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© The Rockefeller University Press,
0021-9525/2001//165 $5.00
The Journal of Cell Biology, Volume 152, Number 1,
, 2001 165-180
Original Article |
The Phagosome Proteome
: Insight into Phagosome Functions
b Département de Pathologie et Biologie Cellulaire, Université de Montréal, Montréal, Quebec, Canada, H3C 3J7
c Neurodégénérescence et Plasticité, Hopital A. Michallon, Centre Hospitalier Universitaire, 38043 Grenoble, France
Département de pathologie et biologie cellulaire, Université de Montréal, C.P. 6128, Succ. Centre ville, Montréal, Québec, Canada, H3C 3J7.(514) 343-2459(514) 343-7250
michel.desjardins{at}umontreal.ca
Phagosomes are key organelles for the innate ability of macrophages to participate in tissue remodeling, clear apoptotic cells, and restrict the spread of intracellular pathogens. To understand the functions of phagosomes, we initiated the systematic identification of their proteins. Using a proteomic approach, we identified >140 proteins associated with latex bead–containing phagosomes. Among these were hydrolases, proton pump ATPase subunits, and proteins of the fusion machinery, validating our approach. A series of unexpected proteins not previously described along the endocytic/phagocytic pathways were also identified, including the apoptotic proteins galectin3, Alix, and TRAIL, the anti-apoptotic protein 14-3-3, the lipid raft-enriched flotillin-1, the anti-microbial molecule lactadherin, and the small GTPase rab14. In addition, 24 spots from which the peptide masses could not be matched to entries in any database potentially represent new phagosomal proteins. The elaboration of a two-dimensional gel database of >160 identified spots allowed us to analyze how phagosome composition is modulated during phagolysosome biogenesis. Remarkably, during this process, hydrolases are not delivered in bulk to phagosomes, but are instead acquired sequentially. The systematic characterization of phagosome proteins provided new insights into phagosome functions and the protein or groups of proteins involved in and regulating these functions.
Key Words: phagosome • proteome • matrix-assisted laser desorption/ionization–time-of-flight–mass spectrometry • membrane fusion • apoptosis
© 2001 The Rockefeller University Press
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Introduction |
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In this study, we present a comprehensive analysis of phagosomes from which >140 proteins have been identified. Furthermore, we built a two-dimensional (2-D) phagosome map that could be used to monitor complex series of modifications occurring during phagosome maturation. This systematic characterization of phagosomes has allowed us to extend greatly our understanding of this organelle and to propose new concepts regarding its biogenesis.
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Materials and Methods |
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In a third set of experiments, the phagosomes were lysed in 1% Triton X-114 to analyze their membrane-associated proteins. Triton X-114 partitioning of phagosome membrane proteins was performed by the method of Bordier 1981. The proteins present in the detergent phase were then separated by SDS-PAGE using standard procedures.
High Resolution 2-D Gel Electrophoresis
Total phagosome proteins were first separated according to their isoelectric point along linear immobilized pH-gradient strips of 18 cm (Amersham Pharmacia Biotech). Sample loading in the first dimension was performed by in-gel reswelling (Pasquali et al. 1997). The strips were then equilibrated in a solution containing 13 mM DTT for 10 min, and then in a solution containing 2.5% iodoacetamide for 5 min. The proteins were then separated according to their molecular mass using standard SDS-PAGE. The large gels (18 x 20 cm) were either silver stained for protein patterns analysis or processed for mass spectrometry (MS) analysis.
For MS analysis, unfixed gels were first incubated in a 1% sodium carbonate solution for 5 min followed by incubation in 0.2 M imidazole/0.1% SDS for 15 min. Gels were then rinsed in ultra pure water for 15 s and incubated in a 0.2-M zinc acetate solution for 45 s. The reaction was then stopped with several washes of ultra pure water.
