Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes.
A multidisciplinary approach was taken to investigate the intracellular locations of the 26-kDa integral membrane protein encoded by the bcl-2 gene. Subcellular fractionation analysis of a t(14;18)-containing lymphoma cell line revealed the presence of Bcl-2 protein in nuclear, heavy-membrane, and light-membrane fractions but not in cytosol. Sedimentation of heavy-membrane fractions in Nycodenz and Percoll continuous gradients demonstrated comigration of p26-Bcl-2 with mitochondrial but not other organelle-associated proteins. Fractionation of light-membrane fractions using discontinuous sucrose-gradients revealed association of Bcl-2 protein primarily with lighter-density microsomes (smooth endoplasmic reticulum) as opposed to heavy-density microsomes (rough endoplasmic reticulum). Immune microscopy studies using laser-scanning microscopy, pre- and postembedding electron microscopic methods, and six different anti-Bcl-2 antibodies demonstrated Bcl-2 immunoreactivity in the nuclear envelope and outer mitochondrial membrane in a patchy distribution. Furthermore, anti-Bcl-2 antibody immunoreactivity generally appeared to directly overlie the nuclear envelope in high magnification electron microscopic studies, reminiscent of nuclear pore complexes. Addition of in vitro translated p26-Bcl-2 to isolated translocation-competent mitochondria revealed transmembrane domain-dependent association of Bcl-2 protein with mitochondria but provided no evidence for import into a protease-resistant compartment, consistent with immunomicroscopic localization to the outer mitochondrial membrane. Taken together, the findings demonstrate that p26-Bcl-2 resides primarily in the nuclear envelope, endoplasmic reticulum, and outer mitochondrial membrane in a nonuniform distribution suggestive of participation in protein complexes perhaps involved in some aspect of transport.
Krajewski
S
S
Oncogene and Tumor Suppression Gene Program, La Jolla Cancer Research Foundation, California 92037.
Tanaka
S
S
Takayama
S
S
Schibler
M J
MJ
Fenton
W
W
Reed
J C
JC
D002458:Cell Fractionation / Q000379:methods; D002499:Centrifugation, Density Gradient; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D016393:Lymphoma, B-Cell; D008928:Mitochondria / Q000378:metabolism* / Q000648:ultrastructure; D009685:Nuclear Envelope / Q000378:metabolism* / Q000648:ultrastructure; D015388:Organelles / Q000378:metabolism* / Q000648:ultrastructure; D014176:Protein Biosynthesis; D011505:Protein-Tyrosine Kinases / Q000032:analysis / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000032:analysis / Q000096:biosynthesis / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2; D011519:Proto-Oncogenes; D013367:Submitochondrial Particles / Q000378:metabolism / Q000648:ultrastructure; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011505:Protein-Tyrosine Kinases