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Cell, Vol. 97, 435447, May 14, 1999, Copyright 1999 by Cell Press

A Coat Protein on Phagosomes Involvedin the Intracellular Survival of Mycobacteria

events along the phagosomal and endocytic pathway

to lysosomes, where such material is degraded (Desjar-

dins et al., 1994; Russell, 1995). In the case of mycobac-

Giorgio Ferrari,* Hanno Langen,

Makoto Naito, and Jean Pieters*

*Basel Institute for Immunology

Grenzacherstrasse 487 teria, early observations suggested that phagosomes

containing living mycobacteria resist fusion with organ-CH-4005 Basel

Switzerland elles of the endosomal/lysosomal system (Armstrong

and DArcyHart, 1971).Thisinhibition depends on active Hoffmann-La Roche

CH-4002 Basel processes exerted by the mycobacteria, as dead myco-

bacteria are readily transported to endosomal/lysosomalSwitzerland Department of Pathology organelles (Armstrong and DArcy Hart, 1971; Barker et

al., 1997; Hasan et al., 1997). This resistance of theNiigata University School of Medicine

Niigata 951-8510 mycobacterial phagosome to fusion with endosomal/

lysosomal organelles is thought to be related to theJapan

ability of mycobacteria to escape from macrophage

bactericidal mechanisms (Small et al., 1994; Russell,

1995).SummaryMorphological characterization of the mycobacterial

phagosome showed that this organelle contains markersMycobacteria are intracellular pathogens that can sur-of the early but lacks markers of later stages of the endo-vive within macrophage phagosomes,therebyevadingcytic pathway, such as late endosomes and lysosomeshost defense strategies by largely unknown mecha-(Sturgill-Koszycki et al., 1994; Clemens and Horwitz,nisms. We have identified a WD repeat host protein1995; Barker et al., 1997). Interestingly, the mycobacte-that was recruited to and actively retained on phago-rial phagosome selectively excludes the proton-ATPasesomes by living, but not dead, mycobacteria. This pro-that is normally responsible for acidification along thetein, termed TACO, represents a component of theendocytic pathway (Mellman et al., 1985; Sturgill-Kos-phagosome coat that is normally released prior tozycki et al., 1994). The resulting limited acidification ofphagosome fusion with or maturation into lysosomes.the mycobacterial phagosome is thought to representIn macrophages lacking TACO, mycobacteria werea key mechanism allowing mycobacterial survival. How-readily transported to lysosomes followed by theirever, the molecular mechanismsby which the mycobac-degradation. Expression of TACO in nonmacrophagesterial phagosome resists fusion with or maturation intoprevented lysosomal delivery of mycobacteria andlysosomes remain largely unknown.prolonged their intracellular survival. Active retention

As an approach toward the identification of compo-of TACO on phagosomes by living mycobacteria thusnents involved in these processes, we have developedrepresents a mechanism preventing cargo delivery toa system in which phagosomes containing living myco-lysosomes, allowing mycobacteria to survive withinbacteria could be physically separated from phago-macrophages.somes containing dead bacilli. We reasoned that bio-

chemical analysis of these two populations of vesicles

might allow the identification of factors that contributeIntroductionto the intraphagosomal survival of mycobacteria. We

here report the identification and characterization of aPathogenicityof microorganismsis oftenrelated to their

WD repeatcontaining molecule, termed TACO (for tryp-ability to survive and replicate within their mammalian

hosts. After entry, a pathogen should find a suitable site tophane aspartatecontaining coat protein), that was

for survival and replication within the cell while at the associated with phagosomes containing live, but not

same time evade or circumvent host defense mecha- with those containing killed, bacteria. TACO representsnisms. As many pathogens have coevolved with the a coat protein that transiently associated with phago-organisms that they infect, they may use host cellular somes but was actively retained on mycobacterial

components for their entry, survival, and growth (Fal- phagosomes as long as the bacteria were viable. Thekow, 1991; Bliska et al., 1993; Beverley, 1996;Galan and capacity of viable mycobacteria to retain TACO on theBliska, 1996; Finlay and Cossart, 1997). phagosomal membrane, thereby preventing their deliv-

One example of a successful exploitation of cellular ery to lysosomes, thus represents a mechanism bymechanisms to survive is provided by the interaction which these pathogens are able to escape host defenseof Mycobacterium species with macrophages. Whereas mechanisms.the normal function of macrophages is to engulf and

destroy microorganisms, mycobacteria have evolved

ways to circumvent the hostile environment of the mac- Resultsrophage (Russell, 1995). Phagocytosed material is nor-

mally transported via a series of fission and fusion Biochemical Characterization of

Mycobacterial Phagosomes

To study the organelles in which mycobacteria reside To whom correspondence should be addressed (e-mail: [email protected]). inside macrophages, the interaction of M. bovis BCG


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Cell436

Figure 1. Detection of a 50 kDa ProteinPres-

ent in Phagosomes Containing Viable Myco-

bacteria

(a and b) Subcellular fractionation of phago-

somes containing viable (a) and heat-killed

(b) mycobacteria. The murine macrophage cell

line J774 was infected for 1 hr with M. bovis

BCG, followed by a 12 (live) or 2 (dead) hr

chase, after which a homogenate was pre-

pared. Organelles were subsequently sepa-

rated by organelle electrophoresis, and the

distribution of the organelle-specific markers

as indicated were determined (upper panels).

From each fraction, the amount of mycobac-

teriawas quantitated bydirect observation by

light microscopy after acid fast staining (lower

panels).

