Differential Compartmentalization of the Calpain/Calpastatin Network with the Endoplasmic Reticulum and Golgi Apparatus*

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.

rabbit), Alexa 594 (anti-mouse and anti-rabbit), and the ProLong antifade 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% CO 2 . 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 ϫ1000 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 (28). 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 ϫ g for 15 min. Cell pellets were resuspended in 4 ml of isotonic cell transfer (ICT) homogenization medium (78 mM KCl, 4 mM MgCl 2 , 8.37 mM CaCl 2 , 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-m clearance. The resulting cell lysate was spun at 1500 ϫ 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 ϫ 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 ϫ g for 20 min. Pellets were washed two times with acetone to remove the trichloroacetic acid and resuspended in 300 l of Laemmli's sample buffer. Samples were heated to 80°C and vortexed three times, and 10 l 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 ϫ g for 15 min. Cell pellets were resuspended in 4 ml of ICT homogenization medium (78 mM KCl, 4 mM MgCl 2 , 8.37 mM CaCl 2 , 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-m clearance. The resulting cell lysate was spun at 1500 ϫ 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 ϫ g in a TLA 100.3 rotor for 1 h at 4°C to pellet organelle microsomes. The microsomal pellet was resuspended in 200 l 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 ϫ 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 l of Laemmli's sample buffer. Samples were heated to 80°C and vortexed three times, and 10 g of each sample was loaded onto a 4 -20% Tris-HCl polyacrylamide gel (Bio-Rad) and analyzed by Western blotting using nitrocellulose membrane. Na 2 CO 3 Treatment 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 ϫ g for 15 min. Cell pellets were resuspended in 4 ml of ICT homogenization medium (78 mM KCl, 4 mM MgCl 2 , 8.37 mM CaCl 2 , 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-m clearance. The resulting cell lysate was spun at 1500 ϫ 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 ϫ g in a TLA 100.3 rotor for 1 h at 4°C to pellet organelle microsomes. The microsomal pellet was resuspended in 200 l of 50 mM Tris buffer, pH 7.4, a protein assay was done, and 0.25 mg of control microsomes were solubilized in 250 l of Laemmli's sample buffer, whereas another set was diluted ϳ30-fold in 0.2 M Na 2 CO 3 , pH 11.5, at 0°C. The microsomes were treated with Na 2 CO 3 for 30 min at 0°C to open the microsomes and strip them of peripherally associated proteins, leaving behind integral membrane proteins. After Na 2 CO 3 treatment, microsomes were spun at 100,000 ϫ g in a TLA 100.3 rotor for 1 h at 4°C. The treated pellet was solubilized in 250 l 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 ϫ g for 20 min at 4°C, the pellets were washed two times with acetone to remove the trichloroacetic acid and resuspended in 250 l of Laemmli's sample buffer. Samples were heated to 80°C and vortexed three times, and 10 g 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 ϫ g for 15 min. Cell pellets were resuspended in 4 ml of ICT homogenization medium (78 mM KCl, 4 mM MgCl 2 , 8.37 mM CaCl 2 , 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-m clearance. The resulting cell lysate was spun at 1500 ϫ 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 ϫ g in a TLA 100.3 rotor for 1 h at 4°C to pellet the microsomes. The microsomal pellet was resuspended in 200 l 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 g/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 ϫ 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 l of Laemmli's sample buffer. Samples were heated to 80°C and vortexed three times, and 10 g 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 (28). 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 ϫ g for 15 min. Cell pellets were resuspended in 4 ml of ICT homogenization medium (78 mM KCl, 4 mM MgCl 2 , 8.37 mM CaCl 2 , 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-m clearance. The resulting cell lysate was spun at 1500 ϫ 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 ϫ 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-l 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 g/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 ϫ g for 20 min. Pellets were washed two times with acetone to remove the trichloroacetic acid and resuspended in 150 l of Laemmli's sample buffer. Samples were heated to 80°C and vortexed three times, and 10 l 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 (29) or the lumenal protein chaperone Grp 78/KDEL (30) (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 COP␤, a COP I vesicle coat protein (31), or P230, a protein associated with the GA (32) (Fig. 2). Cells stained with antibodies to Cp II or COP␤ 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 micros-copy 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 (33). The trans-GA-associated 14-3-3 protein (28) resided in fractions 9 -11 of the gradient, whereas ribophorin II, an ER-resident protein (34), was detected in gradient fractions 3-7. COP␤ was distributed throughout the gradient, with the majority detected in fractions 8 and 9, corresponding to the location of the GA. Because COP␤ 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 (31). Additionally, because the size and buoyancy of vesicles transported by COP␤ vary greatly it is not unreasonable to find COP␤ throughout the gradient. However, for the purposes of analysis, the ERGIC will be defined as the COP␤-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 Ca 2ϩspecific 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.