Protein Digestion
The protein spots of interest were excised from 2-D gels and further washed and analyzed essentially as previously described (Shevchenko et al. 1996). In brief, gel pieces were washed in 25 mM ammonium hydrogenocarbonate (NH4HCO3), pH 8.0, for 30 min, and then in 50% acetonitrile 25 mM NH4HCO3 for another 30 min, and finally with ultra pure water before complete dehydration in a vacuum centrifuge. The 2-D gel pieces were reswollen with a minimum amount of sequenced grade modified porcine trypsin (Promega) solution containing from 0.25 to 0.5 µg of protease, depending on the amount of protein (typically 10 µl of a 0.05-µg trypsin/µl solution, in 25 mM NH4HCO3 containing 10% acetonitrile). When necessary, NH4HCO3 buffer was further added until the gel piece was completely rehydrated. Digestion was performed at 37°C for 3–5 h.
Matrix-assisted Laser Desorption/Ionization-MS Analysis
Mass spectra of the tryptic digests were acquired on a Biflex (Bruker-Franzen Analytik) matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer equipped with a gridless delayed extraction. The instrument was operated in the reflector mode. 0.5 µl of each digest solution (in 25 mM NH4HCO3/10% acetonitrile) was deposited directly onto the sample probe on a dry thin layer of matrix made of 
-cyano-4-hydroxy-trans-cinnamic acid (CCA) mixed with nitrocellulose (mixture 4:3 vol/vol, of a saturated solution of CCA in acetone, and a solution consisting of 5 mg nitrocellulose dissolved in 1 ml isopropanol/acetone, 1:1 vol/vol). Deposits were washed with 5 µl of 0.1% trifluoroacetic acid before the analysis. A mass list of peptides was obtained for each protein digest. This peptide mass fingerprint was then submitted to an appropriate software to identify the proteins (MS-FIT, available online at http://prospector.ucsf.edu/ucsfhtml3.4/msfit.htm, or ProFound, available online at http://129.85.19.192/prowl-cgi/ProFound.exe).
When a protein could not be identified from its tryptic peptide mass map, the tryptic digest was extracted twice with a 50% acetonitrile-25 mM NH4HCO3 solution. The digest solution and the extracts were then pooled, dried in a vacuum centrifuge, and desalted with ZipTip C18 (Millipore) before the nanospray tandem MS analysis.
Nanospray-MS/MS
A quadrupole time-of-flight (Q-TOF) instrument (Micromass) was used with a Z-Spray ion-source working in the nanospray mode. Approximately 3–5 µl of the desalted sample was introduced into a needle (medium sample needle; PROTANA Inc.) to run MS and MS/MS experiments. The capillary voltage was set to an average of 1,000 V, and the sample cone to 50 V. Glufibrinopeptide was used to calibrate the instrument in the MS/MS mode. MS/MS spectra were transformed using MaxEnt3 (MassLynx; Micromass Ltd.), and amino acid sequences were analyzed using PepSeq (BioLynx; Micromass Ltd.). Amino acid sequences, sequence tags, or peptide ion fragments that could be determined were used to screen the protein and expression sequence tag (EST) databases with dedicated software: Pepfrag (http://prowl1.rockefeller.edu/prowl/pepfragch.html), Scan (http://dna.stanford.edu/scan), or BLAST for searching homologies (http://www.ncbi.nlm.nih.gov/blast/blast.cgi).
Analysis of 2-D Gel Spot Patterns
Gels to be compared were always processed in parallel. The same number of phagosomes, determined by the number of latex beads, was loaded in the first dimension. All gels were made in a similar way and stained (silver or Coomassie blue) for the same period of time. For the analysis and comparison of protein patterns, the 2-D gels were scanned using the same scanner set ups. Spot detection and gel alignment were performed using the software package PD-Quest (Bio-Rad Laboratories).