(c) Analysis of proteins present in subcellular

fractions isolated from metabolically labeled

macrophages. From infected macrophages

(upper panels; left, live; right, dead), bacteria

containing fractions after organelle electro-

phoresis were pooled and phagosomes were

prepared by low-speed centrifugation as de-

scribed in Experimental Procedures. Sepa-

rately, from uninfected macrophages a late

endosomal/lysosomal fraction as well as a

lysosomal fraction (from pooling exclusively

the most anodally -hexosaminidase con-

taining shifted membranes) was prepared by

organelle electrophoresis and high-speed

centrifugation (lower panels). Proteins from

phagosomes were separated by two-dimen-

sional IEF/SDSPAGE and analyzed by fluo-

rography. Acidic,left;basic,right. The 50 kDa

protein specifically associated with phago-

somes containing live bacteria is indicated

by an arrow. The major spot in phagosomes

containing viable mycobacteria represents

actin.

with the murine macrophage cell line J774A.1 was investi- by heat treatment prior to uptake by macrophages for

1 hrfollowedby a 2 hrchase (insteadof 12 hr, to preventgated. To analyzethe molecular composition of mycobac-teria-containing phagosomes, subcellular fractionation excessive degradation of the bacilli) resulted in their

recovery in shifted fractions, together with late endo-was utilized. Macrophages that had been metabolicallylabeled with 35S-methionine/cysteine were infected with somal/lysosomal membranes (Figure 1b, Dead, lower

panel). Thus, using this method, vacuoles containinglive BCG for 1 hr, washed, and chased for 12 hr priorto homogenization andsubcellular fractionation. To sep- live mycobacteria could be physically separated from

those containing dead mycobacteria.arate the different subcellular compartments, organelleelectrophoresis was used (Tulp et al., 1994). Duringsuch To analyze the protein composition of the phago-

somes containing viable as well as those containinga separationprocedure, late endosomes and lysosomesshift towardthe anode (Figures 1a and1b, upperpanels), heat-killed mycobacteria, the bacteria-containing frac-

tions were pooled and sedimented at low speed to pre-whereas mycobacterial phagosomes remain in unshiftedfractions, as analyzed by the presence of bacteria (Fig- pare a phagosomal fraction as described in Experimen-

tal Procedures. Separately, a late endosomal/lysosomalure 1a, Live, lower panel). Killing of the mycobacteria


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Mycobacterial Subversion of Phagocytosis437

fraction as well as a lysosomal fraction was preparedfrom uninfected macrophages by organelle electropho-

resis and high-speed sedimentation. Protein patternsfrom these organelles were analyzed by two-dimen-

sional IEF/SDSPAGE followed by fluorography (Figure1c). Most polypeptides present in phagosomes contain-inglivingmycobacteria (Figure1c, Phagosomes(Live))

could also be detected in endosomal/lysosomal organ-elles from uninfected macrophages (Figure 1c, Lateendosomes/Lysosomes). However,in phagosomal mem-

branes containing live mycobacteria, a 50 kDa poly-peptide was present (Figure 1c, arrow) that was notdetected in any of the other subcellular fractions ana-

lyzed, including phagosomes containing killed myco-bacteria (Figure 1c, Phagosomes (Dead)). Phagosomes

containing killed bacteria were enriched for a subsetof lysosomal proteins (compare Figure 1c, right handpanels), allof which were present inlysosomes prepared

froman uninfected homogenate (Figure 1c, Lysosomes).The 50 kDa polypeptide was absent from fluorographsprepared from a similar subcellular fraction in the ab-

sence of infection, as well as from metabolically labeledbacteria (data not shown),and couldbe detectedin total

homogenates from uninfected macrophages, indicatingthat it represented a host protein.

Cloning and Sequencing of the Phagosomal

Protein of 50 kDaTo identify the 50 kDa protein, membrane fractions weresubjected to two-dimensional IEF/SDSPAGE, and after

Coomassie blue staining the 50 kDa polypeptide wasexcised and subjected to lys-C protease digestion and

mass spectrometry (Figure 2a). Nine out of 11 peptidesmatched the digestion profile of entry P31146 (Swiss-Prot), representing a 57 kDa protein previously identified

inhuman cells(Suzukiet al., 1995). Based onthishomol-

ogy, the cDNA encoding the mouse homolog of thisprotein was cloned from a macrophage library as de-scribedin Experimental Procedures. Sequencingof pos-itive clones revealed an open reading frame of 1386

Figure 2. Identification of the 50 kDa Proteinbase pairs encoding a 461 amino acid residue polypep-(a) The polyacrylamide gel piece containing the 50 kDa p rotein (seetide with a predicted sequence shown in Figure 2b. ToFigure 1) was excised and subjected to lys-C digestion and massconfirm that this reading frame encoded the 50 kDa pro-spectrometry. Shown is the spectrum obtained after mass spec-

tein that accumulated in mycobacterial phagosomes, atrometry.

lys-C digest of the polypeptide isolated from the two- (b) Predicted amino acid sequence of the 50 kDa protein. The fivedimensional gel was separated by HPLC. Sequence WD repeats present in the sequence are underlined.

(c) Immunoprecipitation using anti-TACO antibodies of cell lysatesanalysis of the peptide eluting at 21 min by Edman deg-from metabolically labeled macrophages followed by two-dimen-radation revealed the sequence FRHV, matching aminosionalI EF/SDSPAGE. Thegel was coelectrophoresedwith differentacid residues 1114 (Figure 2b). In addition, antibodiesorganelle fractions to allow alignment of the phagosomal 50 kDa

raised against the C-terminal amino acid sequencespot (arrow).

were used to immunoprecipitate the 50 kDa protein

from lysates of noninfected macrophages that hadbeen metabolically labeled. Analysis by two-dimensional

segment bordered by Gly-His (GH) and Trp-Asp (WD) di-IEF/SDSPAGE and coelectrophoresiswith phagosomalpeptide residues, but their function remains unknownorganelles showed that the immunoprecipitated protein(Neer et al., 1994). A BLAST search with the 50 kDamigrated at the identical position as the phagosomal 50proteinsequence revealed homologyto IR10 (40% iden-kDa protein (Figure 2c). Interestingly, several additionaltity), a human WD repeatproteinof unknown function, asmolecules of the same molecular weight but differingwell as to a WD repeat proteinidentified inDictyosteliumin isoelectric point were detected on the fluorograph,discoideum, termed coronin (35% identity), an actin-possibly representing post-translationally modified formsbinding protein involved in actin-based cytoskeletalor, alternatively, closely related proteins.rearrangements. Furthermore, analysis of the primaryThe primary sequence of the 50 kDa polypeptide con-sequence using Prosite revealed the lack of a signaltains five WD repeats (underlined in Figure 2b). WD re-

peats are characterized by a3040 amino acid residue sequence for targeting to the endoplasmic reticulum as


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well as the presence of a coiled-coil region within the

32 C-terminal amino acid residues (Berger et al., 1995).

Because of the presence of the WD repeats as well as

its morphological characterization (cf. later), we have

named this protein TACO for tryptophane aspartate

containing coat protein.

