Differential Compartmentalization of the Calpain/Calpastatin Network with the Endoplasmic Reticulum and Golgi Apparatus
2004; Elsevier BV; Volume: 279; Issue: 41 Linguagem: Inglês
10.1074/jbc.m408100200
ISSN1083-351X
AutoresJoshua L. Hood, William H. Brooks, Thomas L. Roszman,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoCalpain, a calcium-activated cysteine protease, is involved in modulating a variety of cell activities such as shape change, mobility, and apoptosis. The two ubiquitous isoforms of this protease, calpain I and II, are considered to be cytosolic proteins that can translocate to various sites in the cell. The activity of calpain is modulated by two regulatory proteins, calpastatin, the specific endogenous inhibitor of calpain, and the 28-kDa regulatory subunit. Using velocity gradient centrifugation, the results of this study confirm and greatly expand upon our previous finding that the calpain/calpastatin network is associated with the endoplasmic reticulum and Golgi apparatus in cells. Moreover, confocal microscopy demonstrates that calpain II colocalizes with specific proteins found in these organelles. Additional experiments reveal that hydrophobic rather than electrostatic interactions are responsible for the association of the calpain/calpastatin network with these organelles. Treatment of the organelles with Na2CO3 or deoxycholate reveal that calpain I, 78-kDa calpain II, and the regulatory subunit are "embedded" within the organelle membranes similar to integral membrane proteins. Proteinase K treatment of the organelles shows that calpain I and II, calpastatin, and the regulatory subunit localize to the cytosolic surface of the organelle membranes, and a subset of calpain II and the regulatory subunit are also found within the lumen of these organelles. These results provide a new and novel explanation for how the calpain/calpastatin network is organized in the cell. Calpain, a calcium-activated cysteine protease, is involved in modulating a variety of cell activities such as shape change, mobility, and apoptosis. The two ubiquitous isoforms of this protease, calpain I and II, are considered to be cytosolic proteins that can translocate to various sites in the cell. The activity of calpain is modulated by two regulatory proteins, calpastatin, the specific endogenous inhibitor of calpain, and the 28-kDa regulatory subunit. Using velocity gradient centrifugation, the results of this study confirm and greatly expand upon our previous finding that the calpain/calpastatin network is associated with the endoplasmic reticulum and Golgi apparatus in cells. Moreover, confocal microscopy demonstrates that calpain II colocalizes with specific proteins found in these organelles. Additional experiments reveal that hydrophobic rather than electrostatic interactions are responsible for the association of the calpain/calpastatin network with these organelles. Treatment of the organelles with Na2CO3 or deoxycholate reveal that calpain I, 78-kDa calpain II, and the regulatory subunit are "embedded" within the organelle membranes similar to integral membrane proteins. Proteinase K treatment of the organelles shows that calpain I and II, calpastatin, and the regulatory subunit localize to the cytosolic surface of the organelle membranes, and a subset of calpain II and the regulatory subunit are also found within the lumen of these organelles. These results provide a new and novel explanation for how the calpain/calpastatin network is organized in the cell. Calpain (Cp) 1The abbreviations used are: Cp, calpain; Cs, calpastatin; DC, deoxycholate; ER, endoplasmic reticulum; ERGIC, ER to GA intermediate compartment; GA, Golgi apparatus; ICT, isotonic cell transfer; PK, proteinase K; PM, plasma membrane; PNS, postnuclear supernatant; Rs, regulatory subunit; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; COP, coatomer protein; Grp, glucose-regulated protein. 