cells that harbored a certain number of bacteria perphagosome on three coverslips per data point. For Rtn4 localization stud-ies during infection, the cells were fixed and blocked at the time pointsindicated. Staining was performed with rat anti-Legionella serum (1:10,000), Hoechst DNA dye (1:10,000; Invitrogen), rabbit anti-Rtn4 anti-body (1:250; Life Span Biosciences), Texas Red-conjugated anti-mouseantibody (1:500; Invitrogen), and fluorescein isothiocyanate-conjugatedanti-rabbit antibody (1:500; Jackson ImmunoResearch Laboratories).The number of Rtn4-positive vacuoles per strain and time point was de-termined by counting 100 infected macrophages on each of three cover-slips and on each of at least two coverslips for the 40-min time point.Representative images were taken using Zeiss and Nikon fluorescencemicroscopes, and contrast and brightness were adjusted afterward in Pho-toshop (Adobe).

RESULTS

To identify host proteins that are targeted by Ceg9, a plasmid thatplaced 3�FLAG-Ceg9 under cytomegalovirus (CMV) promotercontrol was transfected into HEK293T cells (see Materials andMethods). At 24 h after transfection, extracts were made, andproteins associated with 3�FLAG-Ceg9 were pulled down by im-munoprecipitation with anti-FLAG beads. As a control, extractsfrom cells transfected with the empty 3�FLAG vector were sub-jected to the identical immunoprecipitation strategy. Silver stain-ing of the fractionated samples revealed several bands present inimmunoprecipitates from the 3�FLAG-Ceg9-transfected cellsthat were absent in samples prepared from the empty vector con-trol (Fig. 1A). Of these unique proteins, one comigrated with3�FLAG-Ceg9 on Western blotting, and one was an apparentdegradation product, while the others were larger in apparent mo-lecular weight and were submitted for identification by mass spec-troscopy. Two of the bands generated peptides corresponding tothe Rtn4b and Rtn4d isoforms of the reticulon family (373 and 392amino acids [aa], respectively), while the other proteins identi-fied were the mitochondrial inner membrane proton pumpingATPase and the cytoplasmic AAA ATPase Cdc48/p97 (Fig. 1A).

We first determined the efficiency of Ceg9 association withreticulon family members and with p97. Extracts from HEK293Ttransfectants harboring either the 3�FLAG-Ceg9 plasmid or theempty vector control were immunoprecipitated with anti-FLAGbeads, and precipitates were analyzed by Western blotting withanti-FLAG, anti-Rtn4, or anti-RhoGDI antibody as a negativecontrol. The anti-Rtn4 antibody recognizes three isoforms of thisprotein, but only the 45- to 50-kDa isoforms (Rtn4b and Rtn4d),which are often poorly resolved from each other on SDS gels, areexpressed in this cell line. The procedure efficiently precipitated3�FLAG-Ceg9 and ca. 7.5% of the total cellular Rtn4b/4d foundassociated with the Ceg9-bound beads (Fig. 1B, 3�FLAG-Ceg9).In contrast, no RhoGDI was found to be bead associated, andthere was no precipitation of either 3�FLAG-Ceg9 or Rtn4b/dafter the exposure of beads to extracts from cells transfected withthe empty vector control (Fig. 1B, vector).

To support the model that Rtn4b/d isoforms bind Ceg9, thereverse immunoprecipitation experiment was performed, inwhich extract from cells transfected with 3�FLAG-Ceg9 was ex-posed to anti-Rtn4, and immune complexes were precipitatedwith protein A-Sepharose beads. In this case, the procedure pre-cipitated ca. 7% of the pool of the target Rtn4b and Rtn4d(Rtn4b/d) proteins in the cells, based on comparing the bound tothe unbound fraction of Rtn4 (Fig. 1C, 3�FLAG-Ceg9 and�-Rtn4). Even so, co-association of 3�FLAG-Ceg9 could be ob-served. In contrast, no co-association of another 3�FLAG-taggedIDTS, Ceg19, could be detected in the anti-Rtn4 precipitates (Fig.1C). In addition, binding of Ceg9 was not limited to the Rtn4b/disoforms. Cells cotransfected with the myc-Rtn4a isoform and3�FLAG-Ceg9, followed by extraction in Triton X-100, showedcoprecipitation of Ceg9 and Rtn4a on beads coated with anti-mycantibody, indicating that Ceg9 interacted with multiple Rtn4 iso-forms (see Fig. S1 in the supplemental material).