Tissue Distribution of TACO

Several members of the WD repeat family areinvolved in

regulating cytoskeletal assembly and membrane traffic,

thus carrying out functions that are essential for normal

cellular growth and therefore expected to be expressed

in all cell types (Neer et al., 1994). To analyze the tissue

distribution of TACO, proteins from a homogenate pre-

pared from various tissues were separated by SDS

PAGE and transferred to nitrocellulose (Figure 3a). After

staining of the membrane using anti-TACO antibodies,

immunoreactivity was found to be present in spleen,

lymph nodes, thymus, and brain, as well as a small

amount in lung, but not in any other tissue examined

(Figure 3a, upper panel). As a control, the immunoblot

was stripped and stained for actin (Figure 3a, middlepanel) as well as for tubulin (Figure 3a, lower panel). In

addition, bone marrowderived and peritoneal macro-

phages as well as all macrophage cell lines analyzed

expressed TACO (data not shown), with the notable

exception of Kupffer cells (see later). Thus, TACO is

expressed in cells of the lymphoid/myeloid lineage, con-

sistent with a function in macrophages and macro-

phage-like cells.

Subcellular Localization of TACO

The subcellular distribution of TACO was analyzed mor-

phologically using confocal immunofluorescence mi-

croscopy. In noninfected macrophages, a rat monoclonal

antibody raised against TACO predominantly labeled

the cortex of the cells and colocalized with tubulin, but

not with actin (Figure 3b). Double labeling of cells forFigure 3. Distribution and Localization of TACO

actin and tubulin showed only a partial colocalization(a) Tissue distribution of TACO. The different tissues as indicated

(Figure 3b), indicating the presence of two distinct corti-were isolated from a mouse, homogenized, and total proteins (15

cal cytoskeletal elements in these cells. Together these g) separated by SDSPAGE and immunoblotted using anti-TACOresults suggest that innoninfected macrophages, TACO antiserum (upper panel). After stripping, the blot was reincubated

with anti-actin antibodies (middle panel) or anti-tubulin antibodiesis associated with the cortical microtubule network.(lower panel).

(b) Subcellular localization of TACO in uninfected cells. Macro-Localization of TACO in Infected Cells

phages were grownon coverslips, fixed, permeabilized, and stainedSince TACO was identified as a component of phago- for tubulin (upper left) followed by Texas redlabeled secondarysomes containing viable mycobacteria, the subcellular antibodies or F-actin using phalloidin-Texas red (upper right) and

double labeled for TACO using a rat monoclonal antibody followeddistribution incells infected withmycobacteria was ana-by FITC-labeled secondary antibodies. (Lower left) Localization oflyzed. To visualize mycobacteria-containing phago-actin (Texas red) and tubulin (FITC). (Lower right) Inclusion of thesomes, M. bovisBCG expressing green fluorescent pro-immunizing peptide blocked TACO staining. Bar, 5 m.

tein (BCG-GFP) was utilized. Cells were allowed tointernalize mycobacteria for 1 hr, washed and trypsin-

ized to remove adherent bacteria, and cultured at 37C cortex over the next few hours, resulting in a staining

pattern as in noninfected cells (Figure 4b). Quantitationfor the times indicated in Figure 4a. Immediately after

infection (chase 0 hr), TACO was localized around the of TACO localization at the phagosome revealed that

80%90% of the mycobacterial phagosomes stainedmycobacterial phagosomes, in addition to a cortical lo-

calization. Within the first 2 hr, TACO became almost positive for TACO in case of viable bacilli at all time

points. In contrast, TACO dissociated from phagosomescompletely relocalized to the phagosomal membrane

and remained associated with the phagosome up to containing killed bacilli within the first 2 hr and redistrib-

uted to the cell cortex duringthistime period (Figure 4c).prolonged periods of time (Figure 4a).

When heat-killed bacteria were allowed to be internal- In noninfected macrophages, TACO strongly colocal-

ized with tubulin (Figure 3), and as tubulin has beenized by macrophages, TACO accumulated initially at

the site of bacterial uptake but redistributed to the cell implicated in phagosomelysosome fusion (Desjardins


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Figure 4. Subcellular Localization of TACO in Infected Cells

Macrophages were grown on coverslips and infected with live (a) or heat-killed (b) M. bovis BCG expressing the green fluorescent protein

(BCG-GFP) for 1 hr followed by a chase for the times indicated. After fixation and permeabilization, the cells were stained for TACO using

mAb GK3 followed by Texas redlabeled secondary antibodies. For each field, a bright field image is shown (left side). Bar, 5 m. (c) For

quantitation, cells were scored for the presence of TACO at the mycobacterial phagosome. Shown are mean values ( SD) from three

experiments (n 50). In (d), cells were infected with live (left) or heat-killed (right) mycobacteria followed by fixation and staining using anti-

tubulin antibodies and Texas redlabeled secondary antibodies. Bar, 5 m.


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Figure 5. Effect of Rifampicin and Alkaliniza-

tion on TACO Distribution

(a) Cells were infected for1 hr with live myco-

bacteria, followed by a 2 hr chase in the ab-

sence (upperpanels) or presence (lower pan-

els) of rifampicin (1 mg/ml), fixed, andstained

forTACOas described. Quantitationof TACO

recruitment (as in Figure 4) revealed a strong

reduction of TACO colocalization with myco-

bacteria (right side). The difference in TACO

recruitment between phagosomes containing

heat- and rifampicin-killed mycobacteria might

be due to differences in bacterial treatment.

(b) Cells were alkalinized as described in Ex-

perimental Procedures and subsequently in-

fected with live (upper panels) or heat-killed

(lower panels)mycobacteria for1 hr, followed

by a 2 hr chase. After fixation, cells were

stained for TACO as described. Quantitation

(right side) revealed that whereas 90% of

the phagosomes containing viable bacilli had

recruited TACO, virtually none of those con-

taining killed bacilli colocalized with TACO.