1The abbreviations used are: Cp, calpain; Cs, calpastatin; DC, deoxycholate; ER, endoplasmic reticulum; ERGIC, ER to GA intermediate compartment; GA, Golgi apparatus; ICT, isotonic cell transfer; PK, proteinase K; PM, plasma membrane; PNS, postnuclear supernatant; Rs, regulatory subunit; PMSF, phenylmethylsulfonyl fluoride; PBS, phosphate-buffered saline; COP, coatomer protein; Grp, glucose-regulated protein. is a Ca2+-dependent neutral cysteine protease (1Goll D.E. Thompson V.F. Li H. Wei W. Cong J. Physiol. Rev. 2003; 83: 731-801Crossref PubMed Scopus (2305) Google Scholar). The major isoforms of Cp, Cp I and Cp II, are heterodimers composed of an 80-kDa catalytic subunit and an identical 28-kDa regulatory subunit (Rs). The Cp I and Cp II isoforms differ not only in the amino acid sequence of the catalytic subunits but in their in vitro Ca2+ requirements for activation. Thus, Cp I (μ-calpain) requires 3-50 μm Ca2+, whereas 0.2-1.0 mm of Ca2+ is required for activation of Cp II (m-calpain) (2Croall D.E. DeMartino G.N. Physiol. Rev. 1991; 71: 813-847Crossref PubMed Scopus (776) Google Scholar, 3Mellgren R.L. FASEB J. 1987; 1: 110-115Crossref PubMed Scopus (271) Google Scholar, 4Ono Y. Sorimachi H. Suzuki K. Biochem. Biophys. Res. Commun. 1998; 245: 289-294Crossref PubMed Scopus (106) Google Scholar). In general, these concentrations of Ca2+ are far greater than can be achieved intracellularly (<1 μm) (5Chakrabarti A.K. Dasgupta S. R. H. Sr, Gadsen Hogan E.L. Banik N.L. J. Neurosci. 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FASEB J. 1987; 1: 110-115Crossref PubMed Scopus (271) Google Scholar, 21Inomata M. Hayashi M. Ohno-Iwashita Y. Tsubuki S. Saido T.C. Kawashima S. Arch. Biochem. Biophys. 1996; 328: 129-134Crossref PubMed Scopus (79) Google Scholar, 22Kawasaki H. Kawashima S. Molec. Membr. Biol. 1996; 13: 217-224Crossref PubMed Scopus (76) Google Scholar, 23Perrin B.J. Huttenlocher A. Int. J. Biochem. Cell Biol. 2002; 34: 722-725Crossref PubMed Scopus (225) Google Scholar, 24Sato K. Kawashima S. Biol. Chem. 2001; 382: 743-751Crossref PubMed Google Scholar, 25Rock M.T. Dix A.R. Brooks W.H. Roszman T.L. Exp. Cell Res. 2000; 261: 260-270Crossref PubMed Scopus (52) Google Scholar), where it facilitates reorganization of the actin cytoskeleton by cleaving a variety of cytoskeleton-associated substrates such as talin, FAK, paxillin, and α-actinin (26Glading A. Lauffenburger D.A. Wells A. Trends Cell Biol. 2002; 12: 46-54Abstract Full Text Full Text PDF PubMed Scopus (320) Google Scholar). Although the ability of Cp to move within the cell is well known, the spatial and temporal aspects governing the subcellular positioning of the Cp/Cs network remain undefined. Using confocal microscopy and velocity gradient centrifugation, the current study was undertaken to expand upon our previous observation that Cp, the Rs, and Cs associate with the endoplasmic reticulum (ER) and Golgi apparatus (GA) (27Hood J.L. Logan B.B. Sinai A.P. Brooks W.H. Roszman T.L. Biochem. Biophys. Res. Commun. 2003; 310: 1200-1212Crossref PubMed Scopus (50) Google Scholar). The results show a differential association of Cp I, Cp II, Cs, and the Rs with the ER and GA as a result of hydrophobic rather than electrostatic interactions. Moreover, Cp II and the Rs appear to not only be associated with these cytosolic side of these membranes but also in the lumen of the organelles. Positioning of the Cp/Cs network within the ER and GA affords a focal environment capable of facilitating activation in the presence of elevated [Ca2+]i, providing a means of protease regulation and access to substrates. Cells, Reagents, and Antibodies—Lung adenocarcinoma cells (A-549) were grown in F-12 medium supplemented with 5% fetal bovine serum (Equitech-Bio, Inc., Kerrville, TX), 2 mml-glutamine, 1.5 g/liter NaHCO3, and 100 units/ml (1%) penicillin and streptomycin at 37 °C and 