Acidic pH generated by H+-ATPase pumps triggers the activity of a fusogenic protein associated with rat liver endoplasmic reticulum

Fusogenic protein (FP) is a glycoprotein (approximate to 50 kDa), previousl y purified by us from rat liver endoplasmic reticulum, which explicates fus ogenic activity at acidic pH in vitro. To suggest a possible role of FP in membrane fusion, the topology of the protein in the membrane and the condit ions in:which FP is operating in microsomes have been investigated. Anti-FP polyclonal antibodies inhibited pure FP activity, but not the protein acti vity in microsomes, suggesting interaction of antibodies with a part of FP concealed in intact membranes. FP activity in microsomes was lost after tre atment with Pronase. Western blot analysis of Pronase-treated microsomes sh owed that the proteolysis removed a fragment (approximate to 5 kDa). This f ragment is exposed on the outer surface of microsomes and involved in fusog enic activity, whereas the largest part of FP is embedded in microsomal ves icles. Therefore, FP can be affected by modifications on the cytosolic and luminal sides of microsomal membranes. Indeed, when microsomal lumen was ac idified by Hf-ATPase activity, binding and fusion of fluorescent labelled l iposomes to microsomes occurred. Direct involvement of FP in the fusogenic event was observed by reconstituting pure FP in liposomes with a preformed H+ gradient. FP triggered a fusion process in response to the acidic interi or of liposomes, despite an exterior 7.4 pH unable to promote fusogenic pro tein activity. As intracellular membrane fusion occurs at neutral pH involv ing the cytosolic sides of membranes, FP may participate in this event by e xploiting the acidic pH formed in the lumen of endoplasmic reticulum throug h H+-translocating ATPase activity.