To determine whether there was specific association of Cdc48/p97 with Ceg9, we repeated the immunoprecipitation analysis ofextract from 3�FLAG-Ceg9 transfectants with anti-FLAG beads,probing Western blots with anti-p97 (Fig. 1D; analysis of ATP5A,below; see Fig. 2). Immunoprecipitates from the vector control,

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3�FLAG-Ceg19 and 3�FLAG-Ceg9, showed equivalent amountsof a band that did not comigrate with p97 (Fig. 1D, IP lanes). Incontrast, there was no precipitation of another control protein,ribosomal protein S6. Therefore, there was no evidence for spe-cific p97 association with Ceg9.

The soluble carboxyl-terminal region of Ceg9 is not suffi-cient for Rtn4 binding. Members of the reticulon family have tworegions of high hydrophobicity that are thought to form a pair ofhairpins that promote membrane curvature in the ER membrane(Fig. 2A) (44). The predicted amino acid sequence of Ceg9 has asimilar arrangement of hydrophobic residues, with two 23-resi-due hydrophobic regions that are sufficiently large to span mem-brane bilayers, separated by 15 aa (Fig. 2A). To determine whetherthere are specific domains involved in either promoting associa-tion with Rtn4 isoforms or localizing Ceg9 to the ER, EGFP-Ceg9deletion derivatives were constructed (Fig. 2A). To determine theefficiency of binding, each of the derivatives was expressed inHEK293T cells and immunoprecipitated with anti-GFP from de-tergent-solubilized extracts (Fig. 2B). Although the full-lengthEGFP-Ceg9 bound efficiently to Rtn4b/d, there was little associa-tion of a deletion derivative (aa 121 to 241) that retained the car-boxyl-terminal hydrophilic region but was missing the amino ter-minus and the hydrophobic regions (Fig. 2B). Control GFPfusions that have hydrophobic regions, such as the IDTS fusionGFP-Lpg0107 or the model ER protein ER-GFP (63), did notshow Rtn4b/d association, arguing that the mere presence of hy-drophobic domains was not sufficient to allow binding of proteinsto Rtn4 (Fig. 2B, right panel). Derivatives that retained the Ceg9hydrophobic regions but were missing either the carboxyl- oramino-terminal regions were poorly expressed, making it difficultto evaluate whether membrane targeting was sufficient for inter-action with Rtn4b/d.

The efficiency of interaction with the ATP5A subunit of themitochondrial proton pump was also tested, because peptidescorresponding to this protein were detected in the initial massspectrometry analysis (Fig. 1A). There was little or no coprecipi-tation of ATP5A subunit with any GFP-Ceg9 derivative, furtherindicating that Ceg9 shows specific interaction with Rtn4 isoforms(Fig. 2B).

FIG 1 Ceg9 (lpg0246) binds Rtn4b/d when expressed in human cells. (A)Identification of proteins that coimmunoprecipitate with 3�FLAG-Ceg9.HEK293T cells transfected with 3�FLAG-Ceg9 were extracted in TritonX-100 and immunoprecipitation was performed using anti-FLAG resin (seeMaterials and Methods). After separation by SDS-PAGE and visualization bysilver staining, candidate proteins were excised and analyzed by matrix-as-