The Association of the Cp/Cs Network with Organelles as Peripheral or Integral Membrane Proteins-The next sets of experiments were designed to determine whether the Cp/Cs network associated with organelle membranes as peripheral or integral membrane proteins. Accordingly, microsomes obtained from A-549 cells were treated with Na 2 CO 3 at pH 11.5 (35) and the presence of the GA peripheral protein 14-3-3, the transmembrane protein calnexin, and calreticulin, a lumenal ER protein (36), assessed by Western analysis (Fig. 5). As expected, treatment of microsomes with Na 2 CO 3 removed the peripheral protein 14-3-3 associated with the cytosolic face of the trans-GA as well as the ER lumenal protein calreticulin (Fig. 5A). Re-moval of these ER-and GA-associated proteins was confirmed by the presence of 14-3-3 and calreticulin in the Na 2 CO 3 supernatant isolated subsequent to ultracentrifugation. In contrast, calnexin, a trans-membrane protein associated with the ER, was not removed by carbonate and did not appear in the carbonate supernatant.
Similar experiments were done to determine the association of the Cp/Cs network with organelle microsomes after carbonate treatment as described (Fig. 5B). The results show that Cp I was not susceptible to carbonate treatment; none was removed from the organelles, nor did any appear in the Na 2 CO 3 supernatant. The 78-kDa form of Cp II and the Rs were relatively resistant to carbonate treatment, and only small amounts of either appeared in the carbonate supernatant. In contrast, most of the 80-kDa form of Cp II appeared in the carbonate supernatant with only small amounts remaining associated with the microsomes. Carbonate treatment removed approximately half of all forms of Cs. These results indicate that Cp I, the majority of 78-kDa Cp II, and the Rs are integral membrane proteins or interact with organelles similar to integral membrane proteins. However, Cs and the majority of 80-kDa Cp II appear to be peripherally associated membrane proteins.
Determining the Cytosolic or Lumenal Orientation of the Cp/Cs Network with Organelle Membranes-Additional experiments were performed to determine the cytosolic or lumenal orientation of the Cp/Cs network associated with organelle membranes. Thus, organelle microsomes obtained from A-549 cells were treated with either DC to permeabilize microsomal membranes and remove lumenal contents or PK, which enzymatically cleaves proteins on the cytoplasmic face of microsomes. The presence or absence of the ERGIC vesicle coat protein COP␤, ER trans-membrane protein calnexin, and ER lumenal protein calreticulin subsequent to treatment served as controls (Fig. 6A). Treatment with PK removed COP␤ that is associated with the cytosolic surface and cleaved the cytosolic domain of calnexin, generating an 80-kDa fragment. Calreti-

FIG. 3. Subcellular localization of the Cp and its regulatory subunits to the ER and GA in A-549 cells using velocity gradient centrifugation.
A-549 cells were cultured to confluence and subcultured 24 h before use, and cellular membranes were isolated and separated on Optiprep velocity gradients. Gradient fractions were subjected to SDS-PAGE, and Western analysis was performed using antibodies to ribophorin II, COP ␤, mannosidase II, 14-3-3, Cp I, Cp II, Rs, and Cs. These data are representative of at least three experiments.
culin, an ER lumenal protein, was resistant to PK treatment. Treatment of microsomes with DC resulted in the loss of calreticulin but had no effect on calnexin or COP␤. However, when microsomes were treated with PK and DC, both calnexin and COP␤ were removed, consistent with DC treatment creating access to these moieties for proteolysis by PK.