Antibody Preparation and Immunofluorescence Analysis
A full-length cDNA encoding mouse Alix (Missotten et al. 1999) was cloned into the pGEX-6P-2 (Amersham Pharmacia Biotech) using SmaI-XhoI. This allowed the fusion of Alix with glutathione-S-transferase (GST). Alix-GST fusion protein was affinity purified using glutathione sepharose; Alix was cleaved from the GST using the "precision protease." Purified Alix was dialyzed against PBS and used to immunize rabbits. The antibody, purified on Protein G-sepharose, recognizes one single band migrating at 96 kD in Hek cells transfected with an expression vector encoding Alix.
In addition to Alix, some of the newly identified proteins were further studied by immunofluorescence to confirm their association to phagosomes using standard procedures. Antibodies against Rab7 and the cytoplasmic tail of LAMP1 were kind gifts from Stéphane Méresse (Centre d'lmmunologie de Marsaille, Luminy, France). Anti–flotillin-1 was a kind gift from Rob Parton (University of Queensland, Brisbane, Australia). Anti–14-3-3 was from Santa Cruz Biotechnology, Inc. Anti–GAPDH was from Chemicon.
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Results |
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With the migration conditions used in the present study, the phagosome preparations loaded on 2-D gels yielded patterns displaying a few hundred spots between 15 and 100 kD in size with pI values ranging from 3.0 to 
9.0. Most of these protein spots were not observed in 2-D gels of total cell lysates, demonstrating the ability of our approach to enrich phagosome proteins (not shown). A representative silver-stained gel of phagosomal proteins was used to display the identified proteins and build our database (Fig. 1). Since only a handful of the spots present on phagosome 2-D gels were known at the beginning of the present study (see Desjardins et al. 1994b), we systematically excised the visible spots from zinc acetate–stained gels for mass spectrometry analysis. These analyses allowed the recognition of >140 phagosomal proteins identified, for the most part, by peptide mass fingerprinting (PMF) (Table ).
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MS/MS analyses were conducted on proteins not identified by the PMF approach. In most cases, several peptide sequence tags were easily obtained and the protein was identified by mining protein databases with Pepfrag (PROWL). As an example, the lysosomal acid lipase (10% coverage) was confirmed by tandem MS. In other cases, when the mouse protein was not present in the protein and EST databases, we had to generate amino acid sequences by MS/MS and use BLAST software to look for proteins exhibiting high amino acid sequence homology (for example, GILT was identified from human databases). We could also reach some EST sequences using peptide sequence tags (e.g., ESTAA445025, which shows an homology with cathepsin A) or peptide mass fingerprints (only in the case of low molecular weight proteins; e.g., EST 1447369, similar to CU-Zn SOD), and even reconstruct one protein amino acid sequence by matching a first EST, and then clustering several ESTs [e.g., an ADP-ribosylation factor (ARF)–6 isoform].
Based on these different criteria, we identified 116 proteins by the PMF approach and 7 ESTs. 19 were further analyzed by MS/MS to get unambiguous identifications. All of these were confirmed. Furthermore, 17 protein spots yielded good MS spectra that could not be matched to any entries in current databases. Identification of these potentially novel proteins will require further analysis and sequencing.
Phagosome Membrane-associated Proteins
Separation of membrane proteins is still problematic with 2-D gels (Santoni et al. 2000). Although some of the proteins identified on 2-D gels from our samples are clearly membrane associated, such as flotillin-1, other expected proteins, such as those of the LAMP family, were not identified. Accordingly, to identify as many membrane-associated proteins as possible, we complemented our 2-D gel studies with SDS-PAGE analysis of phagosome membrane proteins obtained after phase partition in Triton X-114. Using this approach, we resolved 24 major bands visualized after zinc acetate staining (Fig. 2). From these bands, we were able to identify 36 proteins, 5 of which had previously been identified from the 2-D gels. Among these proteins were seven members of the ras superfamily: rab2, 3c, 5c, 7, 10, 11, and rab14, as well as rap1b. Interestingly, these included the small GTPases previously shown to be associated to phagosomes; namely, rab5, 7, 11, and rap1 (Desjardins et al. 1994a; Pizon et al. 1994; Cox et al. 2000). Transmembrane proteins were also identified in our phagosome preparations, including LAMP1, LAMP2, lgp110, and LIMPII. LAMP1 was also identified by MS/MS analysis in a band at 44 kD. Other identified membrane proteins included stomatin, Tmp21, and VDAC1.
