Rab7 and Phagosome Maturation 2451

RESEARCH ARTICLE 2449 Rab7 regulates phagosome maturation in Dictyostelium

Adam Rupper1, Bryon Grove2 and James Cardelli1,* 1Department of Microbiology and Immunology and The Feist/Weiller Cancer Center, LSUHSC, Shreveport, LA 71130, USA 2Department of Anatomy and Cell Biology, University of North Dakota, Grand Forks, North Dakota 58202, USA *Author for correspondence (e-mail: [email protected])

Accepted 6 April 2001 Journal of Cell Science 114, 2449-2460 © The Company of Biologists Ltd

SUMMARY

A Dictyostelium Rab7 homolog has been demonstrated to LBC phagosomes isolated from cells overexpressing regulate fluid-phase influx, efflux, retention of lysosomal dominant negative (DN) Rab7 contained very low levels of hydrolases and phagocytosis. Since Rab7 function LmpA (lysosomal integral membrane protein) and α- appeared to be required for efficient phagocytosis, we mannosidase was not detectable. Interestingly, cysteine sought to further characterize the role of Rab7 in proteinases were delivered to phagosomes as apparent pro- phagosomal maturation. Expression of GFP-Rab7 resulted forms in cells overexpressing DN Rab7. Despite these in labeling of both early and late phagosomes containing defects, phagosomes in cells overexpressing DN Rab7 yeast, but not forming phagocytic cups. In order to matured to form multi-particle spacious phagosomes, determine if Rab7 played a role in regulating membrane except that these phagosomes remained significantly more traffic between the endo/lysosomal system and maturing acidic than control phagosomes. These results suggested phagosomes, latex bead containing (LBC) phagosomes that Rab7 regulates both an early and late steps of were purified from wild-type cells at various times after phagosomal maturation, similar to its role in the internalization. Glycosidases, cysteine proteinases, Rab7 endo/lysosomal system. and lysosomally associated membrane proteins were delivered rapidly to nascent phagosomes in control cells. Key words: Rab7, Phagocytosis, Phagosome maturation

INTRODUCTION vacuole (Wichmann et al., 1992; Schimmoller and Riezman, 1993). While most data suggest that Rab7 regulates trafficking The Rab small molecular mass GTPases have taken center from early endosomes to late endosomes, two papers propose stage as master regulators of vesicle trafficking events (Somsel that GTPase inactive forms of Rab7 localize predominantly to and Wandinger-Ness, 2000). These proteins are thought to lysosomes and, in one case, that expression of a dominant regulate formation of transport vesicles from donor negative (DN) form of Rab7 causes lysosomes to disperse compartments, transport of membrane vesicles on cytoskeletal from their perinuclear location in Hela cells (Bucci et al., structures, tethering of vesicles to acceptor compartments and 2000; Meresse et al., 1995). Interactions between late fusion of lipid bilayers. Rab proteins are activated in the GTP- endosomes and lysosomes have been suggested to occur bound state and interact with effectors until GTP hydrolysis through formation of a hybrid organelle, which can then has occurred (Bourne, 1988). undergo fission events to result in reformation of late As the hypothesis that Rab proteins regulate the specificity endosomes and lysosomes (Pryor et al., 2000; Mullock et al., of fusion events would predict (though it is now clear that other 1998). Though evidence points to the involvement of a Rab factors such as membrane tethers act in concert with Rabs to protein, it is not clear whether Rab7 or some other Rab protein provide specificity), individual Rab proteins have been may be involved. localized to specific compartments in the endo/lysosomal A cDNA with 85% identity to human Rab7 has been cloned pathway. For instance, Rab4 and Rab5 have been localized to from Dictyostelium and indirect immunofluorescence has early endosomes. Rab5 regulates fusion of clathrin-coated shown that Rab7 associates with endosomes and post- vesicles with early endosomes and homotypic fusion of early lysosomes (Buczynski et al., 1997). The endo/lysosomal endosomes (Bucci et al., 1992; Barbieri et al., 1994), while system of Dictyostelium consists of a linear pathway including Rab4 regulates recycling of membrane receptors to the plasma endosomes, lysosomes and a terminal secretory compartment membrane (van der Sluijs et al., 1991; van der Sluijs et al., known as a post-lysosome (Padh et al., 1993). Rates of 1992; Daro et al., 1996). Rab7 and Rab9 have been localized phagocytosis and endocytosis are lower in a cell line to late endosomes (Chavrier et al., 1990; Soldati et al., 1995), overexpressing DN Rab7 (T22N mutation) (Buczynski et al., where Rab9 regulates recycling of membranes back to the 1997). In addition, the rate of fluid-phase efflux is slower, Golgi complex (Lombardi et al., 1993). while paradoxically lysosomal enzymes are over-secreted Reports have suggested that Rab7 plays a role in vesicular (Buczynski et al., 1997), and these cells also contain vacuoles transport from early endosomes to late endosomes (Feng et the size of post-lysosomes that are acidic (post-lysosomes are al., 1995; Mukhopadhyay et al., 1997; Vitelli et al., 1997), and normally close to neutral pH). It was hypothesized that Rab7 in yeast (Rab7 homolog Ypt7), from late endosomes to the regulates membrane and hydrolase recycling from post- 2450 JOURNAL OF CELL SCIENCE 114 (13) lysosomes to an earlier endosomal compartment since cells MATERIALS AND METHODS overexpressing wild-type Rab7 retain lysosomal enzymes more efficiently, while rates of fluid-phase efflux are increased. This Cells and culture conditions recycling event may be necessary for proper maturation of D. discoideum, strain Ax4, was grown axenically at 18°C in HL5 post-lysosomes; thus, expression of DN Rab7 slows efflux, and growth medium (1% Oxoid proteose peptone, 1% glucose, 0.5% yeast results in a membrane ‘traffic jam’, which in turn slows the extract, 2.4 mM Na2HPO4, and 8.8 mM KH2PO4, pH 