sisted laser desorption ionization–time of flight mass spectrometry. Vector,elution fraction of immunoprecipitates from extracts of cells harboring the3�FLAG empty vector only; Ceg9, elution fraction of immunoprecipitatesfrom extracts of cells harboring the 3�FLAG-Ceg9 vector. (B) Efficient coim-munoprecipitation of Rtn4 isoform with 3�FLAG-Ceg9. HEK293T cells weretransfected with the 3�FLAG vector control and 3�FLAG-tagged Ceg9, ex-tracted, and subjected to anti-FLAG immunoprecipitation, followed by West-ern blotting with the denoted antibodies. Lanes: Input, 0.87% of the startingmaterial; Un., 0.91% of the unbound supernatant; IP, 12.5% of the immuno-precipitation fractions eluted by boiling in 2� SDS sample buffer (Materialsand Methods). (C) Identification of Ceg9 as an Rtn4-associated protein. Celllysates from HEK293T cells harboring the 3�FLAG vector, as well as3�FLAG-Ceg19 and 3�FLAG-Ceg9, were subjected to anti-Rtn4 immuno-precipitation, and associated proteins were identified by Western blot analysiswith anti-FLAG and anti-Rtn4 antibodies. Lanes: Input, 2% of the startingmaterial; Un., the remaining material after the immunoprecipitation; IP,about 25% of the total immunoprecipitates. (D) Ceg9 does not pull down p97.HEK293T cells were transfected with the 3�FLAG vector control, 3�FLAG-tagged Ceg9, and 3�FLAG-Ceg19, extracted, and subjected to anti-FLAG im-munoprecipitation, followed by Western blotting with the denoted antibodies.Although the 3�FLAG-Ceg9 and Rtn4 isoforms interact, binding of3�FLAG-Ceg9 to p97 could not be confirmed.

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Ceg9 derivatives retaining putative transmembrane regionsform tubular structures associated with the ER. To analyze theconnection between the hydrophobic regions of Ceg9 and its in-tracellular localization, the EGFP-tagged deletion derivatives (Fig.2A) were transfected into the Cos7 cell line and evaluated by flu-orescence microscopy (Fig. 3). Cos7 cells were chosen for micros-copy studies due to their size and highly spread ER morphology.The full-length GFP-Ceg9 showed clear tubular morphology inthe periphery of transfected cells that exhibited either completeoverlap or interdigitation with immunofluorescent staining ofRtn4, a finding consistent with the fusion being localized in theER. Derivatives limited to the amino-terminal region lacking thehydrophobic stretches (aa 1 to 60 [aa1-60]) or the carboxyl-ter-minal region downstream of these stretches (aa 121 to 241)showed diffuse fluorescence throughout the cell and failed to co-localize with Rtn4. Consistent with the low expression levels, Ceg91-120 and Ceg9 61-241 showed much lower levels of transfection(ca. 10%) than full-length Ceg9. Even so, there were distinct lo-calization patterns for each one of these derivatives in the fewtransfectants that could be detected. Derivatives that lacked eitherthe carboxyl-terminal hydrophilic domain of the protein (aa1-120) or the amino terminus (aa61-241), but which still retainedthe pair of hydrophobic stretches, localized to the ER and showedcolocalization with Rtn4 (Fig. 3). It is worth noting that the Ceg961-241 fusion had partial cytoplasmic localization apart from lo-

calizing to the ER, whereas the localization of Ceg9 1-120 wasindistinguishable from that of the full-length fusion. Although farfewer cells showed detectable transfection of the derivatives (aa1-60, aa1-120, and aa161-241) than of full-length Ceg9, the cells thatwere successfully transfected showed levels of expression that weresimilar to those of the wild-type transfectants. Therefore, theseresults are consistent with the hydrophobic regions being neces-sary for Ceg9 colocalization with Rtn4 and may be sufficient forthis localization pattern as well.