Bar, 5 m.

et al., 1994), the distribution of tubulin in macrophages distributionof phagosomes(Heuser, 1989). Whenorgan-

elles from NH4Cl-treated macrophages infected withinfected with live as well as with heat-killed mycobac-

teria was analyzed. As shown in Figure 4d, whereas heat-killed bacteria were separated by organelle elec-

trophoresis,virtuallyall bacilliwere retrieved inunshiftedtubulin remained present at all times around phago-

somes containing killed bacilli, in cells infected with fractions, confirming the ability of NH4Cl to inhibit

phagosomelysosome fusion (data not shown). Immu-live mycobacteria tubulin rapidly dissociated from the

mycobacterial phagosome. Besides tubulin, as ex- nofluorescence analysis of these cells revealed that

while ammonium chloride treatment did not affect TACOpected, also LAMP was recruited to the phagosomes

containing killed bacilli (results not shown), consistent recruitment on phagosomes containing living mycobac-

teria (Figure 5b, upper panels), TACO was not retainedwith a role for microtubules in phagosomelysosome

fusion (Desjardins et al., 1994; Blocker et al., 1998). on phagosomescontaining heat-killed bacilli (Figure 5b,

lower panels). This further indicates that lack of acidifi-To analyze whether the recruitment and retention of

TACO required viability of the mycobacteria, cells in- cation per se is not responsible for TACO retention but

rather processes exerted by the mycobacteria them-fected with live bacteria were incubated for 2 hr in me-

dium containing the anti-mycobacterial drug rifampicin. selves.

Whereas in nontreated cells90% of the mycobacterial

phagosomes contained TACO (Figure 5a, upperpanels),

the in situ killing of the bacteria by rifampicin resulted Localization of Mycobacteria in TACO-NegativeKupffer Cellsin a rapid release of TACO from the phagosomal mem-

brane (Figure 5a, lower panels). In contrast to all other macrophage-containing tissue,

the liver did not show any expression of TACO (FigureRetention of TACO on phagosomes may occur as a

result of the elevated pH in phagosomes as opposed 3a), despite the presence of large numbers of macro-

phages, the Kupffer cells (Wardle, 1987). The absenceto lysosomes (Crowle et al., 1991; Sturgill-Koszycki et

al., 1994) or, alternatively, be dictated by processes ex- of TACO from Kupffer cells was confirmed by immu-

noblotting of a lysate prepared from isolated Kupffererted by viable bacteria. To distinguish between these

possibilities, phagosomes containing heat-killed myco- cells as well as by immunohistochemistry on liver sec-

tions (Figures6a and 6c). To directlyanalyzephagocyto-bacteria formed inthe presenceof NH4Cl were analyzed.

Ammonium chloride inhibits phagosomelysosome fu- sis of mycobacteria in TACO-negative Kupffer cells,

these cells were isolated from the liver and infected withsion (Gordon et al., 1980; Kielian and Cohn, 1982; Hart

and Young, 1991) without altering the morphology or mycobacteria for 1 hr followed by a 2 hr chase. Cells


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Figure 6. TACO Expression and Mycobacterial Entry in Liver Macrophages

(a and b) Localization of mycobacteria in TACO-negative Kupffer cells. (a) Kupffer cells were isolated from liver as described in Experimental

Procedures, lysed, and total proteins (10 g) separated by SDSPAGE followed by immunoblotting using anti-TACO antibodies. As a control,

a J774 lysate (10 g) was used. (b) Isolated Kupffer cells were incubated for 1 hr with M. bovisBCG expressing the green fluorescent protein

(BCG-GFP). Cells were washed and incubated for 2 hr (upper panels) or 16 hr (lower panels) prior to fixation and staining for lysosomal

glycoprotein followed by Texas redlabeled secondary antibodies (LAMP-TR). Bar, 5 m.

(c and d) Immunohistochemistry of TACO expression in mouse liver. Mice were untreated (c) or infected (d) with M. bovis BCG, sacrificed,

and sections were obtained from the liver. Immunohistochemistry was performed using the rat mAb GK3 followed by HRP-coupled secondary

antibodies (left panel), or double labeling was performed using mAb GK3 and Ziehl-Neelsen staining.

were subsequently fixed and stained for lysosomes us- panels). Thus, in macrophages lacking TACO, myco-

bacteria were rapidly taken up in lysosomes, followedingantibodies against lysosomal-associated membrane

glycoprotein(LAMP) andanalyzed by confocal immuno- by a complete destruction of the bacilli.

fluorescence microscopy (Figure 6b, upper panels). All

of the infected Kupffer cells observed contained large Immunohistochemical Analysis of TACO ExpressionAlthough the liver Kupffer cells are known to completelyvesicular structures that strongly stained positive for

LAMP (Figure 6b, LAMP-TR). All phagocytosed bacilli eradicate bacterial infections (North, 1974), one conse-

quence of a mycobacterial challenge in vivo can be thewere present in lysosomal organelles, and no mycobac-

teria could be detected in LAMP-negative vacuoles. In formation of liver granulomas. Granulomas are macro-

phage-rich lesions harboring viable bacilli, thought tofact, in most if not all phagocytosing Kupffer cells, lyso-

somalvacuoles could be found recruited to andconcen- be involved in restricting the bacterial spread (Gordon

et al., 1994). When liver sections obtained from BCG-trated at the site of bacterial uptake. Interestingly,

whereas after a 2 hr chase phagolysosomes could be infected mice were analyzed, the granuloma regions

stained positive for TACO, and these macrophages har-detected harboring both intact bacilli as well as partially

degraded mycobacteria (as judged from the green fluo- bored mycobacteria as revealed by Ziehl-Neelsen stain-

ing (Figure 6d). TACO-positive macrophages representedrescent protein distribution pattern), after an overnight

chase virtuallyallbacilliwere degraded (Figure 6b, lower exclusively infiltrated macrophages, as they stained posi-


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Figure 7. Differential Trafficking and Survival of Mycobacteria in TACO-Transfected Cells

Mel JuSo cells transfected with cDNA encoding TACO in the sense (TACO-transfected) or antisense (Control) orientation were lysed andtotal proteins (10 g) separated by SDSPAGE followed by immunoblotting using anti-TACO antibodies (a). Control (b) or TACO-transfected

(c) cells were incubated with mycobacteria for 1 hr followed by a 3 hr chase, homogenized, and subjected to organelle electrophoresis, and

the distribution of the organelle-specific markers as indicated was determined (b and c, upper panels). From each fraction, the amount of

mycobacteria was analyzed by direct observation by light microscopy after acid fast staining (b and c, lower panels). Shown is a representative

experiment out of three. (d) TACO-transfected or control cells were infected with metabolically labeled mycobacteria for 1 hr at 37C, washed,

and lysed, and the total uptake was determined by liquid scintillation counting of the lysate. (e) TACO-transfected or control cells were

incubated with mycobacteria, washed, and chased. At the chase times indicated, cells were lysed in PBS containing 0.5% Triton X-100, and

bacteria were collected by centrifugation and resuspended in 7H9 medium. Serial dilutions were plated out and the number of colonies

determined from the appropriate dilution. Shown are mean values ( SD) from five experiments (d and e).

tive both for F4/80 and Mac-I antigens, the formerstain- mycobacterial uptake, which was in fact three orders of

magnitude lower when compared to J774 macrophagesing all macrophage cell types, the latter being not reac-

tive with Kupffer cells (Hirsch et al., 1981; Nibbering et (Figure 7d and data not shown). When Mel JuSo cells

were infected for 1 hr with mycobacteria followed by aal., 1989).