6.5) either in internalization pathways. Consistent with these observations is shaking suspension or in tissue culture flasks. Construction of the Ax4 a report demonstrating that Rab7 regulates homotypic fusion strain overexpressing DN Rab7 was described in a previous events between early endosomes (Laurent et al., 1998). Thus, publication (Buczynski et al., 1997). In brief, site-directed mutagenesis was performed to generate a Thr 22 to Asn mutation in Rab7 might direct recycling vesicles to early endosomes and the Rab7 cDNA. The resulting cDNA was cloned into the pDA80-HA may also regulate homotypic interactions between early vector behind and in-frame with the flu hemagglutinin epitope, endosomes. transformed into Dictyostelium and G418 resistant colonies were We were interested in pursuing the role of Rab7 in selected and screened for overexpression by western blot analysis. A phagocytosis and phagosomal maturation, since expression of cell line expressing green fluorescent protein (GFP) fused in-frame to DN Rab7 inhibited phagocytosis of latex beads, and subcellular the N terminus of Rab7 was generated as follows. A cDNA encoding fractionation experiments demonstrated that Rab7 was GFP with the following mutations, F64L and S65T, was cloned into enriched in early phagosomes (Buczynski et al., 1997). A role the KpnI site of pVEII∆ATG (Rebstein et al., 1993). The full-length for Rab7 in phagosomal maturation in mammalian cells has not cDNA encoding wt Rab7 was cloned into the SacI site of the resulting been described, though Rab7 does localize to phagosomes plasmid. The resulting plasmid was purified using (Qiagen Inc., Valencia, CA, USA) Qiafilter Maxi Prep columns and was (Rabinowitz et al., 1992; Desjardins et al., 1994). Rab5 has transformed into D. discoideum, Ax3 strain, by the calcium phosphate been the key protein described to regulate interactions of early precipitation method (Sadeghi et al., 1988). Geneticin (Sigma, St endosomes and late endosomes with phagosomes, though it is Louis, MO, USA) resistant colonies were screened for expression of unclear which Rab might regulate fusion of lysosomes with GFP fluorescence by observation with a fluorescent microscope. phagosomes (Alvarez-Dominguez et al., 1996; Funato et al., Construction of the GFP-ABD expressing cell line was described in 1997; Jahraus et al., 1998). Furthermore, pathogens such as Pang et al. (Pang et al., 1998) and was a kind gift of Dr David Knecht. Listeria and Salmonella have mechanisms to recruit Rab5 to Magnetic purification of the vesicles from the the phagosomal membrane and somehow delay subsequent endo/lysosomal system maturation steps (Hashim et al., 2000; Alvarez-Dominguez et Ax3 cells were harvested during log-phase growth between 3-5×106 al., 1996; Alvarez-Dominguez et al., 1997). Thus, the role of cells/ml, centrifuged at 1000 g for 5 minutes and resuspended at Rab proteins during phagosomal maturation is important and 2.0×108 cells/ml in HL5. The cells were then fed dextran-coated much remains to be discovered. colloidal iron at 3 mg/ml for 60 minutes to load vesicles of the In Dictyostelium, phagosomal maturation consists of an endo/lysosomal system. Iron-containing vesicles were purified as early acidic phase followed by a late non-acidic phase, similar described elsewhere (Rodriguez-Paris et al., 1993). to that seen in the endo/lysosomal system (Rupper et al., 2001). Early phagosomes are acidified to a pH of Purification of latex bead containing phagosomes Control cells or cells overexpressing DN Rab7 were harvested during approximately 5, while late phagosomes have a pH of over 6. × 6 Concomitant with the rise in pH, mature phagosomes fuse to log-phase growth between 3-5 10 cells/ml, centrifuged at 1000 g for 5 minutes and resuspended at 2.5×107 cells/ml in HL5. The cells were give rise to late, spacious phagosomes. We have found that allowed to recover for 10 minutes prior to addition of latex beads. an increase in phagosomal pH and fusion of mature Latex beads (0.8 µm diameter, blue-dyed; Sigma, St Louis, MO, USA) phagosomes requires phosphatidylinositol 3-kinase activity, were added at a concentration of 100 beads/cell and incubated with as cells with a disruption in both ddpik1 and ddpik2 (two the cells for various periods. The cells were then washed 4 times with homologs of mammalian class I PI 3-kinases) and cells cold HL5 by centrifugation at 1000 g for 5 minutes. The cells were inhibited with PI 3-kinase inhibitors were blocked in these then resuspended at 2.5×107 cells/ml in HL5 and chased to various functions (Rupper et al., 2001). Furthermore, a cell line with time points in shaking suspension. At each time point, 5.6×108 cells a genetic disruption of PKB (a Dictyostelium protein kinase were harvested, washed once in cold homogenization buffer (100 mM B homolog) was also blocked in these steps, suggesting that sucrose, 5 mM glycine, pH 8.5) and the pellet was kept on ice. The PI 3-kinases act through a PKB dependent pathway (Rupper cell pellets were resuspended in 2.8 ml of cold homogenization buffer supplemented with protease inhibitors (0.1 mM TLCK, 5 µg/ml et al., 2001). leupeptin, 5 µg/ml pepstatin A, 5 µg/ml aprotinin and 0.5 mM phenyl Other work has demonstrated that proteins from the methyl sulphonyl fluoride, final concentrations; Sigma, St Louis, MO, endo/lysosomal system are rapidly delivered to nascent USA) and the cells were broken by passage through doubled, 5 µm phagosomes in Dictyostelium (Rezabek et al., 1997) and that pore size, polycarbonate filters (Osmonics, Inc.). Protease inhibitors two types of lysosomes may sequentially fuse with nascent were not included in preparations where protease activity was to be phagosomes containing bacteria (Souza et al., 1997). These measured. Phagosomes were prepared from cell homogenates as two types of lysosomes contain hydrolases with distinct sugar described. modifications (see below). Western blot and protease activity analysis In the present study, we sought to answer the following Equivalent amounts of PNS and phagosome protein samples were questions. Does Rab7 direct internal membrane to the loaded onto gels, and separated by SDS-PAGE using the phagosomal cup in Dictyostelium, accounting for its role in discontinuous buffer system of Laemmli (Laemmli, 1970). In the case phagocytosis? Does Rab7 regulate phagosomal pH changes of silver stains, we used the Silver Stain Plus kit from Bio-Rad and or delivery of endo-lysosomal proteins to maturing followed the manufacturer’s recommended protocol. For western phagosomes? blots, proteins were electroblotted to nitrocellulose membranes in Rab7 and phagosome maturation 2451