5% CO2. The A-549 cell line was obtained from the American Type Tissue Culture Collection (Manassas, VA). A-549 cells were passaged approximately once every week using 0.5% trypsin-EDTA. Goat polyclonal anti-ribophorin II, and goat polyclonal anti-calregulin (calreticulin) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Mouse monoclonal anti-P230 was purchased from BD Transduction Laboratories (San Diego, CA). Rabbit polyclonal anti-calpain II 80-kDa subunit was purchased from Chemicon (Temecula, CA). Mouse monoclonal anti-14-3-3 and anti-KDEL were purchased from Stressgen Biotechnologies (Victoria, Canada). Rabbit polyclonal anti-mannosidase II was purchased from U.S. Biological (Swampscott, MA). Mouse monoclonal anti-COPβ was purchased from Sigma. All other primary antibodies were mouse monoclonal and purchased from Chemicon. The secondary antibodies Alexa 488 (anti-mouse and anti-rabbit), Alexa 594 (anti-mouse and anti-rabbit), and the ProLong anti-fade kit were purchased from Molecular Probes, Inc. (Eugene, OR). All other reagents were purchased from Sigma. Confocal Microscopy—Confluent cells were detached from the flask using 0.5% trypsin-EDTA, resuspended in serum-free medium, and pelleted at 1500 rpm at 4 °C. The cells were stained with trypan blue and counted using a hemocytometer. Cells (50,000/ml) were allowed to settle and adhere to glass coverslips in 6-well plates for 24 h at 37 °C under 5% CO2. After incubation, cells were washed three times with PBS (pH 7.4) to remove nonadherent cells and fixed with 4% paraformaldehyde in PBS (pH 7.4) for 30 min at room temperature. After fixation, cells were washed three times in PBS (pH 7.4) to remove excess paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS (pH 7.4) for 10 min at room temperature, washed three times in PBS (pH 7.4) to remove excess Triton X-100, and blocked for 10 min at room temperature in PBS (pH 7.4) containing 1% bovine serum albumin and 4% goat serum. Primary antibodies were added to the cells and incubated for 20 min, cells were washed three times in PBS (pH 7.4) to remove excess primary antibody, and appropriate secondary fluorescent antibodies (Alexa 488 or Alexa 594) were added. Cells were washed three times with phosphate-buffered saline (pH 7.4) to remove background fluorescent staining, and coverslips containing cells were removed from the 6-well plates and mounted on glass slides using the ProLong antifade kit according to the manufacturer's protocol. Coverslips were allowed to dry for 24 h and visualized using a Leica confocal microscope under ×1000 magnification and Leica imaging software. Enrichment of ER and GA Organelles by Optiprep Velocity Gradients—The ER and GA subcellular compartments isolated as previously described were separated on Optiprep (Axis-Shield, Oslo, Norway) velocity gradients (28Tekirian T.L. Merriam D.E. Marshansky V. Miller J. Crowley A.C. Chan H. Ausiello D. Brown D. Buxbaum J.D. Xia W. Wasco W. Mol. Brain Res. 2001; 96: 14-20Crossref PubMed Scopus (10) Google Scholar). Briefly, cells were cultured for 24 h at 37 °C and harvested using 0.5% trypsin EDTA. Cells were resuspended in RPMI 1640 medium and pelleted at 1800 × g for 15 min. Cell pellets were resuspended in 4 ml of isotonic cell transfer (ICT) homogenization medium (78 mm KCl, 4 mm MgCl2, 8.37 mm CaCl2, 10 mm EGTA, 50 mm Hepes/KOH, pH 7.0, 1 mm PMSF, and Complete protease inhibitor mixture (Roche Applied Science)) and passaged 10 times through an ice-cold ball bearing homogenizer with a 12-μm clearance. The resulting cell lysate was spun at 1500 × g for 20 min at 4 °C to remove nuclei and debris. The pellet was discarded, and the postnuclear supernatant (PNS) containing organelles was removed and set aside. A 10-25% continuous Optiprep gradient diluted in 0.75% NaCl, 10 mm Tris, 3 mm KCl, and 1 mm EDTA was generated by a Gradient Master (BioComp Instruments, Inc.). A protein