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Modulation of Phagosome Composition during Phagolysosome Biogenesis
Our 2-D database is a powerful tool that can be used to follow modifications of phagosome composition in various conditions. To illustrate the usefulness of this database, we isolated phagosomes at different time points after their formation in order to monitor the changes occurring to phagosomes during phagolysosome biogenesis. Several proteins were shown to be modulated during this process (Fig. 5, arrowheads), including a variety of hydrolases (Table ). Our results clearly indicate that hydrolases are not acquired simultaneously by phagosomes. While some hydrolases, such as cathepsin A and β-hexosaminidase are already present in a high amount in early phagosomes, others, such as cathepsin S and the cleaved form of cathepsin D, appear at later time points during phagolysosome biogenesis (Fig. 6). Others, such as the recently cloned cathepsin Z (Santamaría et al. 1998), are present at early time points but disappear during phagosome maturation, suggesting that they are either recycled or degraded and that they play a role in the processing of peptides at the early stages of phagolysosome biogenesis.
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Discussion |
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In the present study, we identified >140 of the proteins present on or within latex bead–containing phagosomes. Several of these proteins are expected constituents of an organelle that moves along cytoskeletal elements, interacts and fuses with endovacuolar organelles, acidifies its lumen, and degrades its content. Among these are a series of hydrolases, subunits of the vacuolar proton pump ATPase, molecules of the fusion/fission machinery, various GTPases of the Ras superfamily, as well as coat proteins and cytoskeletal-related proteins. Remarkably, a series of novel proteins, not previously reported to be associated with phagosomes were also identified. The confirmation of the enrichment of several of these proteins on phagosomes by various approaches, including differential 2-D gel display against a macrophage total cell lysate, Western blot analysis, and immunofluorescence localization, allowed us to propose new concepts regarding the molecular mechanisms of phagolysosome biogenesis.
Membrane Fusion and Small GTPases
Phagosomes were shown to fuse sequentially with subsets of endosomes (Desjardins et al. 1997). Thus, it was not surprising to find SNARE molecules and regulatory small GTPases, including Rab4, Rab5, Rab7, Rab11, and Rap1b, previously described on phagosomes (Desjardins et al. 1994a; Pizon et al. 1994; Hackam et al. 1996; Cox et al. 2000). Rab5 and Rab7 allow phagosomes to interact with early and late endosomes, respectively (see Méresse et al. 1999), while the association of Rab4 and Rab11 with recycling endosomes (Sheff et al. 1999; Sonnichsen et al. 2000) suggests that recycling processes might also be important for phagolysosome biogenesis. Three other Rab proteins, Rab 3c, Rab10, and Rab14, have been identified on phagosomes in the present study. Rab3, a small GTPase involved in Ca2+-dependent exocytosis (see Geppert and Sudhof 1998), was also localized to endosomes (Slembrouck et al. 1999). The functions for Rab10 and Rab14 still remain to be elucidated. A novel GTPase of the ARF family was also identified by tandem MS and searches in EST databases (AI006608). Based on the alignment of two overlapping ESTs, this sequence was predicted to code for a 21.6-kD protein (189 amino acids) displaying 64% homology to human and mouse ARF6. Western blot analysis using a polyclonal antibody raised against the COOH-terminal region of the protein indicated that this ARF6-like protein is highly enriched on phagosomes compared with a total cell lysate of macrophages (not shown). Previous studies have shown that ARF6 is involved in actin cytoskeleton organization and recycling process (D'Souza-Schorey et al. 1998; Franco et al. 1999). A similar role on phagosomes remains to be established.