Towbin buffer at 100 V for 1 hour (Towbin et al., 1979). The 60× oil objective. Laser lines of 488 nm and 568 nm of the membranes were blocked overnight with TBSTG buffer (10 mM Tris Krypton/Argon laser were used at 3% laser power to simultaneously base, 150 mM NaCl, 0.05% (w/v) Tween-20, 0.1% (v/v) gelatin). All excite FITC and Crimson Red, respectively. FITC fluorescence was antibodies were diluted with TBSTG buffer. The following antibodies imaged after passing through a 522±35 nm filter and Crimson Red were used in this study: B30.2 (recognizes 70 kDa subunit of the V- fluorescence was imaged after passing through a 605±32 nm filter. ATPase specifically; Fok et al., 1993), 1:100; B832 (recognizes 100 The images were analyzed with IPLAB software (Scanalytics, Inc., kDa subunit of the V-ATPase specifically; Fok et al., 1993), 1:100; Fairfax, VA, USA) and scored for the total number of phagosomes LSU 27-2 (recognizes α-mannosidase specifically; J. Cardelli, labeled by both FITC-bacteria and Crimson Red latex bead unpublished results), 1:5000; LSU 18-2 (recognizes 41 kDa subunit fluorescence per cell. Since FITC was cleaved from the bacterial of V-ATPase specifically; Temesvari et al., 1996), 1:2000; 7-2-4 surface by the end point and thus filled the phagosome with (recognizes Rab7 specifically; Buczynski et al., 1997), 1:200; anti- fluorescence, phagosomes were scored based on colocalization of Cathepsin D (recognizes cathepsin D specifically; Journet et al., FITC and Crimson Red fluorescence. 1999), 1:200; anti-CPp36 (recognizes cysteine proteinase p36 specifically, a kind gift of J. Garin), 1:1000; anti-LmpA (a CD36, Fluorescent labeling of bacteria and yeast LIMP II homologue) (recognizes LmpA specifically; Karakesisoglou E. coli from a stationary-phase culture were washed once with 0.1 M et al., 1999), 1:2000 and anti-Glc-Nac-1-phosphate (Souza et al., NaHCO3, pH 9.0, and resuspended in 0.1 M NaHCO3, pH 9.0, with 1997), 1:1000. After incubation with the antibody of interest and 1 mg/ml FITC Isomer 1 (Sigma) for 1 hour at 25°C with gentle extensive washing, blots were incubated with goat anti-mouse stirring. The labeled E. coli were then washed with PBS until the (BioRad, 1:3000 dilution in TBSTG) or goat anti-rabbit (Sigma, supernatant was free of visible FITC. The bacteria were resuspended 1:30,000 dilution), both alkaline phosphatase-conjugated secondary in a suitable volume of PBS and stored at 4°C until needed. Yeast antibodies. The blots were developed in NBT buffer (100 mM Tris from a stationary-phase culture were washed once with PBS and then base, 100 mM NaCl, 5 mM Mg/Cl2) containing the alkaline heat-killed by boiling for 15 minutes. The yeast were then washed phosphatase substrates BCIP (1.2 mM, Amresco) and Nitro Blue with 0.1 M NaHCO3, pH 9.0, and resuspended in 0.1 M NaHCO3, pH Tetrazolium (0.6 mM, Amresco). 9.0, with 1 mg/ml Rhodamine B isothiocyanate (Sigma) for 1 hour at Cysteine proteinase activity was detected following electrophoretic 25°C with gentle stirring. The labeled yeast were then washed with separation of phagosome protein samples as described above except PBS until the supernatant was free of visible Rhodamine. that the polyacrylamide gels contained 0.2% gelatin (w/v) as a substrate for the proteinases and SDS-PAGE was performed at 4°C to Measurement of phagosomal pH prevent proteinase activity during migration. As a control, some PNS Control cells or cells overexpressing DN Rab7 were harvested in log- and purified phagosomal fractions were incubated with trans- phase growth in shaking suspension and resuspended at 5×106 cells/ml epoxysuccinyl-L-leucylamido-(4-guanadino) butane (E-64, 10 µM) at in HL5 and allowed to recover 10 minutes. Control cells and cells room temperature for 30 minutes prior to being loaded on the overexpressing DN Rab7 were pulsed with FITC-labeled E. coli for polyacrylamide gel. Following electrophoresis, the gel was incubated 10 minutes in shaking suspension. The cells were then diluted with in 2.5% Triton X-100 (v/v) with mild agitation for 30 minutes at room ice-cold HL5 and centrifuged at 1000 g at 4°C; this was repeated 3 temperature, and then in 0.1 M sodium acetate/acetic acid buffer (pH times, followed by resuspension at 5×106 cells/ml in room 4.0) with 1 mM dithiothreitol for 16 hours. The gels were then stained temperature HL5. At each time point 5×106 cells were harvested and with Coomassie Brilliant Blue (0.25% (w/v) in 50% (v/v) methanol washed twice with ice-cold HL5 and once with ice-cold 50 mM MES and 10% (v/v) acetic acid) and destained with a solution of 50% (v/v) (pH 6.5) buffer. The cells were resuspended in 2 ml of MES buffer methanol and 10% (v/v) acetic acid in order to visualize proteolyzed and fluorescence was measured with a Hitachi fluorimeter (Model F- bands. Incubation of the samples with E64 inhibited protease activity 4010) at 520 nm following excitation at 450 nm and 495 nm. The ratio completely except in the top two bands visible in the gel, suggesting of fluorescence emission at 450 nm and 495 nm was calculated and that a majority of the visible activity was due to cysteine proteinases. used to determine the intraphagosomal pH by extrapolation from an in vitro standard curve determined by measuring the fluorescence ratio Immunofluorescence (IF) microscopy of FITC-labeled E. coli in buffers from pH 4-7. 