The effects of PK and DC treatment on the association of the Cp/Cs network with microsomes obtained from A-549 cells are shown in Fig. 6B. Cp I and the majority of the 78-kDa form of Cp II were removed from these microsomes after treatment with PK leaving behind no discernible membrane-associated fragments, suggesting that they are not trans-membrane proteins. However, neither Cp I nor the 78-kDa form of Cp II was susceptible to DC treatment alone. Thus, Cp I and the majority of 78-kDa Cp II are located on the cytosolic surface of the microsomes and behave like traditional integral membrane proteins but are atypical in that PK treatment reveals no discernible trans-membrane segments. In contrast, most of the 80-kDa form of Cp II proved resistant to PK treatment, suggesting that it resides within the lumen of the microsomes. Moreover, microsome permeabilization by DC alone removed most of the 80-kDa form of Cp II from the microsomes, further supporting the presence of 80-kDa Cp II within the microsomal lumen. Similar to Cp I and the majority of the 78-kDa form of Cp II, the Rs and Cs were both resistant to lone DC exposure. Treatment of microsomes with PK removed the majority of the Rs and generated an 18-kDa fragment that remained with the microsome membranes, suggesting that the Rs is or behaves like an integral membrane protein. Similarly, PK treatment also removed all but a small amount of the 25-kDa fragment of Cs from the microsome membranes. The combination of PK treatment with DC membrane permeabilization allowed PK access to the lumenal side of the microsomes, resulting in the complete digestion of Cp I, Cp II, and Cs. However, a signifi-cant portion of the Rs remained associated with the microsomes after the combination treatment, suggesting that the Rs is embedded within the microsomal membrane to such an extent that PK cannot access it from the cytosolic or lumenal side. Collectively, these results indicate that Cp I and Cs are located on the cytosolic face of organelle membranes. In contrast, the Rs and Cp II are oriented to both the cytosolic and lumenal sides of organelle membranes with the majority of the 78-kDa form of Cp II being cytosolic and the majority of the 80-kDa form of Cp II being lumenal.
The cytosolic and lumenal orientation of Cp II and the Rs was further examined using PNS prepared from A-549 cells subjected to Optiprep velocity gradient centrifugation, and the resulting fractions were subsequently treated with PK. Treatment of each fraction with PK revealed obvious differences in the association of 78-kDa Cp II, 80-kDa Cp II, and the Rs with the ER and GA (Fig. 7). Whereas the majority of the 80-kDa Cp II form associated with the ER is resistant to PK treatment, that associated with the GA is not. However, a small amount of the 78-kDa form of Cp II associated with the GA remains resistant to PK. The Rs is found distributed throughout the ER and GA. PK treatment removes all of the Rs associated with the ER (gradient fractions 4 -6), whereas a portion of the Rs associated with the GA is resistant to treatment with this enzyme. These results further show that the organelle-associated 80-kDa form of Cp II is localized to the lumen of the ER and the cytosolic surface of the GA, whereas the 78-kDa Cp II form is oriented to both the cytosolic and lumenal sides of the GA. DISCUSSION Calpain is implicated in a variety of cellular functions including mobility, cell division, and apoptosis. Moreover, this protease has been demonstrated to be particularly relevant in many disease states. Although classically considered to be a

FIG. 4. Electrostatic interactions between Cp, Cs, and the Rs and microsomes obtained from A-549 cells.
A-549 cells were cultured to confluence and subcultured 24 h before use, and the microsomes were isolated by centrifugation. Microsomes (0.25 mg) were washed for 1 h at 37°C with 100 mM NaCl, 100 mM KCl, 10 mM EDTA, 10 mM EGTA, or 10 mM EDTA plus 10 mM EGTA, subjected to SDS-PAGE, and Western analysis was done using antibodies to Cp I, Cp II, Rs, and Cs. cytoplasmic protease, the mechanism through which this protease translocates to sites of action remains to be resolved. The novel observations of this study begin to suggest a paradigm by which the engagement of the Cp/Cs network with organelles determines how Cp may be spatially and temporally regulated.