3 hr chase, after homogenization and subcellular frac-tionation by electrophoresis 15% of the internalizedDifferential Mycobacterial Trafficking and Survival

in Nonphagocytes Expressing TACO bacilli were located in shifted fractions, indicating a lim-

ited ability of these cells to target mycobacteria to lateThe recruitment of TACO on phagosomes containing

live mycobacteria together with the resistance of these endosomal/lysosomal organelles (Figure 7b). However,

in TACO-transfected Mel JuSo cells, transfer of the my-mycobacterial phagosomes to fuse with or mature into

lysosomes suggests that TACO functions as a compo- cobacteria to late endosomes was completely abro-

gated, as shown by the absence of mycobacteria fromnent of the phagosomal coat that, when present, pre-

vents fusion with lysosomes. To directly analyze this, shifted fractions (Figure 7c). In accordance with this, in

controlM el JuSo cells a subset of intracellularly residingthe nonphagocytic melanoma cell line Mel JuSo, which

does not express TACO, was transfected with cDNA bacteria colocalized with LAMP (10%), whereas no

colocalization at all was observed in TACO-transfectedencoding TACO, and stably transfected cell lines were

selected (Figure 7a). Transfection of TACO did not affect cells (data not shown).


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To analyze mycobacterial survival in TACO-trans-

fected versus control Mel JuSo cells, these cells were

infected with mycobacteria for 1 hr, washed, and further

incubated for the times indicated in Figure 7e. At each

time point, cells were lysed and the amount of viable

mycobacteria determined by counting the formation of

colonies after plating out different dilutions on agar.

Strikingly, in TACO-transfected Mel JuSo cells survival

of mycobacteria was 3-fold higher within the first 3 hr

after infection and increased to 8-fold after an over-

night chase (Figure 7e). Thus, also in nonphagocytic but

TACO-expressing cells, mycobacteria have the capacity

to resist their delivery to late endosomal/lysosomal or-

ganelles, thereby prolonging their survival.

Discussion

A hallmark of the mycobacterial phagosome is its resis- Figure 8. Model Describing the Role of TACO in Intracellular Sur-tance to fusion with other stages of the endosomal/ vival of Mycobacterialysosomal pathway (Armstrong and DArcy Hart, 1971; Phagocytosis triggers the recruitment of TACO (yellow) around the

particle to be ingested, possibly as a result of its initial associationClemens and Horwitz, 1995; Russell, 1995). As a result,

with microtubules (black lines) at the cell cortex. In the case ofdelivery of mycobacteria to lysosomes is prevented, viable bacteria (violet), TACO is actively retained on the phagosomalwhichis thought to represent a keymechanismby whichmembrane, while it dissociates from the microtubule network, per-

mycobacteria manage to survive intracellularly. In ahaps as a consequence of a conformational change. In contrast,

search for host cell components that are involved in the during phagocytosis of killed bacteria (black) or after in situ killingintracellular survival of mycobacteria, we have identified of the bacilli, TACO initially associates with phagosomes but is

rapidly released concomitant with lysosomal delivery of the bacilli.TACO, a protein associated with phagosomes con-

taining viable but not killed mycobacteria. Retention of

TACO onthe phagosomalmembrane was strictly depen-

dent on mycobacterial viability and not due to impaired C phosphorylation consensus sites present in TACO areacidification. Moreover, in TACO-negative Kupffer cells possibly involved in such regulation.all bacilli were located in lysosomes followed by their Most studies to date have implicated the actin cy-degradation. Expression of TACO in a nonmacrophage toskeleton in phagocytosis of a wide variety of livingcell line was sufficient to prevent lysosomal fusion of and inert particles (Greenberg et al., 1990; Rathman etthe mycobacterial phagosome, allowing survival of my- al., 1997). In agreement with these reports, we foundcobacteria. Together these results suggest that TACO that phagosomes containing viable as well as thoseis actively retained on mycobacterial phagosomes by containing dead bacilli were associated with actin. Inprocesses exerted by the mycobacteria themselves and contrast to actin, tubulin remained associated exclu-is one of the factors providing a niche for mycobacteria sively with phagosomes containing killed bacteria, con-to survive within macrophages. sistent with previous reports showing that fusion of

phagosomes with lysosomes is a microtubule-depen-

dent process (Desjardins et al., 1994; Blocker et al.,TACO Is a WD Repeat Protein

The predicted amino acid sequence of TACO showed 1998). Both the actin and tubulin cytoskeleton may play

a role during the initial phase of bacterial internalizationthat it is a member of the WD repeat protein family (Neer

et al., 1994; Suzuki et al., 1995). WD repeatcontaining to generate the forces required for the actual internaliza-

tion process. However, after the internalization of livingproteins are involved in a variety of processes, including

signal transduction, motility (King et al., 1995), cytokine- mycobacteria, tubulin rapidly dissociated from TACO-

coated phagosomes containing viable mycobacteria.sis (Shirayama et al., 1998), cytoskeletal organization,

and vesicle fusion (Pryer et al., 1993; Salama et al., Interestingly, TACO shows35% homology to a WD

repeat, actin-binding protein identified in the slime mold1993). Members of this family are present throughout

eukaryotic evolution and thusfar have not beenfound to Dictyostelium discoideum, termed coronin (de Hostoset al., 1991; de Hostos et al., 1993). Deletion of thebe present in prokaryotic organisms (Neer et al., 1994).