1×106 cells were allowed to attach to glass coverslips for 10 minutes and then fixed with 2% formaldehyde in phosphate-buffered saline (PBS) containing one-third strength HL5 and 0.1% DMSO for 5 RESULTS minutes at room temperature, followed by permeabilization at –20°C in 100% methanol containing 1% formaldehyde. LmpA was detected GFP-Rab7 localizes rapidly to nascent phagosomes by staining with a 1:500 dilution of a rabbit polyclonal antibody to as actin is removed LmpA (Karakesisoglou et al., 1999) and α-mannosidase was detected by staining with a mouse monoclonal antibody 2H9 at a dilution of It has been reported that Rab GTPases might regulate 1:50 (Bush and Cardelli, 1989). The primary antibodies were detected, exocytosis of internal membrane to the phagosomal cup and after washing, with Texas Red-labeled donkey anti-mouse or FITC- thus play a direct role in regulating phagocytosis (Bajno et al., labeled donkey anti-rabbit at a 1:100 dilution. Antibodies were diluted 2000; Hackam et al., 1998; Cox et al., 2000). Since expression in PBS containing 2.5 mg/ml bovine serum albumin and 0.1% of DN Rab7 in Dictyostelium causes a decrease in saponin. The cells were imaged with an Olympus BX50 fluorescence phagocytosis (30% of control rate) and Rab7 was found microscope using a CCD camera. enriched in purified phagosomal membranes after a short pulse (2 minutes) with latex beads, we hypothesized that Rab7 Confocal microscopy might regulate delivery of internal membrane to the Cells were allowed to internalize a mixture of FITC-labeled E. coli phagosomal cup (Buczynski et al., 1997). To test this and Crimson Red latex beads (Molecular Probes, Eugene, OR, USA) for 10 minutes in shaking suspension. The cells were then washed hypothesis, we generated a Dictyostelium cell line that twice with growth medium and chased for 120 minutes in shaking expresses a green fluorescent protein N-terminal fusion with suspension. 10 minutes prior to the end point cells were harvested and Rab7 (see Materials and Methods). This cell line was allowed allowed to attach to glass coverslips. Images were captured to internalize live yeast for 10 minutes, then fixed with 1% immediately with a BioRad MRC-1000 confocal microscope using a formaldehyde, or the cells were washed free of uninternalized 2452 JOURNAL OF CELL SCIENCE 114 (13) coverslips and then challenged with heat-killed yeast labeled with rhodamine B isothiocyanate (RITC-yeast). A cell with bright GFP-Rab7 expression was then brought into focus in a plane that also contained RITC-yeast and images were captured every 5 seconds. Contrary to our expectations, no detectable GFP-Rab7 localized to the base of forming phagosomal cups, nor could any association of GFP-Rab7 with the plasma membrane be found, but GFP-Rab7 localized rapidly to phagosomes within 60 seconds following internalization (Fig. 2). We were able to gain more insight into the timing of GFP- Rab7 association with phagosomal membranes by studying the dynamics of association of filamentous actin (F-actin) during particle internalization. Previous studies have shown that F- actin is required for phagocytosis in Dictyostelium and that F- actin-binding proteins transiently associate with early phagosomal membranes (Maniak et al., 1995; Rezabek et al., 1997). We sought to determine if the removal of F-actin coincided with the delivery of GFP-Rab7 to the phagosomal membrane. We again used confocal video microscopy to Fig. 1. GFP-Rab7 localizes to early and late phagosomal membranes. visualize the association of the actin cytoskeleton with forming GFP-Rab7 expressing cells were pulsed with live yeast for 10 phagosomes in a cell line that strongly expresses a GFP fusion minutes and then fixed with 1% formaldehyde and viewed by with the actin-binding domain (GFP-ABD) of ABP-120 (actin fluorescence (A) or DIC (A′) microscopy. The arrow denotes an early binding protein 120), an actin cross-linking protein in phagosome ringed with GFP-Rab7. GFP-Rab7 expressing cells were Dictyostelium. It has been shown that the staining pattern of pulsed with live yeast for 120 minutes then fixed with 1% GFP-ABD mimics that of fluorescent phalloidin, strongly ′ formaldehyde and viewed by fluorescence (B) or DIC (B ) suggesting that GFP-ABD only bound to F-actin in the cell microscopy. The arrow denotes a multi-particle phagosome ringed (Pang et al., 1998). with GFP-Rab7. Bar, 2 µm. By examining images captured by confocal microscopy of GFP-ABD cells internalizing RITC-yeast, we found that GFP- yeast and incubated in yeast-free growth medium for 120 ABD strongly labeled the forming phagocytic cup and minutes before fixation. Images taken by fluorescence completely ringed the newly internalized phagosomes (Fig. 3). microscopy clearly demonstrated that yeast-containing Once the phagosome closed, GFP-ABD was rapidly removed phagosomes were ringed with GFP-Rab7 at both early and late from the phagosomal membrane within 30-60 seconds of time points (Fig. 1). Many other vesicular structures from the phagosomal closure. In some cases it appeared that GFP-ABD endo/lysosomal system were ringed with GFP-Rab7 and this was more rapidly removed from the hemisphere of the labeling pattern agreed nicely with previous published results phagosome facing the cytoplasm than it was from the surface of indirect immunofluorescence using a specific antibody to of the phagosome adjacent to the cell cortex. We cannot Rab7 (Buczynski et al., 1997). conclude from these images that removal of F-actin was These results confirmed that Rab7 localizes to early complete or that F-actin does not reassociate with phagosomes phagosomal membranes. We next determined the kinetics later during maturation. The timing of removal of the actin of delivery of GFP-Rab7 to phagosomal membranes by cytoskeleton from maturing phagosomes coincides with the performing confocal videomicroscopy with living cells. association of GFP-Rab7 with phagosomal membranes, as Cells expressing GFP-Rab7 were allowed to attach to glass described above.