Using confocal microscopy and velocity gradient centrifugation, the current findings extend our previous report by clearly showing that the Cp/Cs network is tightly associated with subcellular organelles (27). Additionally, Cp I, Rs, Cs, and 78-kDa Cp II are associated predominantly with the GA, whereas 80-kDa Cp II is associated with both the ER and GA organelles. Interestingly, Cp I, Cp II, Cs, and the Rs are found on the cytosolic surface of the organelle membranes, whereas a subset of Cp II and Rs also appear to be within the lumen of the organelles. The Rs within the lumen of the organelles is probably associated with the lumenal membrane face of the organelles, whereas Cp II is free within the lumen. These results begin to provide a novel explanation regarding the regulation of Cp, which includes the requirement for greater than 1 M Ca 2ϩ for the initiation of protease activity. The association of the FIG. 5. Treatment of microsomes obtained from A-549 cells with Na 2 CO 3 to determine peripheral versus integral association of Cp, Rs, and Cs with membranes. A-549 cells were cultured to confluence and subcultured 24 h before use, and microsomes were isolated by centrifugation. Microsomes (0.25 mg) were incubated with 0.2 M Na 2 CO 3 , pH 11.5, for 30 min at 0°C and subjected to SDS-PAGE and Western analysis done using antibodies to control proteins 14-3-3, calnexin, and calreticulin (A) or Cp I, Cp II, Rs, and Cs (B).
Cp/Cs network with the ER and GA places it in the vicinity of high [Ca 2ϩ ] i microenvironments (37,38). Additionally, the association of Cp with these subcellular organelles could occur through interactions with phospholipids such as phosphatidylinositol 4,5-bisphosphate, thereby lowering the Ca 2ϩ threshold for activation and facilitating protease activation via an electrostatic switch mechanism involving the C-2-like domain (39). Finally, the ER and GA provide platforms for the Cp regulatory proteins, Cs and the Rs, to spatially and temporally modulate Cp activity as well as facilitate encounters between Cp and strategically placed substrates associated with, synthesized by, or modified by these organelles.
Spatial and temporal modulation of Cp activity can be achieved through differential compartmentalization of Cp and its regulatory proteins on subcellular organelles. For example, the preferential association of Cs for the GA over the ER suggests a greater propensity for Cs to inhibit Cp activity in the GA. Why Cs favors an association with the GA rather than the ER is unclear. One possibility is that the Lys-Lys-Arg-His-Lys-Lys stretch of positively charged amino acids in its domain L (1) FIG. 6. Treatment of microsomes obtained from A-549 cells with PK or DC to determine peripheral versus lumenal orientation of Cp, Rs, and Cs with membranes. A-549 cells were cultured to confluence and subcultured 24 h before use, and organelle microsomes were isolated by centrifugation. Microsomes (0.25 mg) were incubated with PK (0.5 g/ml), DC (0.1%), or both for 20 min at 37°C and subjected to SDS-PAGE and Western analysis done using antibodies to control proteins calreticulin, calnexin, and COP ␤ (A) or Cp I, Cp II, Rs, and Cs (B). confers a greater targeting specificity for the GA than the ER. Regardless, the positively charged amino acids could effectively tether Cs to the cytosolic surface of subcellular membranes, leaving its inhibitory subdomains (A, B, and C) exposed to the cytoplasm. This would strategically position Cs to inhibit Cp activity on the cytosolic surface of the ER and GA.
Thus, a potential site for Cs regulation of Cp activity is created by positioning Cs, Cp I, and potentially active 78-kDa Cp II on the cytosolic face of the GA. However, such is not the case for the 80-kDa Cp II found within the lumen of the ER and GA because of the conspicuous absence of Cs in this locale. How the absence of its endogenous inhibitor influences the activation of lumenal 80-kDa Cp II remains to be determined. Regardless, differential compartmentalization of lumenal 80-kDa Cp II and its endogenous inhibitor Cs represents an additional way of spatially and temporally regulating Cp activity. Moreover, Cs found on the cytosolic face of the GA may be within a different GA subcompartment than Cp I or 78-kDa Cp II. An example of this is the absence of Cs in microdomains containing Cp II isolated from the GA (27). Differential organelle subcompartmentalization of Cp II and Cs within the cell may explain why the concentration of Ca 2ϩ required for Cs to inhibit Cp II activity is less than the concentration of Ca 2ϩ required for Cp II to become active in vitro (1).