Ceg9-Rtn4 binding persists under conditions that destabi-lize other Rtn4 binding partners. Homotypic fusion of ER tu-bules is regulated by the dynamin-like GTPase atlastin, which di-rectly binds to reticulon family members in gentle detergents (62).This indicates that Ceg9 interaction with Rtn4 isoforms could beindirect, as a result of Ceg9 binding atlastin. Arguing against indi-rect interaction, however, is the fact that we could detect Rtn4-Ceg9 binding in the presence of buffers containing Triton X-100,which is known to disrupt Rtn binding to all known protein part-ners (62). To test the possibility that Ceg9 could directly bindatlastin, HEK293T cells were cotransfected with an HA-atlastinplasmid and either the 3�FLAG-Ceg9 construction or a vectorcontrol. Cells were then extracted in Triton X-100 or digitonin,and immunoprecipitation analysis was performed with anti-HAantibody (Fig. 4A and C) or anti-FLAG antibody (Fig. 4B). WhenTriton X-100 extracts were incubated with anti-HA beads, immu-

FIG 2 Association of endogenous Rtn4b and 3�FLAG-tagged Ceg9 depends on the predicted hydrophobic region of Ceg9. (A) Schematic of Ceg9 and Rtn4b/d.Ceg9 is a 241-aa protein with two predicted transmembrane helices spanning approximately aa 57 to 79 and aa 94 to 116 (TMHMM Server v2.0). TruncatedEGFP-tagged derivatives of Ceg9 used in immunoprecipitations and localization studies are displayed below. The reticulon 4 isoforms Rtn4b and Rtn4d have twolong hydrophobic regions (gray boxes) hypothesized to form bent helical structures in membranes (65). Rtn4d splice variant has a 19-aa insertion displayed(white box). (B) Lysates from HEK293T cells transfected with the EGFP fusion vectors displayed were subjected to immunoprecipitations with anti-GFP resin(see Materials and Methods). This was followed by immunoblot analysis with anti-GFP, anti-Rtn4, anti-ATP5A, and anti-RhoGDI antibodies. Lanes: Input, 1.6%of the starting material was loaded; Un., 1.6% of the unbound supernatant was loaded; IP, 12.5% of the elution fraction obtained by boiling in SDS sample buffer.

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noprecipitation of HA-atlastin was quantitative, but there was noevidence of coprecipitation of either 3�FLAG-Ceg9 (Fig. 4A,compare �-HA to �-FLAG blots) or Rtn4. Consistent with thislack of association, anti-FLAG immunoprecipitation of 3�FLAG-Ceg9 failed to coprecipitate HA-atlastin in the presence of TritonX-100 (Fig. 4B). Therefore, under conditions that maintain Ceg9-Rtn4 binding (Fig. 1), atlastin does not associate with Ceg9. Fur-thermore, atlastin does not bind to its known binding partnerRtn4 under these conditions, arguing that Ceg9 binding to Rtn4 isnot via an atlastin-mediated bridge.

Although the extraction conditions used are known to disruptoligomerization of Rtn4 and its binding to atlastin, higher-orderRtn4 interactions can be maintained using gentler conditions,with either digitonin or CHAPS as a solubilizing agent (47, 62, 64).Since the Rtn4-Ceg9 interactions occur in the absence of atlastinbinding, we sought to determine whether binding of Ceg9 and

atlastin to Rtn4 was mutually exclusive or whether the three pro-teins could be present in a co-complex. To test this possibility,extracts from the cotransfectants harboring HA-atlastin and3�FLAG-Ceg9 were made in digitonin-containing buffer, whichmaintains atlastin-Rtn4 binding (Fig. 4C) (62). Immunoprecipi-tation of HA–atlastin-1 under this condition resulted in copre-cipitation of ca. 10% of the total pool of transfected 3 � FLAG-Ceg9, indicating a co-complex between these proteins (Fig. 4C,�-FLAG probe). This association was likely to be indirect, sincethere was efficient coprecipitation of endogenous Rtn4b/d withthe anti-HA(atlastin) beads (Fig. 4C, �-Rtn4 blot). Therefore, inthe presence of digitonin (62) there is potential for formation of atripartite complex, in which atlastin-1 binds to Rtn4b/d, resultingin coprecipitation of the Rtn4-bound 3�FLAG-Ceg9. This resultcontrasts with the simple binary Rtn4b/d-Ceg9 binding observedin Triton X-100.