assay was performed on the PNS, and 1.5 mg of PNS was loaded to the top of the gradient. The gradient was spun in an SW40 Ti rotor for 18 h at 27,700 rpm (100,000 × g) and 4 °C. After centrifugation, 12 1.0-ml fractions (heavy to light density) were collected from the bottom of the gradient using a Dynamax RP-1 peristaltic pump (Rainin). The protein from each fraction was precipitated by incubation with 10% trichloroacetic acid for 30 min at 4 °C. The protein precipitates were recovered by centrifugation at 16,000 × g for 20 min. Pellets were washed two times with acetone to remove the trichloroacetic acid and resuspended in 300 μl of Laemmli's sample buffer. Samples were heated to 80 °C and vortexed three times, and 10 μl of each sample was loaded onto a 4-20% Tris-HCl polyacrylamide gel (Bio-Rad) and analyzed by Western blotting using nitrocellulose membrane. Treatment of Microsomes with NaCl, KCl, EDTA, and EGTA—A-549 cells were cultured for 24 h at 37 °C, and the cells were harvested, resuspended in RPMI 1640 medium, and pelleted at 1800 × g for 15 min. Cell pellets were resuspended in 4 ml of ICT homogenization medium (78 mm KCl, 4 mm MgCl2, 8.37 mm CaCl2, 10mm EGTA, 50 mm Hepes/KOH, pH 7.0, 1 mm PMSF, and Complete protease inhibitor mixture) and passaged 10 times through an ice-cold ball bearing homogenizer with a 12-μm clearance. The resulting cell lysate was spun at 1500 × g for 20 min and 4 °C to remove nuclei and debris. The pellet was discarded, and the PNS containing cytosol and organelles was removed and spun at 100,000 × g in a TLA 100.3 rotor for 1 h at 4 °C to pellet organelle microsomes. The microsomal pellet was resuspended in 200 μl of 50 mm Tris buffer, pH 7.4, and 0.25 mg of control or sample microsomes were resuspended in 1.5 ml of 50 mm Tris buffer, pH 7.4. The following five samples were prepared: 100 mm NaCl, 100 mm KCl, 10 mm EDTA, 10 mm EGTA, or both 10 mm EDTA and 10 mm EGTA. The samples and control microsomes were incubated with rotation at 37 °C for 1 h. After incubation, treated microsomes were spun at 100,000 × g in a TLA 100.3 rotor for 1 h at 4 °C, the supernatant was discarded, and the pellets were rinsed in 50 mm Tris buffer, pH 7.4, and solubilized in 250 μl of Laemmli's sample buffer. Samples were heated to 80 °C and vortexed three times, and 10 μg of each sample was loaded onto a 4-20% Tris-HCl polyacrylamide gel (Bio-Rad) and analyzed by Western blotting using nitrocellulose membrane. Na2CO3Treatment of Organelle Microsomes—A-549 cells were cultured in their respective media for 24 h at 37 °C and harvested using 0.5% trypsin-EDTA. Cells were resuspended in RPMI 1640 medium and pelleted at 1800 × g for 15 min. Cell pellets were resuspended in 4 ml of ICT homogenization medium (78 mm KCl, 4 mm MgCl2, 8.37 mm CaCl2, 10 mm EGTA, 50 mm Hepes/KOH, pH 7.0, 1 mm PMSF, and Complete protease inhibitor mixture) and passaged 10 times through an ice-cold ball bearing homogenizer with a 12-μm clearance. The resulting cell lysate was spun at 1500 × g for 20 min and 4 °C to remove nuclei and debris. The pellet was discarded, and the PNS containing cytosol and organelles was removed and spun at 100,000 × g in a TLA 100.3 rotor for 1 h at 4 °C to pellet organelle microsomes. The microsomal pellet was resuspended in 200 μl of 50 mm Tris buffer, pH 7.4, a protein assay was done, and 0.25 mg of control microsomes were solubilized in 250 μl of Laemmli's sample buffer, whereas another set was diluted ∼30-fold in 0.2 m Na2CO3, pH 11.5, at 0 °C. The microsomes were treated with Na2CO3 for 30 min at 0 °C to open the microsomes and strip them of peripherally associated proteins, leaving behind integral membrane proteins. After Na2CO3 treatment, microsomes were spun at 100,000 × g in a TLA 100.3 rotor for 1 h at 4 °C. The treated pellet was solubilized in 250 μl of Laemmli's sample buffer, and the proteins present in the supernatant were