Fig. 2. GFP-Rab7 associates with phagosomes within 60 seconds of internalization. GFP-Rab7 expressing cells were allowed to attach to glass coverslips and mounted in a chamber to facilitate imaging by confocal microscopy. RITC-labeled yeast were added to the growth media and images were captured every 5 seconds. Selected images of a single experimental series are shown. Note that the newly internalized yeast particle in the right portion of the cell does not receive GFP-Rab7 until at least 20 seconds after uptake (panel labeled 60″). The particle in the left portion of the cell appears to be surrounded by GFP-Rab7 vesicles during internalization but a closer inspection of the cup, particularly at 0 seconds and 40 seconds reveals Rab7-free areas. Bar, 5 µm. Rab7 and phagosome maturation 2453

Fig. 4. Purified phagosomes containing latex beads are free from contamination by endosomes and lysosomes. Cells were incubated with growth medium containing 35S-methionine for 1 hour in order to label proteins. Unlabeled cells (A) were pulsed with latex beads for 20 minutes, washed, mixed with labeled cells and latex bead Fig. 3. GFP-ABD is rapidly removed from nascent phagosomes. containing phagosomes were purified as described in Materials and GFP-ABD expressing cells were allowed to attach to glass coverslips Methods. Labeled cells (B) were treated as described for unlabeled and mounted in a chamber to facilitate imaging by confocal cells in A. Latex beads (C) were added to labeled cell homogenate microscopy. RITC-labeled yeast were added to the growth media and and then purified from the homogenate as in A. Equal protein loads images were captured every 5 seconds. Selected images of a single were separated by SDS-PAGE and 35S-met labeled proteins were µ experimental series are shown. Bar, 5 m. visualized by fluorography. H, cell homogenate; P, purified phagosomal membranes. The positions of marker proteins (kDa) are shown. Endo-lysosomally localized proteins are rapidly delivered to maturing phagosomes labeled proteins (Fig. 4A). This strongly suggested that other Since Rab7 did not appear to play a direct role in the organelles from labeled cells, such as endosomes and internalization of phagocytic particles, we hypothesized that lysosomes, which would have contained 35S-methionine DN Rab7 might slow the rate of phagocytosis by blocking labeled proteins, did not copurify with latex bead-containing phagosomal maturation. Rab7 could be involved in the removal phagosomes. As expected, latex bead-containing phagosomes of actin from the phagosomal membrane, or more likely, from 35S-methionine labeled cells gave an enrichment of involved in the delivery of components from the endo/ protein bands (analyzed by SDS-PAGE) when compared to the lysosomal system to nascent phagosomes. We chose to look at protein banding pattern of whole cell extracts from the same delivery of components from the endo/lysosomal system to cells (Fig. 4B). The pattern of proteins observed was phagosomes by purifying latex bead-containing phagosomes comparable to those in previously published gels. Finally, on sucrose step gradients. when added to broken 35S-labeled cells, latex beads bound Previous studies had demonstrated that latex bead- many labeled proteins, but the protein banding pattern containing phagosomes can be purified to high purity from observed by SDS-PAGE was clearly different from the pattern Dictyostelium and that those membranes were minimally observed from purified latex bead-containing phagosomes contaminated by enzymes from the endoplasmic reticulum and (Fig. 4C). These data suggested that cell breakage did not Golgi complex (Rezabek et al., 1997). Since phagosomes fuse compromise the integrity of the phagosomal membrane and with endosomes and lysosomes, it was difficult to demonstrate that latex bead-containing phagosomes could be purified with that purified phagosomes were free from contaminating minimal contamination from other membranes. This was endosomes and lysosomes or other membranes of the further substantiated by electron microscopy of sections of one endo/lysosomal system. We performed the following of the embedded purified preparations. Fig. 5 demonstrates that experiment to determine if purified latex bead-containing the purified phagosomes were minimally contaminated with phagosomes were indeed free from endosome and lysosome membranes that did not contain an enclosed latex bead. contamination. First, we found that latex bead-containing We next determined the kinetics of delivery of various phagosomes could be prepared from unlabeled cells broken in proteins from the endo/lysosomal system to maturing the presence of 35S-methionine labeled cells (which had not phagosomes in control cells. Western blot analysis (Fig. 6A) ingested beads), without significant contamination from indicated that the following proteins were significantly 2454 JOURNAL OF CELL SCIENCE 114 (13) enriched in lysosomes prepared from control cells by magnetic accumulated to higher levels during the chase (CPp36 and fractionation (see Materials and Methods for details): LmpA cysteine proteinase activity). Interestingly, the proteins (lysosomal integral membrane protein) (Karakesisoglou et al., belonging to the second class were all proteases (CPp36 and 1999), α-mannosidase (lysosomal glycosidase), the 41 and 100 cysteine proteinase activity), while lysosomal membrane kDa vacuolar proton pump subunits (s.u.), Rab7, cathepsin D proteins and α-mannosidase fell into the first class. (proteinase) and CPp36 (cysteine proteinase). Phagosomes Comparable results were observed in three independent were purified from cells pulsed for 5 minutes with latex beads, experiments (results not shown). washed free of non-ingested beads and then chased for up to 2 hours in growth medium (Fig. 6B). Equal amounts of Rab7 function is required for delivery of proteins phagosomal proteins and whole cell homogenate were from the endo/lysosomal system to early separated by SDS-PAGE and then visualized by western blot phagosomes analysis. All of the lysosomally associated antigens tested were In order to test the hypothesis that Rab7 might be required for delivered to maturing phagosomes rapidly, all being enriched trafficking of proteins from the endo/lysosomal system to early even at 0 minutes of chase. The kinetics of delivery of these proteins fell into two classes: (1) proteins that were delivered rapidly and remained at constant levels during the chase period (LmpA, proton pump subunits, α-mannosidase, Rab7 and cathepsin D) and (2) proteins that, though enriched in phagosomes initially at time zero of chase, reproducibly

Fig. 5. Electron microscopic evidence that puriﬁed latex bead- containing phagosomes are free from contamination by other organelles. Latex bead-containing phagosomes were puriﬁed as described in the Materials and Methods section and thin sections (Buczynski et al., 1997) were examined using a transmission electron microscope. Arrow points to a multiparticle phagosome; arrowhead points to a single particle phagosome.