Although the precise function of the WD repeat remains gene encoding this protein in Dictyostelium results in

an impaired functioning of actin-based processes, oneunknown, the almost complete colocalization of TACO

with tubulin in noninfected macrophages suggests that of whichleadsto a reduced capacity to internalize yeast

particles (Maniak et al., 1995). This indicates that in thisat least some part of TACO either directly or indirectly

binds to microtubules. Given the role of several, other- organism, one function of this protein is associated with

particle uptake (Maniak et al., 1995; Rauchenberger etwise distinct WD repeat proteins in processes requiring

microtubule-based activity (Li and Suprenant, 1994; al., 1997). Cellular slime molds such as Dictyostelium

live on soil bacteria, including mycobacteria, and it isWilkersonet al., 1995),it istemptingto speculate that the

WD repeat represents a microtubule-binding domain. conceivable that duringthe 15002000 million yearsthat

separate Dictyosteliumfrom mammals(Knoll, 1992),my-Microtubule binding might be regulated by posttransla-

tional modifications, and the predicted protein kinase cobacteria have acquired the ability to actively recruit


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and retain host cell components, including TACO, that Sathyamoorthy et al., 1997), and copurification may

have resulted from the colocalization of both proteinsensure their survival, rather than being delivered to lyso-

somes for degradation (see also Figure 8). at the cortical cytoskeleton (Woodman et al., 1991).

Retention of TACO by Mycobacteria to Prevent A Balance between the Host and the PathogenLysosomal Delivery Evidence for a role for TACO in vivo in the containment

The distribution of TACO during phagocytosis suggests of mycobacteria in a viable state was provided by thethat it represents a coat protein of the mycobacterial finding that infiltrated macrophages in liver granulomasphagosome. Vesicular coat proteins are transiently re- expressed TACO, whereas the resident liver macro-cruited onto membranes and required in a defined bud- phages, the Kupffer cells, did not. The liver is the primeding or fusion step or during organelle maturation to target for infectious agents that have accessed an or-transfer cargo from one stage to another (Rothman and ganism, but as an organ it is rarely infected (BrandborgWieland, 1996; Schekman and Orci, 1996). Thus far, and Goldman, 1990). Clearance of bacteria in the liverthree types of coats have been defined to function in is attributed to the presence of large amounts of Kupfferdistinct steps of vesicular traffic. Clathrin-based coats cells (North, 1974; Wardle, 1987). Indeed, when isolatedfunction in cargo delivery to the endocytic pathway as Kupffer cells were infected with mycobacteria, all ofwell as to post-Golgi organelles (Kirchhausen et al., the bacilli were rapidly transferred to lysosomes and1997), while COPI- (for coat protein I) and COPII-based degraded. First, this indicates that TACO is not abso-coats are involved in traffic between the endoplasmic lutely essential for phagocytosis. Second, these resultsreticulum and Golgi organelles (Bednarek et al., 1995; suggest that at the level of the whole organism ratherCosson and Letourneur, 1997). than at the single cell, evolutionary pressure may have

Instead of functioning in cargo selection and/or bud- ensured downregulation of TACO expression in Kupfferding processes, TACO might in fact prevent the budding cells to provide an efficient and complete mycobacterialand fusion machinery to be recruited onto the plasma clearance site.membrane and therefore identifythis membrane as non- Why have mammalian cells retained a molecule thatfusogenic with lysosomes. Once material is being allows mycobacteria to survive within macrophages?phagocytosed, release of TACO would be required to One possibility is that TACO performs another, as yettrigger lysosomal fusion. It is precisely at this last step unknown function in macrophages. For example, as thein the normal maturation pathway of phagocytosed ma- processes involved in cell locomotion and phagocytosisterial that mycobacteria intervene. To avoid lysosomal are quite similar (Berlin et al., 1978; Valerius et al., 1981),delivery, mycobacteria have gained the capacity to ac- TACO may be a crucial component of the cytoskeletontively retain TACO on the phagosomal membrane, of highlymotilecells, functioningboth inthe invaginationthereby possibly preventing the recruitment of the ma- of large pieces of plasma membrane, as well as in form-chinery necessary for cargo delivery to lysosomes (Fig- ing protrusions of the plasma membrane involved in cellure 8). As transfection of TACO in nonphagocytic cells locomotion(Valeriuset al., 1981).The homologyof TACOalso inhibits mycobacterial delivery to late endosomes, with Dictyosteliumcoronin would indeed argue in favorthis function of TACO appears to be independent from of such a role. The fact that mycobacteria, in the coursethe phagocytic machinery with which macrophages are of their coevolution with higher eukaryotes, have madeequipped. use of the normal cellular processes involved in their

Interestingly, another WD repeat protein, the beige/ own uptake to circumvent host defense responses isyetlyst gene product, might have a very similar scaffolding another striking example of the ingenuity of pathogenicfunction for lysosomes (Perou et al., 1996). Also this microorganisms.protein colocalizes with microtubules (Faigle et al.,

1998), and cells lacking an intact beige/lyst product are Experimental Procedurescharacterized by the presence of abnormally large lyso-

Cells, Bacteria, and Antibodiessomes whose function is impaired (Burkhardt et al.,The murine macrophage cell line J774A.1 was obtained from ATCC1993). In fact, one function of the WD repeat proteinsand cultured in DMEM containing 10% heat-inactivated fetal calf

might be to provide a scaffoldfor the different organellesserum at 37C in 5% CO2. The human melanoma cell line Mel JuSo

to maintain their integrity. (Johnson et al., 1981) was grownin RPMI-1640 containing 5% heat-Recruitment and retention of TACO on the phagoso- inactivated fetal calf serum. M ice (BALB/c) were obtained from Iffa-

Credo (Basel, Switzerland) or from Charles River, Inc. (Tokyo, Ja-mal membrane might be regulated through transmem-pan). Kupffer cells were isolated according to Lee et al. (1986) withbrane signal transduction processes induced by the liv-the following modifications. Mice were sacrificed and the thoracicing bacteria. How mycobacteria achieve this remainscavity exposed followed by canulation of the inferior vena cava via

to be established, and it is possible that phagosomalthe right atrium. After opening of the portal vein to allow efflux, the

transmembrane proteins are involved in the regulation liver was perfused with 10 ml of PBS followed by perfusion of aof TACO retention. Defining the binding partners of collagenase solution (5 mg/ml collagenase D [Boehringer Mann-

heim] in RPMI-1640, prewarmed at 37C). The liver was removedTACO at the phagosomal membrane as well as in theand mechanically disrupted prior to incubation with 10 ml of 1 mg/cytosol might identify the complexes involved in suchml of the same collagenase solution at 37C. After 15 min, DNAsesignal transduction processes. In human neutrophiles,(Sigma) from a 5 mg/ml stock solution in PBS was added to a

a TACO homolog was found to copurify with the p40final concentration of 0.5 mg/ml. The resulting cell suspension was

protein thought to play a role in the NADPH oxidase filtered throughdressing sponges (Johnson and Johnson, Arlington,(Grogan et al., 1997). However, the contribution of p40 TX), and the filtrate was diluted to 50 ml in RPMI containing 10%

heat-inactivated FCS. Cells were centrifuged five times (30 g, 4inthe NADPH oxidase isunclear (Tsunawaki et al., 1996;