FIG 3 Localization of ectopically expressed EGFP-Ceg9 fusions to an ER-like compartment in Cos7 cells correlates with the presence of the transmembranehelices. The EGFP truncation derivatives described in Fig. 2 were transfected into Cos7 cells, incubated for 24 h, fixed, and probed with anti-Rtn4 to determinethe subcellular localization of EGFP-Ceg9. Insets show higher-magnification regions of interest from each image. Panels: left, EGFP-Ceg9 fusion proteins; center,Rtn4a visualized by anti-Rtn4; right, merged images with anti-Rtn4 staining in red and EGFP in green. Scale bar, 20 �m.

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The formation of a Ceg9/Rtn4/atlastin complex in digitoninextracts could be the consequence of a single Rtn4 moleculebinding two different proteins or the result of Rtn4 self-asso-ciation, allowing the oligomeric reticulon to bind multiplepartners simultaneously. Evidence for self-association in thepresence of mild detergent treatment has been previously es-tablished for multiple reticulon isoforms (64, 65). To deter-mine whether we could reproduce previous data, HEK293T

cells were transfected with myc-tagged Rtn4a, a 160-kDa splicevariant allowing clear differentiation from the endogenous 50-kDa Rtn4b/d (66). Immunoprecipitation of Rtn4a-myc withanti-myc beads resulted in co-association of Rtn4b/d in thedigitonin buffer (Fig. 4D, �-Rtn4 Western blot, 50-kDa spe-cies). This occurred in either the presence or the absence of3�FLAG-Ceg9 (Fig. 4D, compare Vector to 3�FLAG-Ceg9).We observed similar results using CHAPS as the detergent (see

FIG 4 Ceg9 binding to Rtn4 does not require an atlastin bridge. (A) HA-tagged ATL1 does not interact with 3�FLAG-tagged Ceg9 in 1% Triton X-100 extracts.HEK293T cells were cotransfected with plasmids encoding HA-tagged ATL1 and 3�FLAG-tagged Ceg9, as well as with the 3�FLAG vector. 1% Triton X-100lysates were subjected to immunoprecipitation with anti-HA resin and analyzed by Western blotting with anti-HA, anti-FLAG, and anti-RhoGDI antibodies.Lanes: Input, 0.91% of the starting material; Un., 0.91% of the unbound remaining material; IP, 11.1% of the precipitation was loaded. (B) Immunoprecipitationof 3�FLAG-Ceg9 does not result in association with ATL1 in the presence of Triton X-100. Lysates of HEK293T cells transfected with plasmids encodingHA-tagged ATL1 and 3�FLAG-tagged Ceg9, as well as the 3�FLAG vector, were used in immunoprecipitations with anti-FLAG resin. Subsequent Western blotswere carried out with anti-HA, anti-FLAG, and anti-RhoGDI antibodies. In the “Input” lanes, 0.9% of the starting material was loaded, and in the “Un.” lanes,0.9% of the unbound supernatant was loaded. The “IP” lanes contained 11% of the total elution fraction after boiling in 2� SDS sample buffer. (C) HA-taggedATL1 and 3�FLAG-tagged Ceg9 associate in 1% digitonin lysates. HEK293T cells were cotransfected with plasmids expressing HA-tagged ATL1 and 3�FLAG-tagged Ceg9, as well as the 3�FLAG vector control. One percent digitonin cell lysates were subjected to immunoprecipitation with anti-HA resin and analyzedby Western blotting with anti-HA, anti-FLAG, and anti-RhoGDI antibodies. Lanes: Input, loaded with 0.91% of the starting material; Un., loaded with 0.91% ofthe material remaining from the immunoprecipitation. The “IP” lanes contained 11% of the total immunoprecipitation. (D). Rtn4 self-associates in extracts thatallow complex formation with Ceg9 and ATL1. HEK293T cells were cotransfected with plasmids expressing myc-tagged Rtn4a and 3�FLAG-tagged Ceg9, as wellas the 3�FLAG vector control. 1% digitonin extracts were made. The “Input” and “Un.” lanes were loaded with ca. 0.7% of the starting material and the materialremaining from the immunoprecipitation, respectively. “IP” lanes contained 11% of the total immunoprecipitation. Immunoprecipitation with anti-myc(Rtn4a) and described Western blotting followed. (E) Schematic of 3�FLAG-tagged Ceg9 interactions in the presence of 1% Triton X-100 compared to 1%digitonin. Although in 1% Triton X-100 lysates 3�FLAG-tagged Ceg9 interaction with endogenous Rtn4b is maintained, HA-tagged ATL1 does not bind (62).However, in 1% digitonin lysates (62), complex formation of HA-tagged ATL1 and 3�FLAG-tagged Ceg9 can be observed.