Fig. 6. Overexpression of DN Rab7 blocks delivery of α-mannosidase and LmpA to phagosomes. In (A), vesicles of the endo/lysosomal system were purified by pulsing control cells with iron dextran for 60 minutes and purifying iron dextran-containing vesicles using a magnetic column (see Materials and Methods). Western blots of cell homogenate (H) and endo/lysosomal vesicles (E/L) were decorated with antibodies to LmpA (lysosomal integral membrane protein), 100 kDa and 41 kDa subunits of the V-ATPase (100 kDa S.U. and 41 kDa S.U.), α-mannosidase (α-mann.), cathepsin D (Cath. D), cysteine proteinase P36 (CPp36) and Rab7 as described in Materials and Methods. In (B), phagosomes were purified as described in Materials and Methods from cells pulsed with latex beads for 5 minutes and chased for the indicated periods of time. Western blots were performed as in A. Bottom strip, cysteine proteinase activity of phagosomal fractions. In (C), phagosomes were purified from control cells (x4) and cells overexpressing DN Rab7 (7TN) pulsed with latex beads for 15 minutes. Western blots were performed as in A. The positions of marker proteins (kDa) are shown. Rab7 and phagosome maturation 2455 phagosomes, we purified latex bead-containing phagosomes observed with longer chase points, suggesting that delivery of from cells expressing DN Rab7 and compared the protein proteins was not simply delayed (results not shown). content of those phagosomes to phagosomes purified from Though cells expressing DN Rab7 oversecrete α- control cells. Cells were allowed to ingest latex beads for 15 mannosidase (Buczynski et al., 1997), this did not appear to minutes and then latex bead-containing phagosomes were explain the complete lack of α-mannosidase delivery to purified on sucrose step gradients. Equal protein loads were phagosomes in these cells. Immunofluorescence microscopy of separated by SDS-PAGE and analyzed by silver staining. A cells expressing DN Rab7 revealed that no mislocalization or comparison of banding patterns revealed that many bands detectable reduction of levels of α-mannosidase or LmpA was present in control phagosomes were missing from DN Rab7 observed as compared to control cells (results not shown). phagosomes (i.e. bands marked by arrows in Fig. 7). In light of Interestingly, LmpA and α-mannosidase did not colocalize to this result, we tried to identify proteins that were not delivered a large extent, but this is in agreement with previous published to phagosomes in cells expressing DN Rab7 by performing results where LmpA did not colocalize with another lysosomal western blots on purified phagosomes with antibodies to known hydrolase, α-fucosidase (Karakesisoglou et al., 1999). proteins enriched in the endo/lysosomal system. We discovered that delivery of both LmpA and α-mannosidase was greatly Apparent immature forms of cysteine proteinases reduced in cells expressing DN Rab7 (Fig. 6C). Interestingly, and proteins with GlcNac-1-P linked to serine two subunits of the V-ATPase, the 100 kDa and 41 kDa residues are delivered to phagosomes in DN Rab7 subunits, were delivered to phagosomes in DN Rab7- expressing cells overexpressing cells, though the amount of 41 kDa subunit The majority of cysteine proteinases (CPs) in Dictyostelium delivered to phagosomes purified from mutant cells was contain N-acetylglucosamine-1-phosphate (GlcNac-1-P) 68.5±5.3% (mean ± s.d., n=2) the amount delivered to control linked to serine residues (Mehta et al., 1996). These proteins phagosomes. Delivery of the 100 kDa subunit was only slightly lack mannose-6-phosphate (Man-6-P) modifications that are decreased by less than 10%. The higher molecular mass band normally found on lysosomal enzymes such as α-mannosidase, recognized by the Rab7 antibody was the overexpressed and while CPs are enriched in a lysosomal compartment, hemagglutinin-tagged DN Rab7. Identical results were also proteins with Man-6-P oligosaccharides or GlcNac-1-P are found in distinct lysosomal compartments (Souza et al., 1997). We were, therefore, interested to determine if the delivery of CPs to phagosomes was also disrupted by expression of DN Rab7. We first looked at proteinase activity in cellular lysates from control cells and cells expressing DN Rab7. Protease activity was visualized using protease gels, also known as zymograms (see Materials and Methods). Lysates from cells expressing DN Rab7 contained a number of protease activity bands, which were not present in control cell lysates (Fig. 8A). The majority of protease activity less than 35 kDa in molecular weight in these lysates was inhibited by trans-epoxysuccinyl-L- leucylamido-(4-guanadino)butane (E-64), a specific inhibitor of cysteine proteinases. The fact that this activity is present at pH 4.0 and can be inhibited by E-64 strongly suggests that this activity is due to cysteine proteinases. These proteases were enriched in phagosomes from control cells and cells overexpressing DN Rab7, and the differences in bands mimicked the differences seen in the whole cell lysates. DN Rab7 phagosomes yielded protease activity bands at higher molecular masses, such as the top band at the 50.8 kDa marker (Fig. 8A, lane marked 7TNP), which were absent in control phagosomes, while some lower molecular mass bands were absent in DN Rab7 phagosomes or decreased in activity compared to bands from control phagosomes (note bands close to the 28.1 kDa marker). This type of pattern suggests to us Fig. 7. The silver stain profile of proteins from phagosomes isolated that these higher molecular mass bands may be due to from cells overexpressing DN Rab7 lacks bands found in unprocessed proteases and the missing lower molecular mass phagosomes isolated from control cells. Phagosomes were purified as bands would correspond to the proteolytically processed described in Materials and Methods from control cells and cells mature forms of these enzymes that are absent. Note that overexpressing DN Rab7 pulsed with latex beads for 30 minutes. incubation of immature proteinases at acidic pH in renaturing Phagosomal proteins were separated by SDS-PAGE and silver stained as described in Materials and Methods. H, whole cell conditions could result in activation of these enzymes (see homogenate; X4, control phagosomes; DN7, phagosomes from cells Discussion). overexpressing DN Rab7. Arrows denote bands missing from the DN We also performed western blots with a monoclonal Rab7 phagosome protein profile. The positions of marker proteins antibody specific for GlcNac-1-P (AD7.5), a modification (kDa) are shown. found almost exclusively on cysteine proteinases, with similar 2456 JOURNAL OF CELL SCIENCE 114 (13)

Fig. 9. The formation of spacious phagosomes is not blocked by overexpression of DN Rab7. Control cells and cells overexpressing DN Rab7 were fed a mixture of FITC-labeled bacteria and Crimson Red latex beads for 10 minutes, washed and then chased for 120 minutes. During the last 10 minutes of chase, cells were allowed to attach to coverslips and then imaged by LSCM, as described in Materials and Methods. The mean number of phagosomes labeled by both crimson red beads and FITC-bacteria per cell were plotted (n=3). Values are means ± s.e.m. P<0.05 for X4 versus LY294002. X4, control cells; 7TN, cells overexpressing DN Rab7; LY294002, cells incubated in the presence of 20 µM LY294002.

