Membrane fusion underlies hormone secretion, neurotransmission, and all exocytic and endocytic traffic. Its mechanism is conserved from yeast to humans. A current paradigm suggests that membrane proteins termed SNAREs inexorably drive fusion when anchored in apposed membranes as a trans-SNARE complex. While SNAREs are required, genetic studies from yeast to humans show that Rab GTPases, their effectors, SM proteins, and SNARE chaperones are also essential. Our studies of yeast vacuole fusion are providing a new paradigm, illuminating the integrated mechanisms of these other essential proteins and showing that their actions extend beyond regulation of trans-SNARE complex levels. Vacuole fusion studies have progressed from initial genetics through our extensive study of in vitro organelle fusion to proteoliposome fusion, which we have reconstituted with all purified and defined proteins and lipids: SNAREs, SNARE disassembly chaperones Sec18p/Sec17p, the Rab GTPase Ypt7p, a hexameric Rab effector complex HOPS, and lipids which include acidic and bilayer-averse headgroups and specific fatty acyl chains. With a rigorous fusion assay of protected lumenal content mixing, our studies show that fusion is driven by several cooperating factors: bilayer stress from trans-SNARE complex assembly, membrane destabilization by nonbilayer lipids, and bilayer bending through the action of a multisubunit tether. Fusion is blocked by the omission of SNAREs, of nonbilayer-prone lipids, or of tethering factors, even though SNAREs still pair in trans in the latter 2 conditions. Our chemically-defined reconstitution of fusion allows independent variation of SNARE concentration, nonbilayer lipid concentration, and the HOPS and Rab tethering proteins, all while assaying both physical associations and fusion function. Testing and extending this model system is changing our view of fusion. In light of the fundamental role of fusion throughout human physiology, and the central role of human HOPS for cellular infection by Marburg and Ebola viruses and by bacteria such as the pathogen Coxiella burnetii, these studies will be of medical significance as well.