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• W2000715360 abstract "The intracellular trafficking processes controlling phagosomal maturation remain to be fully delineated.Mycobacterium tuberculosis var. bovis BCG, an organism that causes phagosomal maturation arrest, has emerged as a tool for dissection of critical phagosome biogenesis events. In this work, we report that cellubrevin, a v-SNARE functioning in endosomal recycling and implicated in endosomal interactions with post-Golgi compartments, plays a role in phagosomal maturation and that it is altered on mycobacterial phagosomes. Both mycobacterial phagosomes, which undergo maturation arrest, and model phagosomes containing latex beads, which follow the normal pathway of maturation into phagolysosomes, acquired cellubrevin. However, the mycobacterial and model phagosomes differed, as a discrete proteolytic degradation of this SNARE was detected on mycobacterial phagosomes. The observed cellubrevin alteration on mycobacterial phagosomes was not a passive event secondary to a maturation arrest at another checkpoint of the phagosome maturation pathway, since pharmacological inhibitors of phagosomal/endosomal pathways blocking phagosomal maturation did not cause cellubrevin degradation on model phagosomes. Cellubrevin status on phagosomes had consequences on phagosomal membrane and lumenal content trafficking, involving plasma membrane marker recycling and delivery of lysosomal enzymes. These results suggest that cellubrevin plays a role in phagosomal maturation and that it is a target for modification by mycobacteria or by infection-induced processes in the host cell. The intracellular trafficking processes controlling phagosomal maturation remain to be fully delineated.Mycobacterium tuberculosis var. bovis BCG, an organism that causes phagosomal maturation arrest, has emerged as a tool for dissection of critical phagosome biogenesis events. In this work, we report that cellubrevin, a v-SNARE functioning in endosomal recycling and implicated in endosomal interactions with post-Golgi compartments, plays a role in phagosomal maturation and that it is altered on mycobacterial phagosomes. Both mycobacterial phagosomes, which undergo maturation arrest, and model phagosomes containing latex beads, which follow the normal pathway of maturation into phagolysosomes, acquired cellubrevin. However, the mycobacterial and model phagosomes differed, as a discrete proteolytic degradation of this SNARE was detected on mycobacterial phagosomes. The observed cellubrevin alteration on mycobacterial phagosomes was not a passive event secondary to a maturation arrest at another checkpoint of the phagosome maturation pathway, since pharmacological inhibitors of phagosomal/endosomal pathways blocking phagosomal maturation did not cause cellubrevin degradation on model phagosomes. Cellubrevin status on phagosomes had consequences on phagosomal membrane and lumenal content trafficking, involving plasma membrane marker recycling and delivery of lysosomal enzymes. These results suggest that cellubrevin plays a role in phagosomal maturation and that it is a target for modification by mycobacteria or by infection-induced processes in the host cell. The mechanisms of phagosomal biogenesis and maturation into the phagolysosome represent a fundamental biological process reflecting regulatory aspects of membrane trafficking and organelle biogenesis in eukaryotic cells (1.Alvarez-Dominguez C. Barbieri A.M. Beron W. Wandinger-Ness A. Stahl P.D. J. Biol. Chem. 1996; 271: 13834-13843Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 2.Alvarez-Dominguez C. Roberts R. Stahl P.D. J. Cell Sci. 1997; 110: 731-743Crossref PubMed Google Scholar, 