of internalization of bacteria or latex beads (Rupper et al., 2001). We tested whether phagosomes in cells expressing DN Rab7 could undergo homotypic fusion events by performing Fig. 8. Both the banding pattern of cysteine proteinase activity from a laser scanning confocal microscope (LSCM)-based fusion purified phagosomes and the banding pattern of a western blot assay. In this assay cells are fed a mixture of FITC-bacteria decorated with an antibody to GlcNac-1-P suggest that immature and fluorescent red beads for 10 minutes, washed extensively, cysteine proteinases are delivered to phagosomes in cells and then chased for 120 minutes in medium without overexpressing DN Rab7. (A) Proteins from phagosomes purified phagocytic probes. The cells are then imaged by LSCM and from control cells and cells overexpressing DN Rab7 were separated by SDS-PAGE on gels made with 0.2% gelatin. Phagosomes were the images are scored for the number of double-labeled formed by pulsing cells with latex beads for 30 minutes. Cysteine phagosomes/cell. We found that the rate of spacious proteinase activity was visualized as described in Materials and phagosome formation in cells expressing DN Rab7 was not Methods. + indicates samples that were pre-incubated with E-64. H, significantly inhibited (Fig. 9). Previous studies have whole cell homogenate; P, phagosomes; X4, control and 7TN, DN demonstrated that PI 3-kinase inhibitors block spacious Rab7 overexpressors. (B) Western blots of whole cell lysates and phagosome formation, and treatment of control cells with 20 purified phagosomes from control cells and cells overexpressing DN µM LY294002 significantly inhibited phagosome fusion in Rab7 were made using the GlcNac-1-P-recognizing antibody AD7.5. this assay (Rupper et al., 2001). This suggested that adding a Phagosomes were formed by pulsing cells with latex beads for 30 mixture of phagocytic probes did not result in a large number minutes. Abbreviations as for A. The positions of marker proteins of phagosomes, which internalized phagocytic probes of both (kDa) are shown. colors. The progress of phagosomal maturation can be monitored results to those described above. There were a number of by measuring the change in phagosomal pH over time. In higher molecular mass bands present in whole cell lysates of control cells, nascent phagosomes are rapidly acidified and as DN Rab7 overexpressing cells that were absent in control cell they mature the internal pH rises, reaching a plateau after 90 lysates. DN Rab7 phagosomes contained an AD7.5 reactive minutes of maturation. We found that phagosomal pH band at 65-70 kDa that was absent from control phagosomes regulation in cells overexpressing DN Rab7 was aberrant. and DN Rab7 phagosomes lacked a doublet close to 45 kDa Within the first 5 minutes, DN Rab7 phagosomes were that was present in control phagosomes. Again, we believe this acidified to the same level as control phagosomes, but then DN may be due to a defect in processing of proteins that contain Rab7 phagosomes increased in pH more rapidly and were GlcNac-1-P glycosylation, and not mistrafficking of proteins significantly less acidic at 15, 30 and 45 minute time points from the trans-Golgi to phagosomes. (Fig. 10). Interestingly, the phagosomal pH of DN Rab7 Since DN Rab7 phagosomes appeared to be deficient phagosomes reached a plateau of pH 5.9 by 20 minutes of in interactions with the endo/lysosomal system, we maturation and was significantly more acidic than control hypothesized that these phagosomes would be blocked in phagosomes after 90 minutes of chase. This suggests that DN maturation. In Dictyostelium, phagosomal maturation Rab7 phagosomes may indeed be blocked in phagosomal consists of an early acidic phase followed by an increase maturation, but apparently at a later stage than phagosome in phagosomal pH and the formation of large, spacious homotypic fusion. This also correlates with the fact that phagosomes through homotypic fusion events (Rupper et al., subunits of the V-ATPase may not be delivered to early 2001). Spacious phagosomes begin to appear after 90 minutes phagosomes to the same level as is found in control Rab7 and phagosome maturation 2457 phagosomes. We would speculate that removal of the F-actin coat facilitates or triggers the interaction of phagosomes with the endo/lysosomal system. Alternatively, the association of Rab7-containing membranes might trigger the disassociation of F-actin. We cannot conclude that removal of F-actin is complete or that F-actin does not associate with maturing phagosomes to facilitate late processing steps. In fact, others have found that F-actin associates with maturing phagosomes in a bimodal fashion (Defacque et al., 2000). The maturation of phagosomes would appear to be important in regulating internalization of bacteria since phagocytosis rates are lower in cells expressing DN Rab7. The localization of GFP-Rab7 to early phagosomes suggested it played an important role in regulating interactions Fig. 10. The regulation of phagosomal pH is aberrant in cells of early phagosomes with the endo/lysosomal system. As a first overexpressing DN Rab7. Control cells (closed circles, X4) and cells approach, we looked at the time course of delivery of a number overexpressing DN Rab7 (open circles, 7TN) were pulsed for 10 of proteins from the endo/lysosomal system to phagosomes in minutes with FITC-labeled E. coli, washed and then chased to wild-type cells, and found that all of these proteins were various time points in growth medium without bacteria. The delivered rapidly to phagosomes, but did seem to fall into two internalized FITC-bacteria were used as a pH-sensitive probe to α determine the pH of the phagosomes in vivo (see Materials and kinetic classes (Fig. 6). Proteins such as -mannosidase and Methods). Values are means ± s.e.m.; n=4 for X4 and n=6 for 7TN. subunits of the V-ATPase were delivered rapidly and remained P<0.05 for 15, 30, 45 and 90 minute time points. enriched at the same level throughout the chase period, while cysteine proteinase activity and CP36 were enriched to higher levels at later time points in the chase. This pattern of delivery phagosomes (Fig. 6C). This might be an explanation for the is consistent with the hypothesis that cysteine proteinases, rapid increase in pH early on. which have GlcNac-1-P modifications and no Man-6-P modifications, are found in a separate endo/lysosomal compartment than lysosomal enzymes such as α-mannosidase, DISCUSSION which contain Man-6-P modifications (Souza et al., 1997). Together, these data suggest that early endosomal Though interest in the mechanisms regulating phagosomal compartments interact with phagosomes by different maturation has increased, only few Rab GTPases have been mechanisms. A recent report found that lysosomal proteins studied in this process. Rab5, the best characterized GTPase, were also delivered with differing kinetics to phagosomes in plays a pivotal role in mammalian phagosomal maturation by mouse macrophages (Claus et al., 1998). regulating phagosome fusion with early and late endosomes Inhibition of Rab7 function by expression of DN Rab7 (Alvarez-Dominguez et al., 1996; Jahraus et al., 1998) and is blocked delivery of α-mannosidase and LmpA to early also a target for disruption of phagosomal maturation by phagosomes (Fig. 6C). Interestingly, blockage of phagosome intracellular pathogens (Hashim et al., 2000; Alvarez- interaction with the endo/lysosomal system by overexpression Dominguez et al., 1997). Nevertheless, other Rabs surely of DN Rab7 was not complete, as subunits of the V-ATPase contribute to the process of phagosomal maturation and the and cysteine proteinase activity were found in early destruction of internalized pathogens (Funato et al., 1997; phagosomal membranes from these cells. This suggests that Jahraus et al., 1998). In this paper we demonstrate that the Rab7 might regulate interaction of a subset of compartments