IN-VIVO EXPRESSION OF MAMMALIAN BIP ATPASE MUTANTS CAUSES DISRUPTION OF THE ENDOPLASMIC-RETICULUM

BiP possesses ATP binding/hydrolysis activities that are thought to be essential for its ability to chaperone protein folding and assembly i n the endoplasmic reticulum (ER). We have produced a series of point m utations in a hamster BiP clone that inhibit ATPase activity and have generated a species-specific anti-BiP antibody to monitor the effects of mutant hamster BiP expression in COS monkey cells. The enzymatic in activation of BiP did not interfere with its ability to bind to Ig hea vy chains in vivo but did inhibit ATP-mediated release of heavy chains in vitro. Immunofluorescence staining and electron microscopy reveale d vesiculation of the ER membranes in COS cells expressing BiP ATPase mutants. ER disruption was not observed when a ''44K'' fragment of BiP that did not include the protein binding domain was similarly mutated but was observed when the protein binding region of BiP was expressed without an ATP binding domain. This suggests that BiP binding to targ et proteins as an inactive chaperone is responsible for the ER disrupt ion. This is the first report on the in vivo expression of mammalian B iP mutants and is demonstration that in vitro-identified ATPase mutant s behave as dominant negative mutants when expressed in vivo.