3.Alvarez-Dominguez C. Stahl P.D. J. Biol. Chem. 1999; 274: 11459-11462Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 4.Bajno L. Peng X.R. Schreiber A.D. Moore H.P. Trimble W.S. Grinstein S. J. Cell Biol. 2000; 149: 697-706Crossref PubMed Scopus (265) Google Scholar, 5.Botelho R.J. Hackam D.J. Schreiber A.D. Grinstein S. J. Biol. Chem. 2000; 275: 15717-15727Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 6.Defacque H. Egeberg M. Habermann A. Diakonova M. Roy C. Mangeat P. Voelter W. Marriott G. Pfannstiel J. Faulstich H. Griffiths G. EMBO J. 2000; 19: 199-212Crossref PubMed Scopus (146) Google Scholar, 7.Downey G.P. Botelho R.J. Butler J.R. Moltyaner Y. Chien P. Schreiber A.D. Grinstein S. J. Biol. Chem. 1999; 274: 28436-28444Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 8.Fratti R.A. Backer J.M. Gruenberg J. Corvera S. Deretic V. J. Cell Biol. 2001; 154: 631-644Crossref PubMed Scopus (423) Google Scholar, 9.Jahraus A. Tjelle T.E. Berg T. Habermann A. Storrie B. Ulrich O. Griffiths G. J. Biol. Chem. 1998; 273: 30379-30390Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). These processes are sometimes targeted by a class of microbes capable of affecting phagosome integrity or maturation (8.Fratti R.A. Backer J.M. Gruenberg J. Corvera S. Deretic V. J. Cell Biol. 2001; 154: 631-644Crossref PubMed Scopus (423) Google Scholar, 10.Hackstadt T. Traffic. 2000; 1: 93-99Crossref PubMed Scopus (75) Google Scholar, 11.Cossart P. Bierne H. Curr. Opin. Immunol. 2001; 13: 96-103Crossref PubMed Scopus (87) Google Scholar, 12.Smith G.A. Portnoy D.A. Trends Microbiol. 1997; 5: 272-276Abstract Full Text PDF PubMed Scopus (34) Google Scholar, 13.Teysseire N. Boudier J.A. Raoult D. Infect. Immun. 1995; 63: 366-374Crossref PubMed Google Scholar, 14.Chakraborty P. Sturgill-Koszycki S. Russell D.G. Methods Cell Biol. 1994; 45: 261-276Crossref PubMed Scopus (44) Google Scholar, 15.Meresse S. Steele-Mortimer O. Finlay B.B. Gorvel J.P. EMBO J. 1999; 18: 4394-4403Crossref PubMed Scopus (202) Google Scholar, 16.Meresse S. Steele-Mortimer O. Moreno E. Desjardins M. Finlay B. Gorvel J.P. Nat. Cell Biol. 1999; 1: E183-E188Crossref PubMed Scopus (301) Google Scholar, 17.Russell D.G. Xu S. Chakraborty P. J. Cell Sci. 1992; 103: 1193-1210Crossref PubMed Google Scholar, 18.Scidmore M.A. Fischer E.R. Hackstadt T. J. Cell Biol. 1996; 134: 363-374Crossref PubMed Scopus (130) Google Scholar, 19.Via L.E. Deretic D. Ulmer R.J. Hibler N.S. Huber L.A. Deretic V. J. Biol. Chem. 1997; 272: 13326-13331Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). Interference with the normal pathway of phagosomal maturation into the phagolysosome enables such intracellular pathogens to avoid microbial growth control or killing by the host phagocytic cells or to avert efficient antigen presentation to the effectors of adaptive immune response in the host (1.Alvarez-Dominguez C. Barbieri A.M. Beron W. Wandinger-Ness A. Stahl P.D. J. Biol. Chem. 1996; 271: 13834-13843Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 3.Alvarez-Dominguez C. Stahl P.D. J. Biol. Chem. 1999; 274: 11459-11462Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 7.Downey G.P. Botelho R.J. Butler J.R. Moltyaner Y. Chien P. Schreiber A.D. Grinstein S. J. Biol. Chem. 1999; 274: 28436-28444Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 20.Vazquez-Torres A. Fantuzzi G. Edwards III, C.K. Dinarello C.A. Fang F.C. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 2561-2565Crossref PubMed Scopus (89) Google Scholar, 21.Ullrich H.J. Beatty W.L. Russell D.G. J. Immunol. 