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

Abstract

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 Na 2 CO 3 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)1 is a Ca 2ϩ -dependent neutral cysteine protease (1). 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 Ca 2ϩ requirements for activation. Thus, Cp I (-calpain) requires 3-50 M Ca 2ϩ , whereas 0.2-1.0 mM of Ca 2ϩ is required for activation of Cp II (m-calpain) (2-4). In general, these concentrations of Ca 2ϩ are far greater than can be achieved intracellularly (Ͻ1 M) (5), suggesting that additional factors, such as binding to phospholipids (6, 7) or activation proteins (8, 9), are required for in vivo activation of both isoforms. The specific endogenous inhibitor of Cp I and Cp II is calpastatin (Cs).The Cp/Cs network is ubiquitously expressed in all vertebrate cells (10 -12) and is involved in a number of important cellular processes, including cell adherence, shape change, migration, and apoptosis (13). Activation of Cp during these processes requires increases in intracellular calcium ([Ca 2ϩ ] i ) as a consequence of receptor-ligand binding or the addition of Ca 2ϩ ionophores (14 -20). These increases in [Ca 2ϩ ] i also induce translocation of Cp from the cytosol to the plasma membrane (PM) and focal adhesions, contacts, or clusters (3, 21-25), where it facilitates reorganization of the actin cytoskeleton...

μ-Calpain binds to lipid bilayers via the exposed hydrophobic surface of its Ca2+-activated conformation

“…Ca 2q -dependent binding of the fulllength small subunit to phospholipids had only a four-to eight-fold lower affinity than the Ca 2q -dependent binding of the heterodimer to phospholipids (Table 3). These in vitro results are consistent with our previous observation of the Ca 2q -dependent translocation of an overexpressed small subunit to cellular membranes (Gil-Parrado et al, 2003) and the recently reported association of the small subunit with the endoplasmic reticulum and the Golgi apparatus (Hood et al, 2004). The ;600-fold reduced phospholipid affinity of the N-terminally truncated single small subunit, (DV)S, clearly underlines the essential contribution of domain V to lipid binding of the small subunit.…”

“…Recent work revealed an increased association of calpains with the endoplasmic reticulum and the Golgi apparatus after stimulation of cells with laminin. In these studies m-calpain co-localised with phosphatidylinositol-4,5-bisphosphate (PIP 2 ) and with membrane lipid rafts (Hood et al, 2004). However, Zalewska et al (2004) reported that inside-out erythrocyte vesicles did not lower the Ca 2q requirement for autolysis of m-and m-calpain.…”

“…The lack of headgroup selectivity supports a membrane-binding mechanism involving hydrophobic interactions with the non-polar moieties of phospholipid vesicles. Indeed, recent work using velocity gradient centrifugation suggested that the large and small subunits of m-and m-calpain may be embedded within the organelle membranes, similar to integral membrane proteins (Hood et al, 2004).…”

An increase in intracellular Ca2+ is required for the activation of mitochondrial calpain to release AIF during cell death

“…This association is hydrophobic; therefore, calpains reside mainly on the cytosolic side of the organelle membranes. [28][29][30][31] However, recent findings indicate that calpain 1 is also present within the mitochondria. 23 It has been suggested that OMM permeabilization should precede AIF processing during apoptosis, as the overexpression of Bcl-2 and Bcl-X L prevented both AIF cleavage and release.…”

Selenoprotein K Is a Novel Target of m-Calpain, and Cleavage Is Regulated by Toll-like Receptor-induced Calpastatin in Macrophages

“…In early studies, calpain and calpastatin were identified in the ER isolated from A549 lung adenocarcinoma cells (8). Topological studies suggested that m-calpain was oriented with the catalytic 80-kDa subunit mostly exposed to the cytolosic side of the ER membrane, along with the regulatory regions of calpastatin (9).…”