Abstract
Past experiments with reconstituted proteoliposomes, employing assays that infer membrane fusion from fluorescent lipid dequenching, have suggested that vacuolar SNAREs alone suffice to catalyze membrane fusion in vitro. While we could replicate these results, we detected very little fusion with the more rigorous assay of lumenal compartment mixing. Exploring the discrepancies between lipid-dequenching and content-mixing assays, we surprisingly found that the disposition of the fluorescent lipids with respect to SNAREs had a striking effect. Without other proteins, the association of SNAREs in trans causes lipid dequenching that cannot be ascribed to fusion or hemifusion. Tethering of the SNARE-bearing proteoliposomes was required for efficient lumenal compartment mixing. While the physiological HOPS tethering complex caused a few-fold increase of trans-SNARE association, the rate of content mixing increased more than 100-fold. Thus tethering has a role in promoting membrane fusion that extends beyond simply increasing the amount of total trans-SNARE complex.
Highlights
A hallmark of eukaryotic cells is their internal organization by distinct, highly specialized, membraneenclosed compartments
The two fluorophores are subject to fluorescence resonance energy transfer (FRET), so that the fluorescence emission of the ‘donor’ fluorophore is quenched due to its close proximity to the ‘acceptor’ fluorophore
FRET efficiency varies with the 6th power of the average distance between fluorophores (Förster, 1948); fusion to unlabeled proteoliposomes, causing a twofold dilution of fluorescent lipids and thereby an increased average distance between fluorophores, results in a remarkable loss of quenching, and the ensuing increase in donor fluorescence is taken as a measure of fusion
Summary
A hallmark of eukaryotic cells is their internal organization by distinct, highly specialized, membraneenclosed compartments. Vesicular traffic functionally connects these individual compartments while preserving their integrity. The constant vectorial exchange between organelles is ensured by a complex machinery that facilitates the formation of vesicles with selective cargo, vesicle movement, specific tethering of each vesicle at its correct target membrane, and fusion of the vesicle and organelle membranes. Selective tethering and fusion are sequential events, but it remains unclear how tightly they are coupled. Tethering is commonly performed by specific proteins, often in large multi-subunit complexes that bind Rab family GTPases (Stenmark, 2009; Yu and Hughson, 2010). Fusion is mediated by soluble N-ethylmaleimide-sensitive attachment protein receptor (SNARE) proteins (Jahn and Scheller, 2006) and Sec1/Munc (SM) proteins (Rizo and Sudhof, 2012)
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