2000; 165: 6073-6080Crossref PubMed Scopus (51) Google Scholar). Consequently, investigations of microbial interference with the trafficking processes in the host cells may provide a means for dissecting the phagosomal maturation pathway. In this context, we have been using Mycobacterium tuberculosis, a highly adapted pathogen that parasitizes macrophages, as a tool to study fundamental phagosome maturation processes (reviewed in Ref. 22.Fratti R.A. Vergne I. Chua J. Skidmore J. Deretic V. Electrophoresis. 2000; 21: 3378-3385Crossref PubMed Scopus (39) Google Scholar). It has been established that M. tuberculosis, M. tuberculosisvar. bovis BCG, and Mycobacterium avium reside in specialized phagosomes that afford protection from acquisition of the characteristics of the terminal lysosomal organelles (23.Xu S. Cooper A. Sturgill-Koszycki S. van Heyningen T. Chatterjee D. Orme I. Allen P. Russell D.G. J. Immunol. 1994; 153: 2568-2578PubMed Google Scholar, 24.Clemens D.L. Horwitz M.A. J. Exp. Med. 1995; 181: 257-270Crossref PubMed Scopus (574) Google Scholar, 25.de Chastellier C. Lang T. Thilo L. Eur. J. Cell Biol. 1995; 68: 167-182PubMed Google Scholar, 26.Deretic V. Fratti R.A. Mol. Microbiol. 1999; 31: 1603-1609Crossref PubMed Scopus (137) Google Scholar), a phenomenon termed in classical microbiological literature as the inhibition of phagosome-lysosome fusion (27.Armstrong J.A. Hart P.D. J. Exp. Med. 1971; 134: 713-740Crossref PubMed Scopus (697) Google Scholar). The unique distribution of markers on M. tuberculosis phagosomal compartments (MPC) 1The abbreviations used are: MPCM. tuberculosis phagosomal compartment(s)BFAbrefeldin ALBClatex bead phagosomal compartment(s)NSFN-ethylmaleimide-sensitive factor, PNS, postnuclear supernatant(s)SNAPsoluble NSF adapter proteinSNARESNAP receptor(s)TGNtrans-Golgi networkTfRtransferrin receptorVAMPvesicle-associated membrane proteinBCGM. tuberculosis var. bovis BCG PasteurANOVAanalysis of variancem.o.i.multiplicity of infections suggests that there may be discrete alterations in MPC interactions with other intracellular organelles (22.Fratti R.A. Vergne I. Chua J. Skidmore J. Deretic V. Electrophoresis. 2000; 21: 3378-3385Crossref PubMed Scopus (39) Google Scholar). Mycobacterial phagosomes display reduced clearance of early phagosomal proteins such as TACO (28.Ferrari G. Langen H. Naito M. Pieters J. Cell. 1999; 97: 435-447Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar), better known as Coronin (22.Fratti R.A. Vergne I. Chua J. Skidmore J. Deretic V. Electrophoresis. 2000; 21: 3378-3385Crossref PubMed Scopus (39) Google Scholar, 28.Ferrari G. Langen H. Naito M. Pieters J. Cell. 1999; 97: 435-447Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar), and several plasma membrane markers (24.Clemens D.L. Horwitz M.A. J. Exp. Med. 1995; 181: 257-270Crossref PubMed Scopus (574) Google Scholar); they manifest impaired acidification due to the paucity of H+ATPase (29.Sturgill-Koszycki S. Schlesinger P.H. Chakraborty P. Haddix P.L. Collins H.L. Fok A.K. Allen R.D. Gluck S.L. Heuser J. Russell D.G. Science. 1994; 263: 678-681Crossref PubMed Scopus (1077) Google Scholar); and they show limited acquisition of late endosomal/lysosomal proteins (23.Xu S. Cooper A. Sturgill-Koszycki S. van Heyningen T. Chatterjee D. Orme I. Allen P. Russell D.G. J. Immunol. 1994; 153: 2568-2578PubMed Google Scholar, 30.Sturgill-Koszycki S. Schaible U.E. Russell D.G. EMBO J. 1996; 15: 6960-6968Crossref PubMed Scopus (300) Google Scholar, 31.Malik Z.A. Iyer S.S. Kusner D.J. J. Immunol. 