Maturation of Phagosomes Is Accompanied by the Sequential Acquisition of Hydrolases
We have highlighted in the present study the presence of several hydrolases in phagosomes, including six members of the cathepsin family as well as some of their cleaved forms. In addition, 12 other hydrolases have been identified, including legumain, shown to be involved in the processing of microbial peptides with affinity for major histocompatibility complex (MHC) class II molecules (Manoury et al. 1998), and GILT, a gamma interferon-induced lysosomal thioesterase (Arunachalam et al. 2000).
Using our 2-D gel database, we were able to show that phagosome composition is modulated during phagolysosome biogenesis (Fig. 5). An interesting observation was that hydrolases are not delivered simultaneously to phagosomes (Fig. 6). Instead, they appear sequentially, at different time points during phagosome maturation. Cathepsin A, an exopeptidase required for the activity of other hydrolases, is already present at its highest detectable level in early phagosomes, at a time point when cathepsins D and S are barely detectable. The appearance of the 46-kD form of cathepsin D in phagosomes occurs before the detection of the 32-kD cleaved form of this protein. While most of the hydrolases seem to reach their highest level 6 h after phagosome formation, the levels of few others, such as cathepsin S, continue to increase as late as 24 h after phagosome formation. Since phagolysosome biogenesis is accompanied by the sequential fusion of phagosomes with early endosomes, late endosomes, and lysosomes (Desjardins et al. 1997), our results support the hypothesis that hydrolases are heterogeneously distributed along the degradative pathway (Munier-Lehmann et al. 1996). The sequential appearance of hydrolases in phagosomes might regulate the correct processing and presentation of antigens by MHC class II molecules (Villadangos and Ploegh 2000). These results indicate that our 2-D gel database is a unique tool to monitor changes occurring to phagosome proteins in various conditions.
Contaminants or Genuine Phagosomal Proteins?
Several proteins not previously shown to be associated with endocytic or phagocytic compartments were also identified in our phagosome preparations. These proteins were rather localized to other organelles, including the plasma membrane (flotillin-1), mitochondria (ATP synthase, prohibitin and VDAC-1), the cytoplasm (GAPDH, 14-3-3), and the endoplasmic reticulum (calreticulin, calnexin, GRP78, endoplasmin, Erp29). The simplest explanation for the presence of these proteins is that our phagosome preparations are contaminated by other organelles, a limitation of most subcellular fractionation approaches. However, unlike most approaches relying on the isolation of organelles based on their intrinsic density, isolation of latex bead–containing phagosomes is facilitated by the low buoyant density of latex. Thus, phagosomes are floated in a region of the sucrose gradient where other cellular organelles are not detected. Previous morphological and biochemical analysis of latex bead phagosome preparations indicated the virtual absence of contamination by mitochondria, Golgi vesicles, endosomes, and the plasma membrane, while low levels of ER elements were found (Desjardins et al. 1994b). Based on all these observations, it is worthwhile to consider that the presence in our preparations of molecules previously identified in other compartments is perhaps representative of more than a simple contamination.
Four mitochondrial proteins were identified in our phagosome preparations, which are ATP synthase, prohibitin, HSP60, and VDAC-1. Interestingly, two of these proteins have been shown to be present on other compartments in the cell. Prohibitin was found to be associated with receptors at the cell surface (see below), while VDAC-1, a type 1 porin molecule of the outer mitochondrial membrane, was shown to be present on endosomes by immunofluorescence and immunogold electron microscopy (Reymann et al. 1998). VDAC-1 was also shown to be present at the cell surface, where it is targeted through the expression of an alternative first exon (Buettner et al. 2000). The potential roles of these molecules on extramitochondrial compartments are not known. An additional finding suggesting that the detection of prohibitin and VDAC-1 in our phagosome preparation is not due to mitochondrial contamination is the fact that these proteins were not identified as major constituents of mitochondria in a recent in-depth proteomic study (Rabilloud et al. 1998). Furthermore, at least two of the most prominent spots present on 2-D gels of mitochondria, fumarate hydratase and aconitase, are not present at their migration coordinates in our phagosome gels. If mitochondria were contaminating our preparations, one would assume that these spots would also be present in our gels. These data support the concept that at least some of the mitochondrial proteins present in our preparations might be genuine constituents of phagosomes.
