M. Monni et al., Acidic pH generated by H+-ATPase pumps triggers the activity of a fusogenic protein associated with rat liver endoplasmic reticulum, EUR J BIOCH, 268(7), 2001, pp. 2020-2027
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.