2001; 166: 3392-3401Crossref PubMed Scopus (155) Google Scholar). M. tuberculosis phagosomal compartment(s) brefeldin A latex bead phagosomal compartment(s) N-ethylmaleimide-sensitive factor, PNS, postnuclear supernatant(s) soluble NSF adapter protein SNAP receptor(s) trans-Golgi network transferrin receptor vesicle-associated membrane protein M. tuberculosis var. bovis BCG Pasteur analysis of variance multiplicity of infections Membrane trafficking, organelle biogenesis, and maintenance of compartmental integrity are highly regulated processes and depend on a multicomponent vesicle docking and fusion machinery. The basal apparatus needed in the final stages of membrane fusion is composed of SNARE proteins forming a four-helix trans-SNARE bundle contributed by the donor and acceptor membranes (32.Weber T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Sollner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2021) Google Scholar). Prior to a fusion event, the pre-existing cis-SNARE complexes are disassembled and primed by the action of the ATPase NSF and α−SNAP, preparing the fusion proteins for the formation of trans-SNARE pairing (33.Block M.R. Glick B.S. Wilcox C.A. Wieland F.T. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7852-7856Crossref PubMed Scopus (402) Google Scholar, 34.Clary D.O. Griff I.C. Rothman J.E. Cell. 1990; 61: 709-721Abstract Full Text PDF PubMed Scopus (404) Google Scholar). Purified, fusion competent SNAREs (32.Weber T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Sollner T.H. Rothman J.E. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2021) Google Scholar, 35.Sollner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2637) Google Scholar, 36.Fukuda R. McNew J.A. Weber T. Parlati F. Engel T. Nickel W. Rothman J.E. Sollner T.H. Nature. 2000; 407: 198-202Crossref PubMed Scopus (193) Google Scholar, 37.Parlati F. McNew J.A. Fukuda R. Miller R. Sollner T.H. Rothman J.E. Nature. 2000; 407: 194-198Crossref PubMed Scopus (209) Google Scholar, 38.McNew J.A. Parlati F. Fukuda R. Johnston R.J. Paz K. Paumet F. Sollner T.H. Rothman J.E. Nature. 2000; 407: 153-159Crossref PubMed Scopus (534) Google Scholar), in combination with other tethering and regulatory factors such as Rab GTPases and their effectors (39.Sogaard M. Tani K. Ye R.R. Geromanos S. Tempst P. Kirchhausen T. Rothman J.E. Sollner T. Cell. 1994; 78: 937-948Abstract Full Text PDF PubMed Scopus (443) Google Scholar, 40.Lupashin V.V. Waters M.G. Science. 1997; 276: 1255-1258Crossref PubMed Scopus (183) Google Scholar, 41.McBride H.M. Rybin V. Murphy C. Giner A. Teasdale R. Zerial M. Cell. 1999; 98: 377-386Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar, 42.VanRheenen S.M. Cao X. Sapperstein S.K. Chiang E.C. Lupashin V.V. Barlowe C. Waters M.G. J. Cell Biol. 1999; 147: 729-742Crossref PubMed Scopus (107) Google Scholar, 43.Cao X. Barlowe C. J. Cell Biol. 2000; 149: 55-66Crossref PubMed Scopus (118) Google Scholar), define the permissive fusion events in vivo. In this work, we extended our analyses of the phagosomal organelles to the v-SNARE cellubrevin. Cellubrevin (also known as VAMP3) is a SNARE molecule involved in the recycling of plasma membrane markers from the endosome (44.Galli T. Chilcote T. Mundigl O. Binz T. Niemann H. De Camilli P. J. Cell Biol. 1994; 125: 1015-1024Crossref PubMed Scopus (196) Google Scholar) and has been implicated in trafficking from the TGN to the endocytic pathway through interactions with the TGN SNARE Syntaxin 6 (45.Bock J.B. Klumperman J. Davanger S. Scheller R.H. Mol. Biol. Cell. 1997; 8: 1261-1271Crossref PubMed Scopus (248) Google Scholar). Here we report the degradation of cellubrevin on the M. tuberculosis phagosome, as a mechanism contributing to altered trafficking from and to the mycobacterial phagosome. The murine macrophage-like cell line, J774, was maintained in Dulbecco's modified Eagle's medium (BioWhittaker) supplemented with 4 mml-glutamine and 5% fetal bovine serum (HyClone). M. tuberculosis var. bovis BCG Pasteur (BCG) harboring phsp60-gfp was grown in Middlebrook 7H9 broth (46.Dhandayuthapani S. Via L.E. Thomas C.A. Horowitz P.M. Deretic D. Deretic V. Mol. Microbiol. 