precipitated by incubation with 10% trichloroacetic acid for 30 min at 4 °C. The precipitates were recovered by centrifugation at 16,000 × g for 20 min at 4 °C, the pellets were washed two times with acetone to remove the trichloroacetic acid and resuspended in 250 μl of Laemmli's sample buffer. Samples were heated to 80 °C and vortexed three times, and 10 μg of each sample was loaded onto a 4-20% Tris-HCl polyacrylamide gel (Bio-Rad) and analyzed by Western blotting using nitrocellulose membrane. Proteinase K (PK) and Deoxycholate (DC) Treatment of Organelle Microsomes—A-549 cells were cultured for 24 h at 37 °C and harvested using 0.5% trypsin-EDTA. Cells were resuspended in RPMI 1640 medium and pelleted at 1800 × g for 15 min. Cell pellets were resuspended in 4 ml of ICT homogenization medium (78 mm KCl, 4 mm MgCl2, 8.37 mm CaCl2, 10 mm EGTA, 50 mm Hepes/KOH, pH 7.0, 1 mm PMSF, and Complete protease inhibitor mixture) and passaged 10 times through an ice-cold ball bearing homogenizer with a 12-μm clearance. The resulting cell lysate was spun at 1500 × g for 20 min and 4 °C to remove nuclei and debris. The pellet was discarded, and the PNS containing cytosol and organelles was removed and spun at 100,000 × g in a TLA 100.3 rotor for 1 h at 4 °C to pellet the microsomes. The microsomal pellet was resuspended in 200 μl of 50 mm Tris buffer, pH 7.4, the protein assay was done, and 0.25 mg of control microsomes were resuspended in 1.5 ml of 50 mm Tris buffer, pH 7.4, whereas 0.25 mg of sample microsomes were resuspended in 1.5 ml of 50 mm Tris buffer at a PK concentration of 0.5 μg/ml or 0.1% DC or both. The samples and control were incubated with rotation at 37 °C for 20 min. After incubation, reactions were quenched with 1 mm PMSF to inhibit PK. The treated microsomes were spun at 100,000 × g in a TLA 100.3 rotor for 1 h at 4 °C to generate a sample microsome pellet, which was rinsed with 50 mm Tris buffer, pH 7.4, and solubilized in 250 μl of Laemmli's sample buffer. Samples were heated to 80 °C and vortexed three times, and 10 μg of each sample was loaded onto a 4-20% Tris-HCl polyacrylamide gel (Bio-Rad) and analyzed by Western blotting using nitrocellulose membrane. PK Treatment of ER and GA Organelles Separated on Optiprep Velocity Gradients—The ER and GA subcellular compartments, isolated as previously described, were separated on Optiprep velocity gradients (28Tekirian T.L. Merriam D.E. Marshansky V. Miller J. Crowley A.C. Chan H. Ausiello D. Brown D. Buxbaum J.D. Xia W. Wasco W. Mol. Brain Res. 2001; 96: 14-20Crossref PubMed Scopus (10) Google Scholar). Briefly, cells were cultured for 24 h at 37 °C and harvested using 0.5% trypsin-EDTA. Cells were resuspended in RPMI 1640 medium and pelleted at 1800 × g for 15 min. Cell pellets were resuspended in 4 ml of ICT homogenization medium (78 mm KCl, 4 mm MgCl2, 8.37 mm CaCl2, 10 mm EGTA, 50 mm Hepes/KOH, pH 7.0, 1 mm PMSF, and Complete protease inhibitor mixture (Roche Applied Science)) and passaged 10 times through an ice-cold ball bearing homogenizer with a 12-μm clearance. The resulting cell lysate was spun at 1500 × g for 20 min at 4 °C to remove nuclei and debris. The pellet was discarded, and the PNS containing organelles was removed and set aside. A 10-25% continuous Optiprep gradient diluted in 0.75% NaCl, 10 mm Tris, 3 mm KCl, and 1 mm EDTA was generated by a Gradient Master (continuous gradient maker, BioComp Instruments, Inc.). A protein assay was performed on the PNS, and 1.5 mg of PNS was loaded to the top of the gradient. The gradient was spun in an SW40 Ti rotor for 18 h at 27,700 rpm (100,000 × g) and 4 °C. After centrifugation, 12 1.0-ml fractions (heavy to light density) were collected from the bottom of the gradient using a Dynamax RP-1 peristaltic pump. Each fraction was divided into two 500-μl samples, one control and one to be treated with PK. Both control and PK samples were