Abstract
The endoplasmic reticulum (ER) is a highly dynamic organelle, continuously undergoing membrane fusion and fission. We have measured homotypic fusion between ER vesicles isolated from Chinese hamster ovary cells kinetically in vitro, using an assay based on the metabolic incorporation of pyrene-labeled fatty acids into the phospholipids of cellular membranes. An increase in pyrene-monomer fluorescence was observed after mixing labeled and unlabeled ER vesicles in the presence of ATP and GTP. The protein, temperature, and nucleotide dependence of the increase indicated that it was caused by membrane fusion rather than molecular transfer of labeled lipids to unlabeled membranes. This assay allowed the first kinetic measurements with virtually nonexchangeable probes of a homotypic membrane fusion event. At 37 degrees C, fusion started off immediately at a rate of 1.14 +/- 0.29%/min and reached a half-maximal level after 56 min. In the presence of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), or after treatment of the membranes with N-ethylmaleimide, fusion was reduced but not completely inhibited. Addition of GTP during a fusion reaction immediately accelerated, and GTPgammaS immediately slowed down the fusion reaction. Thus, these kinetic measurements indicate that G-proteins might act to rapidly enhance fusion beyond a basic level.
Highlights
The molecular mechanism of membrane fusion is best known for fusion induced by the hemagglutinin protein of influenza virus
TLC analysis of lipids extracted from the vesicles showed that pyrene was incorporated efficiently into the phospholipids, similar to what was reported for enveloped viruses [16]. 44.7 Ϯ 4.0% of the pyrene was found in phosphatidylcholine and 34.0 Ϯ 12.4% in phosphatidylethanolamine, whereas 11.9 Ϯ 3.7% was still in fatty acids
The hydrolysis of GTP was required for this stimulation, and we found that the addition of GTP or guanosine 5-O-(3-thiotriphosphate) (GTP␥S) had an immediate effect on the reaction, suggesting that G-proteins might be involved in this process
Summary
We have measured homotypic fusion between ER vesicles isolated from Chinese hamster ovary cells kinetically in vitro, using an assay based on the metabolic incorporation of pyrene-labeled fatty acids into the phospholipids of cellular membranes. The protein, temperature, and nucleotide dependence of the increase indicated that it was caused by membrane fusion rather than molecular transfer of labeled lipids to unlabeled membranes This assay allowed the first kinetic measurements with virtually nonexchangeable probes of a homotypic membrane fusion event. We have isolated ER vesicles from CHO cells labeled with pyrene phospholipids through the metabolic incorporation of pyrene fatty acids Fusion of these vesicles with unlabeled ER vesicles was measured by an assay based on the concentration-dependent formation of pyrene excimers. ER Fusion (GTP␥S) during the reaction had an immediate effect on the rate of fusion, indicating that a GTPase might act as a fusion switch
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