1995; 17: 901-912Crossref PubMed Scopus (164) Google Scholar). Single cell suspensions were generated using a Tenbroek tissue grinder, followed by a 5-min pulse in a water bath sonicator. Remaining bacterial aggregates were removed by low speed centrifugation. For experiments examining brief infection periods (<1 h), infections were synchronized as described previously (8.Fratti R.A. Backer J.M. Gruenberg J. Corvera S. Deretic V. J. Cell Biol. 2001; 154: 631-644Crossref PubMed Scopus (423) Google Scholar). Synchronization of latex beads and BCG phagocytosis was achieved by allowing latex beads or BCG to attach to macrophages cooled to 4 °C without allowing uptake. Samples were shifted to 37 °C for various periods of time and phagosomes isolated as described previously. For longer phagosome chase experiments, BCG or latex beads were taken up by macrophages for 1 h, followed by removal of unphagocytosed particles and fresh media added for 1 to 24 h (19.Via L.E. Deretic D. Ulmer R.J. Hibler N.S. Huber L.A. Deretic V. J. Biol. Chem. 1997; 272: 13326-13331Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). Phagosomes were isolated and characterized for purity as described previously (19.Via L.E. Deretic D. Ulmer R.J. Hibler N.S. Huber L.A. Deretic V. J. Biol. Chem. 1997; 272: 13326-13331Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). Briefly, cells were mechanically lysed in 8.5% sucrose (w/w in homogenization buffer: 1 mm HEPES, pH 7.0, 0.5 mm EGTA, 1 mm EDTA, 1 mg/ml bovine gelatin) supplemented with protease inhibitors. After cellular debris and nuclei were removed by low speed centrifugation to generate post-nuclear supernatants (PNS), PNS from different samples were sedimented by centrifugation through a sucrose step gradient of 8.5, 15, and 50% sucrose (w/w in homogenization buffer). The sediment collected from the 15/50% interface was loaded on a linear 32–53% sucrose gradient (w/w in homogenization buffer). After isopycnic separation of organelles, fractions were collected from the top of the gradients, and membranes were pelleted. The fraction containing purified MPC were characterized for purity as described previously (19.Via L.E. Deretic D. Ulmer R.J. Hibler N.S. Huber L.A. Deretic V. J. Biol. Chem. 1997; 272: 13326-13331Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar). For latex bead phagosome purification, J774 cells were infected with latex beads and incubated as indicated above. Latex bead phagosomal compartments (LBC) were isolated from PNS by flotation and characterized for purity as described previously (47.Desjardins M. Huber L.A. Parton R.G.F. Griffiths G. J. Cell Biol. 1994; 124: 677-688Crossref PubMed Scopus (561) Google Scholar). Prior to infection, cells were treated with 5 μg/ml brefeldin A (Epicentre Technologies) (48.Lippincott-Schwartz J. Yuan L. Tipper C. Amherdt M. Orci L. Klausner R.D. Cell. 1991; 67: 601-616Abstract Full Text PDF PubMed Scopus (683) Google Scholar), 25 nm bafilomycin A (Sigma) (49.Via L.E. Fratti R.A. McFalone M. Pagan-Ramos E. Deretic D. Deretic V. J. Cell Sci. 1998; 111: 897-905Crossref PubMed Google Scholar) or 10 μmnocodozole (Sigma) (50.Advani R.J. Yang B. Prekeris R. Lee K.C. Klumperman J. Scheller R.H. J. Cell Biol. 1999; 146: 765-776Crossref PubMed Scopus (156) Google Scholar) for 1 or 2 h at 37 °C or 20 min at 4 °C, respectively. All treatments remained throughout the experiment. Equivalent amounts of MPC and LBC (5 μg of protein per well) were separated by 12.5% SDS-PAGE and were transferred to Immobilon-P membranes by electroblotting. For immunoblots, membranes