resuspended in 1 ml of 50 mm Tris buffer, pH 7.4. The PK sample was supplemented with 0.5 μg/ml PK. The control and PK samples were incubated with rotation at 37 °C for 20 min. After incubation, reactions were quenched with 1 mm PMSF to inhibit PK. The protein from each sample was precipitated by incubation with 10% trichloroacetic acid for 30 min at 4 °C. The protein precipitates were recovered by centrifugation at 16,000 × g for 20 min. Pellets were washed two times with acetone to remove the trichloroacetic acid and resuspended in 150 μl of Laemmli's sample buffer. Samples were heated to 80 °C and vortexed three times, and 10 μl of each sample was loaded onto a 4-20% Tris-HCl polyacrylamide gel and analyzed by Western blotting using nitrocellulose membrane. Subcellular Localization of Cp II in Cells as Determined by Confocal Microscopy—The association of Cp II with subcellular organelles was determined by confocal microscopy experiments utilizing A-549 lung adenocarcinoma cells cultured for 24 h on glass slides and subsequently immunostained. The initial series of experiments examined the relationship between Cp II and the ER using the trans-membrane protein chaperone calnexin (29Bergeron J.J.M. Brenner M.B. Thomas D.Y. Williams D.B. Trends Biochem. Sci. 1994; 19: 124-128Abstract Full Text PDF PubMed Scopus (455) Google Scholar) or the lumenal protein chaperone Grp 78/KDEL (30Cotner T. Pious D. J. Biol. Chem. 1995; 270: 2379-2386Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) (Fig. 1). Cells probed with antibodies to Cp II and calnexin showed diffuse staining throughout the cell but with considerable colocalization of these two proteins (Fig. 1A). Conversely, colocalization of Cp II and Grp 78/KDEL is more focal and intense, particularly in the perinuclear region (Fig. 1B). These results suggest that Cp II is associated with the cytoplasmic face of the ER as well as the ER lumen in A-549 cells. Similar experiments were performed to determine the relationship between Cp II with either COPβ, a COP I vesicle coat protein (31Duden R. Mol. Membr. Biol. 2003; 20: 197-207Crossref PubMed Scopus (120) Google Scholar), or P230, a protein associated with the GA (32Kjer-Nielsen L. van Vliet C. Erlich R. Toh B.-H. Gleeson P.A. J. Cell Sci. 1999; 112: 1645-1654Crossref PubMed Google Scholar) (Fig. 2). Cells stained with antibodies to Cp II or COPβ show diffuse staining with extensive colocalization. (Fig. 2A). The most prominent feature of this colocalization was the appearance of intensely stained vesicles. This suggests that Cp II associates with the COP I/II vesicle system comprising the ER to GA intermediate compartment (ERGIC) and provides a potential means for Cp II transport between the ER and GA. Cp II also colocalizes with the GA marker P230 (Fig. 2B). The P230 staining of the GA appears perinuclearly with prominent punctuate colocalization. Collectively, these results indicate that Cp II associates with the ERGIC and GA in A-549 cells. Association of Cp and Its Regulatory Proteins with the ER and GA as Determined by Velocity Gradient Centrifugation—To confirm and extend the results obtained with confocal microscopy indicating the association of Cp with subcellular organelles, PNS prepared from A-549 cells was subjected to Optiprep velocity gradient centrifugation. The resulting organelle fractions obtained from the gradient were subjected to Western analysis using antibodies to proteins associated with the ER, ERGIC, and GA (Fig. 3). The results show that the GA resides primarily in gradient fractions 8 and 9 as demonstrated by reactivity to antibodies against mannosidase II, a specific marker for the medial to trans-GA (33Moremen K.W. Biochim. Biophys. Acta. 2002; 1573: 225-235Crossref PubMed Scopus (91) Google Scholar). The trans-GA-associated 14-3-3 protein (28Tekirian T.L. Merriam D.E. Marshansky V. Miller J. Crowley A.C. Chan H. Ausiello