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Fig. S2 in the supplemental material). Therefore, althoughthere are several explanations for the apparent association ofCeg9 with atlastin-1, the presence of multimeric Rtn4 in digi-tonin extracts indicates that co-association is likely to be indi-rect and due to simultaneous association of both ATL1 andCeg9 to multimeric Rtn4 (Fig. 4E).

Reticulon associates with the LCV independently of Ceg9function. Consistent with our previous work in which we deter-mined the phenotypes of insertion mutations in every knownIDTS during intracellular growth (40), the �ceg9 mutant was pro-ficient at growth within bone marrow-derived macrophages (seeFig. S3B in the supplemental material). To determine whethersubtle defects could be observed in replication vacuole formationthat could not be detected in standard assays for colony forma-tion, we assayed for the bacterial content of individual vacuolesafter 8 h of growth. The distribution of replication vacuole sizes inmacrophages challenged with the �ceg9 mutant was almost indis-tinguishable from the results seen with the wild-type strain (seeFig. S3 in the supplemental material).

Binding of Rtn4 with Ceg9 pointed to another possibility,which is that Rtn4 may directly associate with the LCV duringintracellular growth. To test this model, we performed immuno-fluorescence microscopy analysis of mouse bone marrow-derivedmacrophages challenged either with a wild-type strain (dotA�), anisogenic Icm/Dot-defective mutant (dotA3), or a �ceg9 strain (Fig.5A). After 1 h of incubation with the wild-type strain, the LCVshowed robust accumulation of Rtn4 after permeabilization withTriton X-100, whereas the compartment surrounding a dotA3mutant was totally devoid of Rtn4 (Fig. 5A). At this time point, thelocalization of Rtn4 to the LCV in �ceg9 mutant-infected cells wasalmost indistinguishable from the results seen with the wild-typestrain (Fig. 5A). To determine whether loss of Ceg9 activity re-sulted in a kinetic defect in Rtn4 recruitment about the LCV, atime course experiment was performed. Most notable from thisanalysis was that there was clear colocalization of Rtn4 with bac-teria within 10 min of incubation, and by 20 min postinfection,almost 50% of the bacteria showed Rtn4 colocalization duringdotA� strain infection (Fig. 5B). The strain lacking Ceg9 was only

mildly delayed in Rtn4 accumulation. It was clear, however, thatin macrophages incubated for 10 min with the mutant strain,LCVs that accumulated Rtn4 could be easily detected (Fig. 5B).Therefore, Ceg9 does not appear to be the primary bacterial de-terminant of Rtn4 accumulation around the LCV, and reticulonaccumulation occurs with remarkably fast kinetics.