Dictyostelium Rab7 homolog plays an important role in from the endo/lysosomal system (possibly lysosomes), which phagosomal maturation, probably regulating both early (select interact early with phagosomes; an observation that is protein delivery) and late (pH regulation) steps. Rab7 consistent with our data suggesting that a cysteine proteinase associates rapidly with nascent phagosomes and regulates the and cysteine proteinase activity are delivered more slowly to delivery of a subset of lysosomal enzymes and membrane phagosomes than proteins such as α-mannosidase and the V- proteins to early phagosomes (the early step). Furthermore, ATPase. apparently immature forms of cysteine proteinases are The complete absence of α-mannosidase in DN Rab7 delivered to phagosomes in cells overexpressing DN Rab7, and phagosomes is not due to missorting of α-mannosidase, as cells although phagosomal maturation and fusion proceed, overexpressing DN Rab7 process α-mannosidase normally phagosomal pH regulation was aberrant in these cells (the late (Buczynski et al., 1997). The cells do over-secrete the mature step). form of α-mannosidase, but substantial amounts of the While GFP-Rab7 did not associate with the forming protein remain within the cells, as observed by indirect phagosomal cup, thus potentially accounting for its role in immunofluorescence and western blotting (data not shown). regulating phagocytosis, it did rapidly associate with Although cysteine proteinase activity was delivered to internalized phagosomes (Fig. 2). This confirms biochemical phagosomes in cells overexpressing Rab7, the activity-banding data that Rab7 was enriched on latex bead-containing pattern was different from that of control cells (Fig. 8). The phagosomes after a 2-minute pulse with latex beads presence of higher molecular mass bands and the absence of (Buczynski et al., 1997). Interestingly, the arrival of GFP-Rab7 or decreased activity of lower molecular mass bands found in coincides with the removal of GFP-ABD from nascent phagosomes from DN Rab7 cells as compared to phagosomes 2458 JOURNAL OF CELL SCIENCE 114 (13) from control cells suggested that immature forms of cysteine proteases were being delivered to phagosomes in cells overexpressing DN Rab7. This pattern of cysteine proteinases was confirmed by western blots of control versus DN Rab7 phagosomes with an antibody to GlcNac-1-P. Though other proteins may have GlcNac-1-P modifications, it has been demonstrated that cysteine proteinases in Dictyostelium contain this as their sole sugar modification. Cysteine proteinases are synthesized as pre-pro-forms, where the pre-peptide serves as a target for translocation into the ER and is cleaved during this process (Turk et al., 2000). Once these pro-enzymes have reached their targeted compartment, cellular proteases such as cathepsin D, or auto- proteolysis through an intermolecular mechanisms, results in cleavage of the pro-peptide and activation of the protease (Rowan et al., 1992; Kawabata et al., 1993; Podobnik et al., 1997). Presumably, the pro-enzyme becomes activated in the protease gel by incubation at low pH, which results in cleavage of the pro-peptide by other molecules of the same protease. For this reason we see activity in the gel where the pro-enzyme has migrated to. There are two possible explanations for why the pro- Fig. 11. A model proposing the role of Rab7 in regulating enzymes might be unprocessed. First, in DN Rab7 phagosomal maturation. The model depicts that Rab7 is responsible overexpressing cells, the pro-enzymes might not be targeted for transporting vesicles containing glycosidases and LmpA from to the proper endo/lysosomal compartment where they are post-lysosomes (the most distal endosomal compartment in normally processed. This seems unlikely because these Dictyostelium formed by lysosome fusion) to either early endosomes enzymes get delivered to phagosomes. Though it is possible or newly formed phagosomes. Transport to phagosomes might be that pro-cysteine proteinases could be delivered directly direct (route 2) or indirect (route 1) where the glycosidases and LmpA are transported to early endosomes that in turn fuse with from the trans-Golgi to phagosomes, the same machinery newly formed phagosomes. Rab7 is proposed not to regulate delivery would also transport pro-cysteine proteinases to their of cysteine proteinases that reside in compartments distinct from endo/lysosomal compartment, thus, we would not expect those containing glycosidases. Rab7 also does not appear to regulate transport to phagosomes without transport to the fusion of phagosomes to form spacious phagosomes, a role played by endo/lysosomal system. It is likely that the bulk of cysteine PI 3-kinase and PKB. Endo, endocytosis of fluid; Exo, exocytosis of proteinases get delivered to phagosomes by interaction with fluid; E/L=endo/lysosomal vesicles; PL, post-lysosomes; EP, early the endo/lysosomal system. phagosomes; MP, mature phagosomes; LP, late spacious The second possibility is that the conditions in the phagosomes; CP, cysteine proteinases; G, glycosidases; PI3K, endo/lysosomal system are not correct for proper pro-enzyme phosphatidylinositol 3-kinase; PKB, protein kinase B. processing in cells expressing DN Rab7. This could be due to one of the following reasons: (1) morphologic defects in the shown in Fig. 11 proposes that during the early phagosomal endo/lysosomal system, (2) the possibility that a cellular factor maturation step, Rab7 regulates fusion of vesicles that that catalyzes processing is over-secreted and not present at primarily contain hydrolases (with Man-6-P modifications) and high enough concentrations in endosomes and lysosomes, or LmpA. These vesicles are either early endosomes (route 1) or (3) defects in pH regulation of the endo/lysosomal system. The vesicles recycling from post-lysosomes (route 2). This is endo/lysosomal system of cells expressing DN Rab7 is consistent with the proposed role for Rab7 in endosome fusion morphologically different from that in control cells, consisting events (Laurent et al., 1998) and endosomal recycling of large, acidic compartments the size of post-lysosomes. (Buczynski et al., 1997). Fusion of a second endosomal Control cells have both acidic endosomes and lysosomes and compartment, which primarily contains proteins with GlcNac- non-acidic post-lysosomes. Cells expressing DN Rab7 also 1-P modifications such as cysteine proteinases, and homotypic oversecrete lysosomal enzymes. It may be that a combination fusion of phagosomes, do not rely on Rab7 function. The of oversecretion of lysosomal enzymes and increased regulation of phagosomal pH in cells expressing DN Rab7 is compartment volume causes defects in proteolytic processing also aberrant although the delivery of the proton pump appears of cysteine proteinases. In this situation, lysosomal enzymes normal. Perhaps Rab7 regulates the delivery of a protein would not be concentrated sufficiently for normal processing regulating the proton pump or ion channels that is not delivered and lysosomal acidification might not be as great as in control correctly. Future experiments are being designed to further test cells. Once cysteine proteinases are delivered to phagosomes, these hypotheses. they still do not get processed correctly. The above arguments probably also apply to DN Rab7 phagosomes because some The published research was supported by an NIH grant DK 39232 lysosomal enzymes are not present and pH regulation of DN to J.C. The authors would like to thank Dr Michael Schleicher for the Rab7 phagosomes is clearly aberrant. antibodies to LmpA, Dr Jerome Garin for the antibody to cathepsin In conclusion, we propose that Rab7 regulates both an early D and the cysteine p36 protein, and Dr Hudson Freeze for the antibody and a late event during phagosomal maturation. The model to Glc-Nac-1-phosphate. Rab7 and phagosome maturation 2459

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