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min) at room temperature, and the pelletswere discarded. After two In short, excised spots were destained with 30% acetonitrile in 0.1

M ammonium bicarbonate and washed with water. The gel piecesfinal washes in RPMI-FCS, cells were plated out, left to adhere for

60 min at 37C, after which the nonadherent cells were removed by were reswollen with 5 l of 3 mM Tris-HCl (pH 8.8), containing 1 g

of endopeptidase Lys-C (Wako)after drying in a speedvac evapora-five washes in RPMI. The resulting cell population consisted of

90% macrophages as revealed by F4/80 staining. tor. The digestion was performed for 312 hr at room temperature.

Three microliters of 30% acetonitrile containing 0.1% trifluoroaceticMycobacterium bovis BCG, strain M ontreal was kindly provided

by Dr. Thole (TNO, Leiden), and M. bovis BCG harboring phsp60- acid was added, and the content was vortexed, centrifuged for 2

min, and sonicated for 5 min. One microliter from the separatedgfp (BCG-GFP) (Dhandayuthapani et al., 1995) was kindly provided

by Dr. Deretic (University of Michigan, Ann Arbor, MI) and propa- liquid was applied onto the dried matrix spot. The matrix consisted

of 15 mg of nitrocellulose (Bio-Rad) and 20 mg of -cyano 4-hydroxy-gated in7H9 mycobacterial medium(Difco) supplemented with10%

OADC Middlebrook supplement (Difco). Killing of mycobacteria was cinnamic acid (Sigma) dissolved in 1 ml of acetone isopropanol 1:1

(v/v). Matrix solution (0.5 l) was applied on the sample target. Thecarried out byheat treatment for 30 min at 80C. Infection of macro-

phages was essentially as described (Hasan et al., 1997). samples were analyzed in a time-of-flight Perseptive Biosystems

mass spectrometer (Voyager Elite) equipped with a reflector andQuantitation of the total mycobacterial uptake was performed

by incubating cells for 1 hr with 35S-methionine/cysteine-labeled delayed extraction. An acceleration voltage of 20 kV was used.

Calibration was internal to the samples with des-Arg-1 Bradykininmycobacteria. After extensive washing, cells were lysed in Dulbec-

cos phosphate-buffered saline (D-PBS)containing 1% Triton X-100 (Sigma) and adrenocorticotropic hormone fragment 18-39 (Sigma).

For the protein search, monoisotopic peptide masses were used,and analyzed by liquid scintillation counting.

Mycobacterial survival was analyzed by lysing cells in D-PBS and a mass tolerance of 0.006% was allowed.

containing 0.5% Triton X-100 and sedimentation of the bacteria at

2700 g for 10 min. The pellet was resuspended in 7H9 medium,cDNA Cloning and Sequencing

and different dilutions were plated out on 7H11 agar (Difco), supple-To obtain the cDNA encoding TACO, a mouse macrophage ZAP

mented with 10% OADC Middlebrook and glycerol (5 ml/l). After 3II library (from peritoneal macrophages; Stratagene) was screened

weeks, the number of colonies formed was determined from thewith a probe amplified from human spleen cDNA (Clonetech) using

appropriate dilutions.

the following primers: 5-CCAGTGCTATGAAGATGTGCGCG-3 (for-In vivo infection was performed by injecting 8-week-old BALB/cward primer) and 5-ATGATGCGCACGCGCTTGTCACGGC-3 (re-

mice intravenously with 0.2 ml of M. bovis BCG (Japan BCG Inc.,verse primer). Positive clones were expanded, and the pBluescript

Tokyo, Japan), dissolved in physiological saline at a concentrationphagemid containing the cloned DNA inserts was excised by coin-

of 10 mg/ml. The liver was removed at various intervals after BCGfection with helper phage in XL1-Blue MRF to yield TACOpBL-SK.

injection and processed for immunohistochemistry.Sequencing was performed using the ABI PRISM Dye terminator

Polyclonal antisera were raised against KLH-coupled peptides incycle sequencing reaction (Perkin Elmer, Foster City, CA) and ana-

New Zealand white rabbits, and antibodies were affinity purified bylyzed using an ABI 373 DNA sequencer. For the construction of a

affinity chromatography using the immunizing peptides as ligands.eukaryotic expression construct, a TACO-encoding fragment was

Monoclonal antibodies were generated by immunizing rats (Wistar)subcloned into pBL-SK after digestion of TACOpBL-SK with XhoI

with the same peptides and fusion of the spleen and lymph nodesand XbaI, and the purified fragment ligated into pBK-CMV (Strata-

with SP2/0 myeloma cells. Antibodies against actin (clone C4;gene)digested withthe same enzymes. A TACO-encoding fragment

mouse IgG1) and-tubulin (mouse IgG1) were obtained from Boeh-was generated using EcoRI and ligated into the mammalian expres-

ringer Mannheim and Amersham, respectively. Rat anti-Mac-1 anti-sion vector pUC-CAGGS (Niiwa et al., 1991) either in the antisense

body (IgG2b) was from CALTAG (San Francisco, CA), and the rator sense orientation. cDNAs were introduced into Mel JuSo cells

mAb F4/80 (IgG2b) was kindly provided by Dr. S. Gordon (Oxford,by electroporation using the BioRad Gene Pulser (400 V/125 F),

UK). The rat anti-murine LAMP-1 antibody 1D4B (IgG2a) was devel-and clones were selected using G418.

oped by T. August and obtained from the Developmental Studies

Hybridoma Bank at the University of Iowa.Alkalinization was performed as described in Heuser (1989). Metabolic Labeling and Immunoprecipitation

Briefly, cells were washed three times in Ringers solution without Priorto labeling, macrophages weregrownfor30 minin methionine/NH4Cl, followed by equilibration of the cells (two times for 15 min cysteine-free medium containing 10% dialyzed FCS. Labeling waseach) in Ringers solution containing 30 mM NH4Cl (alkalinization performed by placing the cells in methionine/cysteine-free mediummedium). Bacteria were washed in the same alkalinization medium, containing 10% dialyzed FCS and 0.15 mCi/ml 35S-methionine/cys-and the infection was performed as described above. teine mix (Promix, Amersham). Mycobacteria were labeled in 7H9

medium supplemented with 10% OADC M iddlebrook containing 5

Ci/ml 35S-methionine/cysteine mix (Promix). ImmunoprecipitationsSubcellular Fractionation and Phagosome Isolationwere performed as described (Pieters et al., 1991).Cells were homogenized and organelles prepared essentially as

described (Tulp et al., 1994; Ferrari et al., 1997; Hasan et al., 1997).

