Ultrastructural localization of bcl-2 protein.

Abstract

Previous cell subfractionation studies have indicated that bcl-2 is an inner mitochondrial membrane protein. We have sought to determine the ultrastructural localization of bcl-2 protein in lymphoma and breast carcinoma cell lines and biopsy material known to overexpress bcl-2 using immunoelectron microscopy. To avoid the possibility of processing artifacts, samples were prepared by three different methods: progressive lowering of temperature, cryosectioning, and freeze-substitution. In all instances the labeling of bcl-2 protein was relatively weak but the distribution the same. In both lymphoma and breast carcinoma tissues, bcl-2 protein was detected on the periphery of mitochondria: little labeling of either the mitochondrial matrix or cristae could be detected. Labeling was also detected on the perinuclear membrane and throughout the cytoplasm, as also indicated by confocal microscopy. These data therefore indicate that bcl-2 protein can be detected at several intracellular sites and that at the likely functional destination, the mitochondria, there appears to be, contrary to expectations, a preferential association with the outer membrane.

Evidence against a functional site for Bcl-2 downstream of caspase cascade in preventing apoptosis

“…However, since caspases possessing substrate speci®city similar to that of caspase-3 likely function most downstream in the caspase cascade, we conclude that there are no functional sites of Bcl-2 and Bcl-x L downstream of the caspase cascade. Although several groups have shown that Bcl-2 protein localizes in multiple membrane compartments (Hockenbery et al, 1990;Monaghan et al, 1992;Jacobson et al, 1993;Krajewski et al, 1993;Akao et al, 1994), it has not been convincingly determined in which subcellular compartment Bcl-2 acts to prevent cell death. Since active caspase-3 induces apoptotic changes of nuclei in vivo when microinjected into the cytoplasm (Yasuhara et al, 1997, and this study) and also in vitro when incubated with naked nuclei and cell lysates (Enari et al, 1996), caspase-3-mediated death signals or the caspase itself must be transferred from the cytoplasm to the nucleus to induce apoptotic nuclear changes.…”

“…Thus, Bcl-2 acts at least upstream of the activation of caspases. Bcl-2 localizes in multiple membrane compartments, including the nuclear envelope, endoplasmic reticulum and mitochondrial membranes (Hockenbery et al, 1990;Monaghan et al, 1992;Jacobson et al, 1993;Krajewski et al, 1993;Akao et al, 1994), raising the possibility that Bcl-2 also acts downstream of caspases. Indeed an anti-cell death role for Bcl-2 exerted downstream of active ICE family proteases has been suggested by the observations that (1) using in vitro apoptosis system, apoptotic DNA ladder formation is prevented by a Bcl-2-containing cell lysate, even after an e ector(s) downstream of caspases is activated, as judged by the failure of caspase inhibitors to prevent apoptotic changes in naked nuclei (Enari et al, 1995) and (2) apoptosis induced by transfection with caspase genes which encode precursor forms of proteases is prevented by overexpressed Bcl-2 (Miura et al, 1993;Wang et al, 1994).…”

The Bax pore in liposomes, Biophysics

“…[1][2][3] The function of Bcl-2-family of proteins is to govern the central decision point within the mitochondrial (intrinsic) apoptotic pathway. [4][5][6][7][8][9][10] This protein family includes both proapoptotic (e.g. Bax, Bak, Bcl-x s , Bad, Bik, Bid, Hrk, Bim, Noxa) and antiapoptotic (e.g.…”

Overexpression of Bcl-2 inhibits alveolar cell apoptosis during involution and accelerates c-myc-induced tumorigenesis of the mammary gland in transgenic mice

“…Bcl-2 is an integral membrane protein localised to mitochondria, the endoplasmic reticulum and the nuclear envelope (Monaghan et al, 1992). The biochemistry of its functioning is still unknown.…”