Insights into New Phagosome Functions
ER Recruitment to Phagosomes.
Our phagosome analysis allowed us to identify several molecules normally associated with the endoplasmic reticulum. Although this might represent a contamination, further data suggest that ER elements could interact directly with phagosomes. The pronase experiments have shown that all the lumenal ER proteins identified in our 2-D gels were not affected by the pronase treatment, indicating, at least, that they are not simply associated with ER elements entrapped in the cytoskeletal matrix associated to the phagosomes and floated during the isolation procedure. A further indication of the close association of ER with phagosomes came from Western blot analysis showing the enrichment of calnexin in our phagosome preparations compared with the total cell lysate. Furthermore, pre-embedding immunocytochemical analysis using an antibody against the cytosolic portion of calnexin demonstrated the presence of this protein on the phagosomal membrane, while an antibody against the lumenal portion of the molecule gave no signal (Gagnon, E., and M. Desjardins, manuscript in preparation). The presence of ER molecules on phagosomes is not surprising. Recent studies have shown that microorganisms such as Legionella and Brucella reside within their host cells in compartments displaying ER features (see Méresse et al. 1999). In our experiments, it was not uncommon to observe macrophages that had engulfed >25 latex beads. Despite the important surface of plasma membrane needed for the engulfment of these particles, the cell does not consume itself, but rather maintains a relatively stable size. This suggests that while plasma membrane recycles rapidly back to the cell surface (Pitt et al. 1992), membrane from the ER could be recruited to keep the particles within closed compartments.
Another interesting link between phagolysosomes and ER elements comes from the finding that calreticulin, a chaperone of the ER identified in our phagosome preparations, is also present in the lysosome-like lytic granules of T lymphocytes (Dupuis et al. 1993). There, it controls the lytic activity of perforin by stabilizing membranes to prevent polyperforin pore formation (Fraser et al. 2000). Although perforin has not been identified in our phagosome preparations, a newly identified protein, MPS1, was shown to display a certain homology with perforin (Spilsbury et al. 1995). The MPS1 gene was first identified as being upregulated during monocyte-to-macrophage differentiation (Spilsbury et al. 1995), as well as during prion infection, along with other lysosomal hydrolases (HEXA and HEXB) (Kopacek et al. 2000). The coded protein appears to share distant ancestry to perforin, although its property to form pores, yet alone to polymerize or span a membrane, has not been shown. Further analyses should clarify whether the ER is a contaminant or if it is recruited to form phagosomes.
Lipid Rafts.
Among the new phagosomal proteins found in our study is flotillin-1. This protein was reported to be associated with caveolae or other subdomains of the plasma membrane (Bickel et al. 1997; Lang et al. 1998). Its identification by mass spectrometry on phagosomes, further confirmed by Western blot and immunofluorescence analyses, suggests that lipid rafts could also be present on phagosomes, and not solely on the Golgi apparatus or the plasma membrane (Simons and Ikonen 1997). A second molecule recently shown to be associated to lipid rafts, stomatin (Snyers et al. 1999), is also present on phagosomes. Although stomatin was first proposed to be involved in the maintenance of the structural integrity of the red blood cell membrane, this function was ruled out after the observation that red blood cells from stomatin knockout mice were not altered (Zhu et al. 1999). Of further interest, flotillin-1 and stomatin share a homologous domain with prohibitin, a molecule also present on lipid rafts (Terashima et al. 1994), identified in our phagosome preparations. Although prohibitin is present in mitochondria (Ikonen et al. 1995), reports indicate that it may associate with certain receptors present at the cell surface (Terashima et al. 1994), a phenomenon that could explain its presence on phagosomes. The potential involvement of prohibitin in the regulation of mitochondrial membrane protein degradation (Steglich et al. 1999) may be relevant to some of the phagolysosome degradative functions. Altogether, these results strongly suggest that specialized subdomains or lipid rafts might be present on the phagosome membrane. Lipid rafts have been implicated in many important cellular processes, such as polarized sorting of apical membrane proteins in epithelial cells and signal transduction (for review, see Kurzchalia and Parton 1999). Interestingly, molecules involved in signal transduction, such as the , β1, and β2 subunits of trimeric G-protein, as well as annexin II, are present on phagosomes (Desjardins et al. 1994b; Berón et al. 1995).