were incubated with antibodies against globular actin (Sigma), AP3 (ς3 subunit; from J. Bonifacino and E. Dell'Angelica, National Institutes of Health, Bethesda, MD), cellubrevin (from P. DeCamilli, Yale University, New Haven, CT), LAMP 2 (Developmental Hybridoma Bank), NSF (from S. Whiteheart, University of Kentucky, Lexington, KY), Rab 5 (from L. Huber, Research Institute of Molecular Pathology, Vienna, Austria), SNAP 23 (from P. Roche, NIH, Bethesda, MD), α-SNAP (StressGen), Syntaxin 13 (from R. Scheller and R. Prekeris, Stanford University, Palo Alto, CA), Coronin/TACO (from J. Pieters, Basel Institute, Basel, Switzerland), transferrin receptor (BIOSOURCE International), and VAMP1–3 (VAMP c10.1; Synaptic Systems GmbH). Bound antibodies were visualized using the ECL Western blotting system (PerkinElmer Life Sciences). When comparing MPC and LBC on discontinuous membranes, antibody incubations and ECL reactions were performed simultaneously. Membranes were exposed to film simultaneously, and identical exposure times were used. All statistical analyses were calculated using Fisher's Protected LSD post hoc test (ANOVA) (SuperANOVA 1.11, Abacus Concepts, Inc.). p values of ≤0.05 were considered significant. Purified phagosomes containing M. bovis BCG (BCG) or latex beads were prepared and characterized as described previously (8.Fratti R.A. Backer J.M. Gruenberg J. Corvera S. Deretic V. J. Cell Biol. 2001; 154: 631-644Crossref PubMed Scopus (423) Google Scholar, 19.Via L.E. Deretic D. Ulmer R.J. Hibler N.S. Huber L.A. Deretic V. J. Biol. Chem. 1997; 272: 13326-13331Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar) and examined for the presence and dynamics of endosomal SNAREs. The most striking observation was made with the v-SNARE cellubrevin (VAMP3). Purified LBC were found to contain and accumulate cellubrevin over time (Fig. 1). Surprisingly, purified MPC, while containing cellubrevin (14 kDa), also displayed a lower molecular mass band (12.2 kDa) (Fig. 1 A) that reacted with the cellubrevin antibody directed against the cytoplasmic N terminus of cellubrevin (44.Galli T. Chilcote T. Mundigl O. Binz T. Niemann H. De Camilli P. J. Cell Biol. 1994; 125: 1015-1024Crossref PubMed Scopus (196) Google Scholar). The identity of the lower band as a VAMP was confirmed (Fig. 1 B) by using the antibody anti-VAMP c10.1, which recognizes the cytoplasmic α-helix of VAMP3 (cellubrevin), as well as VAMP1 and VAMP 2 (51.McMahon H.T. Ushkaryov Y.A. Edelmann L. Link E. Binz T. Niemann H. Jahn R. Sudhof T.C. Nature. 1993; 364: 346-349Crossref PubMed Scopus (403) Google Scholar). We conclude that the lower molecular mass polypeptide corresponds to a removal of a 1.8-kDa fragment from the C terminus of cellubrevin. The truncated polypeptide, referred to as Δcellubrevin, was not present on LBC. A decrease in the amount of full-length cellubrevin was seen in PNS from BCG-infected cells with the effect depending on the multiplicity of infection (Fig. 1, C and D). Cellubrevin levels were significantly reduced in macrophages infected at a multiplicity of infections (m.o.i.) of 50 or 100 bacilli per cell (Fig. 1, C and D) (p < 0.005 for m.o.i.50; p = 0.0001 for m.o.i.100, relative to maximum cellubrevin levels; ANOVA). Actin levels were unaffected at all m.o.i. values (Fig. 1 D). ΔCellubrevin in PNS observed from BCG-infected cells could only be detected when membranes were pelleted from PNS and overloaded on SDS-PAGE gels (data not shown). To test whether BCG infections can affect cellubrevin in trans, e.g. on latex bead phagosomes in the same macrophages, cells were infected with BCG for 24 h prior to superinfecting with latex beads for an additional 24 h after which LBC were purified