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 ( approximately 50 kDa), previously purified by us from rat liver endoplasmic reticulum, which explicates fusogenic 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 conditions in which FP is operating in microsomes have been investigated. Anti-FP polyclonal antibodies inhibited pure FP activity, but not the protein activity in microsomes, suggesting interaction of antibodies with a part of FP concealed in intact membranes. FP activity in microsomes was lost after treatment with Pronase. Western blot analysis of Pronase-treated microsomes showed that the proteolysis removed a fragment ( approximately 5 kDa). This fragment is exposed on the outer surface of microsomes and involved in fusogenic activity, whereas the largest part of FP is embedded in microsomal vesicles. Therefore, FP can be affected by modifications on the cytosolic and luminal sides of microsomal membranes. Indeed, when microsomal lumen was acidified by H+-ATPase activity, binding and fusion of fluorescent labelled liposomes 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 interior of liposomes, despite an exterior 7.4 pH unable to promote fusogenic protein activity. As intracellular membrane fusion occurs at neutral pH involving the cytosolic sides of membranes, FP may participate in this event by exploiting the acidic pH formed in the lumen of endoplasmic reticulum through H+-translocating ATPase activity.
Laboratory of Biochemistry, Department of Internal Medicine, University of Perugia, Italy.
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Monni
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Roberti
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Corazzi
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000906:Antibodies; D015153:Blotting, Western; D004721:Endoplasmic Reticulum / Q000201:enzymology*; D006023:Glycoproteins / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008081:Liposomes / Q000378:metabolism; D008099:Liver / Q000378:metabolism*; D008862:Microsomes, Liver / Q000737:chemistry; D008970:Molecular Weight; D011402:Pronase / Q000378:metabolism; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D011817:Rabbits; D051381:Rats
D000906:Antibodies; D006023:Glycoproteins; D008081:Liposomes; D000255:Adenosine Triphosphate; D006026:Glycoside Hydrolases; D011402:Pronase; D006180:Proton-Translocating ATPases