DISCUSSION

Biogenesis of the L. pneumophila replication vacuole is thought toinvolve hijacking of membrane material that exits from the endo-plasmic reticulum on its route to the Golgi compartment (13).Evidence in support of this model includes the results that inter-ference with ER budding and biogenesis of vesicles involved in theER to Golgi membrane depresses the formation of replication vac-uoles (13, 22, 67). Furthermore, a large collection of IDTS targetproteins are involved in both tethering and fusion of ER-derivedvesicles to the Golgi compartment (68). There is evidence, how-ever, that L. pneumophila may hijack ER-plasma membrane inter-actions or induce novel pathways to direct peripheral ER to inter-face directly with the plasma membrane during the early stages ofvacuole formation. First, entry of the bacteria takes place at thecellular periphery, the site of tubular ER interaction with theplasma membrane, a finding consistent with the first microbialencounter being with tubular peripheral ER (69). Second, mem-brane fusion events in eukaryotes are promoted by pairing ofSNARE proteins on the vesicle and target membranes, with theseproteins having distinct compartment specificities (70). It hasbeen demonstrated that L. pneumophila is able to bypass this com-partment specificity by promoting the noncognate pairing of anER-derived SNARE protein, normally involved in traffic to theGolgi compartment, with partners on the plasma membrane (71,72). Therefore, it seems likely that the earliest critical event invacuole formation is the commandeering of tubular ER associatedwith the plasma membrane.

Within 20 min of challenging bone marrow-derived macro-phages with L. pneumophila, we observed a Icm/Dot-dependentaccumulation of reticulon 4 (Rtn4) around the bacterium in al-most 50% of the cells, forming structures that included tubule-like

FIG 5 Rtn4 localizes to the LCV during infection of A/J mouse bone marrow-derived macrophages independently of Ceg9. (A) Representative examples forvisualization of Rtn4 during infection of A/J mouse bone marrow-derived macrophages with denoted L. pneumophila strains by immunofluorescence micros-copy (see Materials and Methods). Panels: left, L. pneumophila; center, Rtn4; right, merge images with L. pneumophila strains in red and Rtn4 in green. (B) Hostcells were challenged with the wild-type strain Lp02, the dotA-deficient strain Lp03, and a ceg9 deletion mutant at an MOI of 1. At the denoted time points, thecells were fixed and probed for Legionella and Rtn4 as described previously (see Materials and Methods). The phagosomes surrounded by aggregates of Rtn4 witha signal above background were enumerated for each time point. The data represented are the averages and standard errors of three coverslips per data point.

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formations (Fig. 5A). The rapid recruitment appears with kineticssimilar to those observed previously for proteins involved in ERvesicular traffic (67). Therefore, the initial events involved in in-tracellular replication may be wholesale manipulation and dock-ing of tubular ER during the entry process, followed by continuedvesicular transit to support the formation of the nascent replica-tion vacuole. Interestingly, although peripheral tubular ER is nor-mally considered to be smooth ER and devoid of ribosomes, thereis no question that rough ribosome-rich ER is tightly associatedwith the replication vacuole during the later stages of infection(15, 73). Perhaps the early recruitment of ER is an initial step inwhich tubule-encompassed compartments mature into rough ER,which is often linked to the formation of sheet-like structures. Inspite of the demonstration that rough ER recruitment is an earlystep during intracellular replication, Ceg9 appears to be at least thesecond example of an L. pneumophila IDTS that localizes to tubu-lar structures that are largely smooth ER. It was recently shownthat IDTS PieE, when overexpressed in mammalian cells, localizesto smooth ER and appears to organize tubules in stacks (74).There may be a group of IDTS that are translocated into tubulestructures and act to signal downstream from these structures tocontrol maturation of the replication vacuole. Rtn4 has previouslybeen reported to be excluded from phagosomes containing poly-styrene beads, indicating that the recruitment of Rtn4 to the LCVmay be specific for LCVs and play a unique role during intracel-lular growth (75).