Phagosomes and Apoptosis.
Using immunofluorescence analysis, we were able to demonstrate that 14-3-3 is effectively associated with phagosomes. In adrenal chromaffin cells, 14-3-3 proteins were shown to regulate secretory vesicle exocytosis by reorganizing the cortical actin barrier (Morgan and Burgoyne 1992), possibly by interacting with annexin II (Roth et al. 1993), a protein also present on phagosomes (Desjardins et al. 1994b). Recently, dominant-negative forms of 14-3-3 were used to disrupt 14-3-3 function in cultured cells and transgenic animals. Fibroblasts transfected with these mutants exhibited markedly increased apoptosis, suggesting that a primary function of mammalian 14-3-3 proteins is to inhibit apoptosis (Xing et al. 2000). Interestingly, other molecules related to apoptosis have been identified in our study. These are Alix, annexin 5, TRAIL, and galectin3. All of these proteins are prominent spots in 2-D gels of phagosomes, but not detectable in gels from total cell lysates, demonstrating their enrichment on phagosomes (not shown). Intracellular TRAIL/Apo2L has been demonstrated in macrophages, but, to our knowledge, this is the first report of its enrichment within phagosomes. This finding is noteworthy in view of the recent report that thymocytes from ICAD-Sdm mice only develop oligonucleosomal DNA degradation inside macrophages (McIlroy et al. 2000), and that the emergence of TUNEL-positive cells is prevented in Caenorhabditis elegans Ced-7 mutants, in which engulfment is impaired (Wu et al. 2000). These observations suggest that phagolysosomes may induce apoptosis within macrophages through an unknown mechanism. Intraphagosomal TRAIL binding to its receptor on the phagocytosed cell could be part of such a mechanism. A role for lysosomes in cell-autonomous apoptosis has also been suggested since autophagy or cathepsin D translocation to nonlysosomal structures can be instrumental during the death of nonphagocytic cells (see Ferri and Kroemer 2000). Our findings showing that proteins with a demonstrated function in apoptosis like galectin 3, Alix, VDAC1, GAPDH, or 14-3-3 are enriched in phagosomes reinforces this hypothesis and may open new avenues in understanding the role of phagolysosomal compartments in apoptosis.
Phagolysosome biogenesis is obviously a complex process made possible by the contribution of a large number of molecules. In the last few years, in-depth studies of some of the phagosome proteins have allowed us to significantly increase our knowledge of this organelle's functional properties. In the present study, we used a proteomic approach to gain a global view of the composition of phagosomes and their potential functions. The confidence level attained by the identification of several key phagosomal components and the immunofluorescent localization of some of the unexpected proteins to phagosomes, together with the low contamination of our preparations by other organelles, allows us to consider most of the proteins identified in this study as genuine constituents of phagosomes. This wide body of data provides new insights into the molecular mechanisms governing phagosome functions and phagolysosome biogenesis (Fig. 7). This proteomic approach is likely to become extremely powerful as we learn to fully use all of its strengths.
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Acknowledgments |
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This work was supported by grants from the Medical Research Council (MRC) of Canada (GX-15526 and MT-12951) and from FCAR Equipe. J.-F. Dermine is the recipient of a studentship from Natural Sciences and Engineering Research Council of Canada, S. Duclos is the recipient of a studentship from the MRC, and M. Desjardins is a Scholar from Fonds de la recherche en santé du Quebec.
Submitted: 15 September 2000
Revised: 9 November 2000
Accepted: 10 November 2000
Drs. Garin and Diez contributed equally to this work and should be considered co-first authors.
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