Although the cDNA of Cp I or II does not reveal any known signal sequence to target Cp to the ER or GA (1), a number of potential mechanisms can be offered. For instance, both Cp I and Cp II contain C2-like domain acidic loops (39 -41) similar to the C2 domain acidic loop of phospholipase A2 (42). The binding of Ca 2ϩ to the C2 domain acidic loops of phospholipase A2 targets this enzyme to the ER and GA (42). In a similar fashion, Cp also may be targeted to subcellular organelles. This may also explain why Cp I preferentially associates with the GA, whereas Cp II associates with both the ER and GA. Thus, one possibility for the difference in the targeting of Cp I and Cp II to organelles may reside in the structure of their acidic loops. The Cp I molecule contains eight widely spaced negative charges within its acidic loop, whereas the Cp II molecule contains 10 more closely spaced negative charges (39). Consequently, the total negative charge of the acidic loop of the Cp I molecule is less than that of the Cp II molecule as well as distributed over a larger area. The resultant reduction in charge repulsion could allow the Cp I molecule to come into closer proximity to a negatively charged phospholipid surface. Importantly, the lower number of negatively charged residues in the acidic loop of the Cp I molecule, versus those in the Cp II molecule, would require fewer Ca 2ϩ ions to neutralize the net negative charge repulsion between the acidic loop of the Cp I molecule and membrane phospholipids than for the Cp II molecule. However, if the Cp II molecule maximizes its ability to bind Ca 2ϩ within its acidic loop, it is conceivable that the Cp II molecule will have a greater attractive force for membrane phospholipids than the Cp I molecule. The overall result would be that the acidic loop of the Cp II molecule would become more positively charged than that of the Cp I molecule and be able to more deeply penetrate a membrane surface by pushing past negatively charged phospholipid head groups. This provides an explanation for the differential organelle distribution of Cp I and II as well as their Ca 2ϩ requirements for activation. Consequently, differences in the targeting specificity of Cp I and Cp II molecules initially may be dependent on electrostatic interactions, in the presence or absence of Ca 2ϩ , with certain membrane phospholipids. After initial electrostatic recruitment of Cp I and Cp II molecules to their respective organelles, differences in hydrophobicity between these molecules may lead to more precise localization to the ER or GA.
Interestingly, some of the Cp II is found in the lumen of the ER and GA despite the absence of a known N-terminal signal FIG. 7. Gradient fractions obtained by velocity gradient centrifugation of A-549 cell lysates treated with PK to determine peripheral versus lumenal orientation of Cp II and the Rs with the ER and GA. A-549 cells were cultured to confluence and subcultured 24 h before use, and cell lysates were prepared and separated on Optiprep velocity gradients. Gradient fractions were incubated with or without PK (0.5 g/ml) for 20 min at 37°C and subjected to SDS-PAGE, and Western analysis was performed using antibodies to Cp II and the Rs. Organelle distribution corresponds to those shown in Fig. 3. These data are representative of at least three experiments. peptide capable of targeting Cp II into the ER lumen (43). Therefore, the entry of this molecule into the lumen of the ER must occur by some other means. One possibility is that Cp II possesses an internal topogenic signal sequence that assembles post-translationally through intramolecular conformational changes, and it is this new topology that targets Cp II into the ER lumen. A number of proteins utilize internal rather than N-terminal signal sequences to enter the ER lumen (44 -47). To resolve how Cp II gains entry into the ER will require deletion or substitution mutations to attempt to identify an internal topogenic signal sequence. Whereas the functional significance of Cp II residing in the lumen of the ER remains to be established, a number of advantages are imparted to the cell. For example, this may provide an opportunity for Cp II to cleave substrates shortly after their synthesis and insertion into the ER membrane as well as those that accumulate in the ER lumen. Another potential role for intralumenal Cp II may be to aid in the removal of misfolded proteins. Finally, Cp II in the ER lumen may not be active and may ultimately be moved from the ER through the GA and secreted. It has been observed that exteriorized Cp II was required for the degradation of the extracellular matrix protein fibronectin in myoblasts undergoing fusion (48). Additionally, the presence of Cp II in the extracellular matrix of MC3T3-E1 cells appears to be dependent on the secretion of matrix and media vesicles containing this protease (43).