The identification of Ceg9 as an interacting partner of Rtn4represents the first demonstration that L. pneumophila proteinsmay interface with ER structural proteins involved in morpholog-ical transformations. The nature of Ceg9-Rtn4 interaction, in fact,is unusual. Previously, it had been shown that self-interaction be-tween reticulon monomers, as well as Rtn4-ATL1 binding, oc-curred in defined gentle detergents (62, 64, 65). We were able toreproduce these results and showed that we could disrupt thesebinding events in the presence of Triton X-100 detergent (Fig. 1and 4). Ceg9, however, bound to Rtn4b/d in all detergent condi-tions tested, indicating that it may show more stable binding totarget than other Rtn4 partners. This result allowed us to demon-strate that binding of Ceg9 to the Rtn4b/d isoforms is likely to bedirect and does not occur via an ATL1 bridge or require Rtn4b/dmultimerization. This does not eliminate the possibility, however,that there could be some uncharacterized mammalian proteinthat bridges Ceg9 to reticulon that we did not detect in our initialpulldown experiments (Fig. 1). Demonstrating a direct interac-tion will require purification of reticulons from other host com-ponents, which has yet to be accomplished after years of study ofthis protein family.

There is no evidence that Ceg9 causes direct disruption of re-ticulon function, and the ER morphology in host cells harboringCeg9 appeared normal (Fig. 3). Overexpression of Ceg9 alignedwith structures in mammalian cells that clearly mimicked tubularER, extending from a perinuclear site out toward the periphery ofhost cells. In these structures, the protein either colocalized withRtn4 isoforms or appeared interdigited. Therefore, we do not fa-vor a model in which Ceg9 directly misregulates reticulon to gen-erate novel ER topology. Rather, the interaction with reticulonmay be an important localization signal that allows Ceg9 to beimmobilized within ER tubules and maintain a position withinthis site. Alternatively, regions of the ER that result in high levels ofCeg9 insertion could generate a stable local structure in which

reticulon and Ceg9 collaborate to form a scaffold for the position-ing of other IDTS.

In spite of the tight association of Ceg9 with Rtn4 isoforms, itsabsence has little effect on the high density of Rtn4 observedaround the replication vacuole relative to that in the remainder ofthe host cell. Presumably, there are multiple L. pneumophila IDTSthat target reticulons, and at least one of these is responsible for theaccumulation of Rtn4. Based on previous work with other IDTS,the ability to bind a target host protein is rarely sufficient to pro-mote localization of the protein. This point can be illustrated byanalyzing the determinants of recruitment of the small GTPaseRab1 to the replication vacuole. One of the tightest Rab-bindingproteins in the literature is the L. pneumophila LidA protein, butbacterial mutants lacking this protein show only mild defects inRab1 recruitment about the replication vacuole (23). Instead,SidM/DrrA, a protein that charges Rab1 with GTP and modifies itso that it is locked in an active form, is the primary bacterial de-terminant responsible for Rab1 recruitment (23, 24, 76). There-fore, it is reasonable to assume that Rtn4 recruitment is similarly aconsequence of enzymatic modification of proteins that are in-volved in controlling ER tubular structure. How Ceg9 interfaceswith the machinery that is responsible for Rtn4 recruitment iscritical for understanding the relationship between topologicaltransitions in the ER proximal to the vacuole and the ability tosupport a functional compartment that supports intracellular rep-lication.

ACKNOWLEDGMENTS

R.R.I. is an investigator of the Howard Hughes Medical Institute (HHMI),E.H. was supported by the German Academic Exchange Service (DAAD),and V.R. and M.I.H. were supported by an American Heart AssociationPostdoctoral Fellowship and a National Institutes of Health (NIH) NRSAFellowship, respectively. This study was supported by the HHMI.

We thank C. Blackstone (NIH, Bethesda, MD), S. Strittmatter (YaleUniversity School of Medicine, New Haven, CT), and Tom Rapoport(Harvard Medical School, Boston, MA) for plasmids and reagents. Wealso thank Kim Davis, Edward Geisinger, Andrew Hempstead, Seblewon-gel Asrat, and Kristin Kotewicz for review of the manuscript.

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