Protein dissociation from GRP78 and secretion are blocked by depletion of cellular ATP levels.

Secretory proteins expressed in Chinese hamster ovary (CHO) cells interact to various degrees with glucose-regulated protein 78 (GRP78), a resident protein of the endoplasmic reticulum. von Willebrand factor (vWF) and wild-type tissue plasminogen activator (tPA) are efficiently secreted and exhibit a slight transient association with GRP78. Factor VIII and unglycosylated tPA are inefficiently secreted and display a more stable association with GRP78. We have studied the effect of ATP depletion mediated by carbonyl cyanide 3-chlorophenylhydrazone (CCCP) treatment on GRP78 association and secretion of factor VIII and vWF that are coexpressed in CHO cells. Low concentrations of CCCP in the medium prevented disassociation of factor VIII from GRP78 and blocked its secretion. In the same cells, higher concentrations of CCCP were required to block secretion of vWF. Thus, the block to factor VIII secretion at low CCCP concentrations did not result from a general defect in secretion. Secretion of unglycosylated tPA but not wild-type tPA from CHO cells was also blocked by low concentrations of CCCP. The increased sensitivity to CCCP concentration observed for secretion of factor VIII and unglycosylated tPA compared to wild-type tPA and vWF correlates with their degree of interaction with GRP78. In vivo, dissociation from GRP78 may be a primary ATP-dependent step in transport from the endoplasmic reticulum. ATP requirements for secretion of various proteins may vary.