MAMMALIAN-CELLS EXPRESS 2 DIFFERENTLY LOCALIZED BAG-1 ISOFORMS GENERATED BY ALTERNATIVE TRANSLATION INITIATION

The Bcl-2 oncoprotein is a key regulator of apoptosis and the Bag-1 pr otein interacts with Bcl-2 and cooperates with Bcl-2 to suppress apopt osis. The human Bag-1 cDNA is essentially identical with a previously described cDNA encoding RAP46, which interacts with activated steroid hormone receptors. However, there is considerable confusion over the s tructure of Bag-1/RAP46 proteins and their relationship to endogenous Bag-1 proteins. Here we have characterized Bag-1 expression in mammali an cells. We demonstrate that, in addition to the previously identifie d 32 kDa murine and 36 kDa human Bag-1 proteins, cells express a secon d 50 kDa Bag-1 isoform. In some murine cell lines p50 is expressed at the same level as p32 Bag-1, and p50 and p32 Bag-1 proteins have disti nct subcellular localizations, suggesting that they are functionally d istinct. The published mouse Bag-1 cDNA is partial, and sequencing of additional murine Bag-1 RNA 5' sequences demonstrated that human and m urine Bag-1 cDNAs contain longer open reading frames than originally s uspected. We determined which open reading frames gave rise to the Bag -1 isoforms in human cells. Suprisingly, translation of neither protei n initiated at the first in-frame methionine, and cells do not express Bag-1/RAP46 proteins with the previously proposed structures; p50 Bag -1 initiates at an upstream CUG codon, whereas p36 Bag-1 initiates at a downstream AUG codon. Therefore, cells express two differently local ized Bag-1 isoforms generated by alternative translation initiation, a nd Bag-1 proteins may play a dual role in regulating apoptosis and ste roid hormone-dependent transcription.