as described under “Experimental Procedures.” We found that conditioning cells with BCG at levels that did not reduce overall cellular cellubrevin levels in infected cells (Fig. 2, PNS), did not significantly alter cellubrevin levels on LBC (Fig. 2, LBC). Furthermore, Δcellubrevin was absent from LBC isolated from BGC-conditioned macrophages (Fig. 2). This suggests that the effects of BCG on cellubrevin are not exerted globally within BCG-infected macrophages, but represent a phenomenon specific for the mycobacterial phagosome. We next examined whether the generation of Δcellubrevin on MPC is only a secondary phenomenon caused by phagosome maturation arrest or could actively contribute to the arrest. This was addressed by inducing a phagosome maturation block using pharmacological inhibitors of membrane trafficking. We found that treatment with bafilomycin A and nocodozole, two known inhibitors of cellular functions essential in endosome maturation (acidification and microtubule based vectorial transport, respectively) (52.van Deurs B. Holm P.K. Kayser L. Sandvig K. Eur. J. Cell Biol. 1995; 66: 309-323PubMed Google Scholar, 53.van Weert A.W. Dunn K.W. Gueze H.J. Maxfield F.R. Stoorvogel W. J. Cell Biol. 1995; 130: 821-834Crossref PubMed Scopus (302) Google Scholar), did not cause reduction in cellubrevin levels on LBCs (Fig. 3 A). Thus, induction of a maturation block alone, by causing stagnation of latex bead phagosomes, did not suffice to cause the appearance of ΔCellubrevin on LBC (Fig. 3 A) or reduction of cellubrevin levels in PNS. In contrast to bafilomycin A and nocodozole, treatment with brefeldin A (BFA), an inhibitor of ARF GTPase-based vesicular trafficking within the secretory pathway, including the trafficking from the TGN, decreased levels of cellubrevin on LBC (Fig. 3, A and B). The effect was specific, as levels of SNAP 23 on LBC were not affected in BFA-treated cells (Fig. 3 A). Cellubrevin levels were reduced on LBC by 75% in cells treated with BFA (p = 0.0001; ANOVA) (Fig. 3 B). However, a cellubrevin degradation product was not detected on latex bead phagosomes in BFA-treated cells (Fig. 3 A; Δcellubrevin on MPC is shown for reference). Although cellubrevin levels were significantly reduced on LBCs in BFA-treated macrophages, overall cellular cellubrevin levels were not affected (Fig. 3 A; PNS). It is possible that BFA effects on phagosome-associated cellubrevin were due to either its decreased delivery or increased removal from phagosomes. We favor the former possibility based on the following considerations: (i) cellubrevin has been implicated in TGN-to-endosome trafficking (45.Bock J.B. Klumperman J. Davanger S. Scheller R.H. Mol. Biol. Cell. 1997; 8: 1261-1271Crossref PubMed Scopus (248) Google Scholar); (ii) it has been shown that the TGN t-SNARE syntaxin 6 can interact with cellubrevin (45.Bock J.B. Klumperman J. Davanger S. Scheller R.H. Mol. Biol. Cell. 1997; 8: 1261-1271Crossref PubMed Scopus (248) Google Scholar); (iii) we have also found that syntaxin 6 is excluded from MPC, a phenomenon linked to the block in delivery of lysosomal effectors (e.g. H+ATPase and immature cathepsins) to phagosomes. 