D. Brown D. Buxbaum J.D. Xia W. Wasco W. Mol. Brain Res. 2001; 96: 14-20Crossref PubMed Scopus (10) Google Scholar) resided in fractions 9-11 of the gradient, whereas ribophorin II, an ER-resident protein (34Fu J. Pirozzi G. Sanjay A. Levy R. Chen Y. De Lemos-Chiarandini C. Sabatini D. Kreibich G. Eur. J. Cell Biol. 2000; 79: 219-228Crossref PubMed Scopus (20) Google Scholar), was detected in gradient fractions 3-7. COPβ was distributed throughout the gradient, with the majority detected in fractions 8 and 9, corresponding to the location of the GA. Because COPβ is involved in the trafficking of proteins and lipids between the ER and GA, it is expected that this protein should be found in both the ER and GA (31Duden R. Mol. Membr. Biol. 2003; 20: 197-207Crossref PubMed Scopus (120) Google Scholar). Additionally, because the size and buoyancy of vesicles transported by COPβ vary greatly it is not unreasonable to find COPβ throughout the gradient. However, for the purposes of analysis, the ERGIC will be defined as the COPβ-containing compartment, fractions 7 and 8, with a buoyant density between that of the ER and GA. Fraction 12 is defined as containing those cellular compartments, vesicles/PM, with buoyant densities less than that of the trans-GA. The presence of Cp and its regulatory proteins in the gradient fractions was determined by Western analysis (Fig. 3). The results show that Cp I is found predominantly in fraction 9, with smaller amounts detected in fractions 7, 8, 10, and 11, indicating that Cp I is primarily associated with the GA. The distribution of Cp II is quite different in that this isoform is detectable throughout the gradient. Interestingly, the 80-kDa form of Cp II is found in all gradient fractions, whereas the 78-kDa form is predominant in fractions 6-12. Moreover, the ratio of the 80-kDa to the 78-kDa forms of Cp II is dependent on their association with the different organelles. Accordingly, only the 80-kDa Cp II is detected in the densest region of the gradient (fractions 1-3). Appreciable amounts of the 78-kDa form of Cp II initially are observed in the ERGIC (fractions 7 and 8) and exceed that of the 80-kDa form in the GA and vesicle/PM fractions. These data suggest that the 80-kDa form of Cp II may undergo conversion to the 78-kDa form within the GA. Both the Rs and Cs are found distributed throughout the gradient, but the greatest concentration of these proteins extends from the ERGIC to the vesicle/PM fractions of the gradient (fractions 7-12). It is noteworthy that the Rs and Cs also are present in gradient fractions 7-12 concurrent with the appearance of the 78-kDa form of Cp II, thereby suggesting a potential site for regulation of Cp activity. Electrostatic Association of Cp and Its Regulatory Proteins with Organelle Membranes—Having demonstrated differential compartmentalization of Cp and its regulatory proteins with the ER, ERGIC, and GA, the next set of investigations were designed to begin to determine the mechanism(s) of this association. To explore the possibility that the association of Cp, Rs, and Cs with the ER and GA is dependent on electrostatic interactions, microsomes were prepared from A-549 cells and treated with either NaCl or KCl or the divalent metal ion chelators EDTA or EGTA (Fig. 4). The results reveal no observable changes in binding of Cp I, Cp II, or the Rs to microsomes after treatment with NaCl or KCl. Similarly, the association of these proteins with microsomes was not altered after treatment with the divalent metal chelator EDTA or the more Ca2+-specific chelator EGTA. Interestingly, a modest decrease in the 110-kDa form of Cs was noted in all treatment groups but not for the other breakdown products of Cs. These results indicate that the association of the Cp/Cs network with organelles is not dependent on electrostatic interactions. T
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