The results indicate that the Rs is found on the cytosolic side of the ER and GA as well as in the lumen. This prompts several questions, such as how the Rs associates with organelles and gains entry into the lumen of organelles. An important functional auxiliary question is whether the Rs is required for Cp activity or functions more in the capacity of a Cp-specific chaperone or docking site for Cp in these organelles. A docking role for the Rs is supported by the fact that the Rs proved to be more difficult to remove from organelles by biochemical or proteolytic means than Cp I, Cp II, or Cs. This would suggest that the Rs   FIG. 8. A model for the association of the Cp/Cs network with the ER and GA obtained from A-549 cells. A, the association of the Cp/Cs network with the ER. A portion of the 80-kDa Cp II localizes to the lumen of the ER, whereas the remainder is "embedded" on the cytosolic side of the membrane. The Rs is found only in the lumen, and Cs associates with the ER as a cytosolic peripheral protein. B, the association of the Cp/Cs network with the GA. Both the 80-and 78-kDa forms of Cp II are found within the lumen as well as "embedded" on the cytosolic surface of the GA. Conversely, Cp I is only associated with the cytosolic side of the GA as an "embedded" protein. The Rs is "embedded" within the cytosolic and lumenal surfaces of the GA as well as between these surfaces. Finally, Cs associates with the GA as a cytosolic peripheral protein. Orange triangle, Cp I; red triangle, Cp II; dark blue rectangle, Cs; green circle, Rs. has a higher affinity for organelle membranes than Cp I, Cp II, or Cs and because of this strong association aids in the docking of Cp.
Structurally, the Rs is composed of two domains, D-V and D-VI. D-VI contains an EF-hand pentamer and thus is capable of associating with membranes through these hydrophobic EFhands (49). D-V of the Rs is unusual in that it contains 40 glycine residues that are moderately hydrophobic, 30 hydrophobic residues, 5 prolines, and 26 polar residues (1). It could be envisioned that the polyglycine repeats within Rs D-V confer a high degree of flexibility to the molecule, whereas the hydrophobic residues allow for strong associations between the Rs and membranes. Therefore, the binding of the Rs to ER and GA membranes would provide a platform for Cp binding to the ER and GA through hydrophobic interactions between EF hands of Cp D-IV and Rs D-VI. Thus, Cp could associate with the Rs in the cytosol and "dock" as a heterodimer to the ER and GA or Cp could "dock" as a monomer to previously bound Rs. This allows for a degree of flexibility in the regulation of the Cp/Cs network and both forms of docking may occur depending on the physiology of the cell. Once docked to organelles, the Rs may facilitate the conformational changes that take place in the Cp molecule leading to its activation but may not be required subsequent to activation. In this context, the Rs is serving three functions: (a) to act as a chaperone for Cp in the cytosol, (b) to aid in its association with organelles, and (c) to assist in its activation.
The mechanism for how Cp is spatially and temporally regulated in the cell remains largely unknown. The observations reported here begin to offer insight into these mechanisms. These studies suggest a new and novel paradigm as to how the Cp/Cs network operates by demonstrating that the Cp/Cs network is associated with the cytosolic surface and the lumen of the ER and GA (Fig. 8). The findings that Cp, Cs, and the Rs associate with the ER and GA begin to explain the coordinated actions of this complex protease system. The association of Cp with the ER and GA places it in the vicinity of high [Ca 2ϩ ] i microenvironments as well as providing it with convenient proximity to potential substrates. Finally, the relationship between Cp and subcellular organelles insures efficient regulation of Cp activity by Cs and the Rs.