Themembranepellet was resuspended in 6% Ficoll-70 inhomogeni- Immunofluorescence Microscopy and Immunohistochemistryzation buffer, and organelle electrophoresis as well as the determi- Cells were grown on glass coverslips and infected as describednation of organelle specific markers was carried out as described above. After incubation, cells were fixed in 3% paraformaldehyde(Ferrari et al., 1997). The presence of mycobacteria was determined (at 37C) for 10 min, washed in PBS, and permeabilized in 0.1%by centrifugation of the fractions onto glass coverslips followed by saponin in PBS for 30 min at room temperature. Saponin (0.1%)acid fast staining (Difco) and quantitation by direct observation of

was included in all subsequent incubations. Alternatively, cells werethe coverslips. Phagosomes were isolated afterpoolingof the bacte- fixed in methanol (4 min at 20C). Coverslips were incubated withria-containing fractions followed by sedimentation at 100,000 g. primary antibodies for 40 min at room temperature, washed in PBSThe membrane pellet was resuspended in homogenization buffer, containing 2% BSA, and then incubated with FITC and Texas redand phagosomes were collected by low-speed centrifugation (30 coupled goat anti-rabbit and/or goat anti-mouse antibodies (South-min, 380 g). ern Biotechnology Associates, Birmingham, AL). Actin was visual-

ized using phalloidin-Texas red (Molecular Probes, Leiden, The

Netherlands). Coverslips were mounted in FluoroGuard AntifadeProtein Analysis and Mass Spectrometry

Proteins were analyzed using one-dimensional (10%) SDSPAGE Mounting Reagent (Bio Rad) and analyzed using a confocal laser

scanning microscope system (MRC-60 0; Bio Rad laboratories, Her-according to Laemmli (1970) or two-dimensional IEF/SDSPAGE

as described (Lefkovits et al., 1985). For mass spectrometry, two- cules, CA)attached to an Axiovert 35M microscope (Carl Zeiss, Inc.,

Thornwood, NY). For each condition as well as for quantitation, 150dimensional gels were fixed and stained using colloidal Coomassie

(Novex, San Diego CA). The protein spot of interest was excised, bacteria-containing cells were analyzed, and a representative field

is shown.digested in situ, and characterized by matrix-assisted laser desorp-

tion ionization mass spectrometry (Fountoulakis and Langen, 1997). For immunohistochemistry, the liver was fixed for 4 hr at 4C in


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Cell446

periodate-lysine-paraformaldehyde, washed for 4 hr with phos- de Hostos, E.L., Bradtke, B., Lottspeich, F., Guggenheim, R., and

Gerisch, G. (1991). Coronin, an actinbindingproteinof Dictyosteliumphate-buffered saline (PBS) containing 10% , 15% , and 20% su-

discoideum localized to cell surface projections, hassequencesimi-crose, embedded in OCT compound (Miles, Elkhart, IN), frozen inlarities to G protein beta subunits. EMBO J. 10, 40974104.dry ice-acetone, and cut by a cryostat (Bright, Huntington, UK) into

6m thicksections. Afterinhibitionof endogenous peroxidaseactiv- de Hostos, E.L., Rehfuess, C., Bradtke, B., Waddell, D .R., Albrecht,ity as described by Isobe et al. (1977), immunohistochemistry was R., Murphy, J., and Gerisch, G. (1993). Dictyosteliummutants lackingperformed using the antibodies described above, with anti-rat Ig or the cytoskeletal protein coronin are defective in cytokinesis and cellsheep anti-rabbit Ig-horseradish peroxidase-linked F(ab)2 fragment motility. J. Cell Biol. 120, 163173.(Amersham,Little Chalfont, UK)as secondaryantibodies. Antibodies Desjardins, M., Huber, L.A., Parton, R.G., and Griffiths, G. (1994).were visualized with 3,3-diaminobenzidine (DAB; Dojin Chemical, Biogenesis of phagolysosomes proceeds through a sequential se-Kumamoto, Japan), and nuclei were stained with methylene green ries of interactions with the endocytic apparatus. J. Cell Biol. 124,or hematoxyline. 677688.

Dhandayuthapani, S., Via, L.E., Thomas, C.A., Horowitz, P.M., De-Acknowledgments retic, D., andDeretic, V.(1995).Greenfluorescent proteinasa marker

for gene expression and cell biology of mycobacterial interactions

with macrophages. Mol. Microbiol. 17, 901912.We are grateful to L. Kuhn and I. Lefkovits for support in the two-

dimensional gel analysis and M . Dessing for his help in confocal Faigle, W., Raposo, G., Tenza, D., Pinet, V., Vogt, A.B., Kropshofer,microscopy. We thank T. Miyazaki for his advice on cloning and V. H., Fischer, A., de Saint-Basile, G., and Amigorena, S. (1998). Defi-Deretic, Z. Hasan, and J. Thole for discussion as well as bacterial cient peptide loading and MHC class II endosomal sorting in astrains. We thankT. Miyazakiand M. Kopf fordiscussionand critical human genetic immunodeficiency disease: the Chediak-Higashievaluation of the manuscript. The Basel Institute for Immunology syndrome. J. Cell Biol. 141, 11211134.was founded and is supported by F. Hoffmann-La Roche and Co., Falkow, S. (1991). Bacterial entryintoeukaryoticcells. Cell65, 1099Ltd., Basel, Switzerland. 1102.

Ferrari, G., Knight, A.M., Watts, C., and Pieters, J. (1997). Distinct

Received November 25, 1998; revised April 7, 1999. intracellular compartments involved in invariant chain degradationand antigenic peptide loading of major histocompatibility complex

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