2R. A. Fratti and V. Deretic, unpublished results. As BFA has been shown to cause the tubulation and fusion of endosomes and TGN (48.Lippincott-Schwartz J. Yuan L. Tipper C. Amherdt M. Orci L. Klausner R.D. Cell. 1991; 67: 601-616Abstract Full Text PDF PubMed Scopus (683) Google Scholar), this opens the theoretical possibility that BFA-mediated alterations in the protein profiles of LBC may be linked to mixing of compartments. However, since we observed a reduction rather than increase in phagosomal acquisition of cellubrevin, we can exclude the possibility that BFA in our experiments caused indiscriminant fusion of phagosomes with endosomes or TGN. Instead, the observed reduction of cellubrevin acquisition by LBC in BFA-treated cells is most likely associated with the disruption of phagosomal interactions with the endosomal and biosynthetic compartments. We next tested whether alterations in cellubrevin levels affected recycling of markers from the phagosome. The effect of BFA (which lowers cellubrevin on phagosomes; Fig. 3) on recycling of transferrin receptor (TfR) from model phagosomes was examined. LBC were isolated from BFA-treated macrophages at 30 min post-phagocytosis. LBC from BFA-treated cells retained significantly more TfR relative to LBC from untreated control macrophages (p < 0.05; ANOVA) (Fig. 4, A and B). BFA treatment also caused increased levels of AP3 (p < 0.05; ANOVA), an adapter protein involved in TGN and endosomal trafficking (54.Faundez V. Horng J.T. Kelly R.B. Cell. 1998; 93: 423-432Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar) (Fig. 4, A and B). Similarly, levels of coronin, an actin-binding, general phagocytosis protein (also termed TACO, proposed to accumulate on mycobacterial phagosomes (28.Ferrari G. Langen H. Naito M. Pieters J. Cell. 1999; 97: 435-447Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar)), was moderately increased on LBC upon BFA treatment (p< 0.01; ANOVA) (Fig. 4, A and B). In contrast, levels of the early endosomal SNARE syntaxin 13 were not affected by BFA, suggesting that the effects seen with TfR, AP3, and coronin (Fig. 4, A and B) were not a result of indiscriminate membrane mixing. After a round of membrane fusion is completed, SNARE proteins remain tightly associated in a cis-SNARE complex. SNARE molecules stored in cis complexes are made available for subsequent fusion events only upon a priming cycle, which involves an ATP-dependent disruption of the cis-SNARE aggregate. The priming process is dependent on the ATPase NSF association with SNARE complexes, via interactions that include another factor, α-SNAP (55.Banerjee A. Barry V.A. DasGupta B.R. Martin T.F.J. J. Biol. Chem. 1996; 271: 20223-20226Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar). Upon ATP hydrolysis, the cis-SNARE complex is dissociated, and the primed free SNARE proteins become available for new docking and fusion events. Isolated phagosomes were probed for the presence of NSF and α-SNAP. Both MPC and LBC displayed membrane bound NSF and α-SNAP throughout the experiment with some evidence of reduced amounts as the maturation progressed (Fig. 5 A). When NSF was examined at 24 and 72 h post-infection, we found that MPC retained significant levels of NSF, while most of it was lost from LBC (Fig. 5 B). LBC isolated from BFA-treated cells showed an increase in NSF levels (200%) relative to those isolated from untreated cells (p < 0.05; ANOVA) (Fig. 6, A and B). Inhibiting H+ATPase activity with bafilomycin A had a similar effect (p < 0.05; ANOVA) (Fig. 6, Aand B). Nocodozole did not have an effect on NSF increase, and, if anything, it caused a slight reduction in NSF levels. Accumulation of NSF on LBC isolated from BFA-treated macrophages (Fig. 6) correlates with a marked reduction in cellubrevin levels (Fig. 3), while retention of NSF on MPC (Fig. 5) correlates with cellubrevin degradation. We interpret these observations as an impeded release of NSF from MPC due to disfunctional SNARE priming associated with either reduced levels of cellub" @default.
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• W2000715360 title "Cellubrevin Alterations and Mycobacterium tuberculosis Phagosome Maturation Arrest" @default.
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