Homotypic fusion of early endosomes: SNAREs do not determine fusion specificity

Abstract

Membrane fusion in the secretory pathway is mediated by soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins. Different fusion steps are thought to be effected by independent sets of SNAREs, but it is unclear whether specificity is determined by an intrinsic specificity of SNARE pairing or by upstream factors. Using a newly developed microscopy-based assay, we have investigated the SNARE specificity of homotypic early endosomal fusion. We show that early endosomes contain multiple sets of SNAREs, including, in addition to the putative early endosomal SNAREs, those involved in exocytosis and in fusion of late endosomes. We demonstrate that fusion is largely mediated by a complex formed by syntaxin 13, syntaxin 6, vti1a, and VAMP4, whereas the exocytic and late endosomal SNAREs play little or no role in the reaction. In contrast, proteoliposomes reconstituted with early endosomal SNAREs promiscuously fuse with liposomes containing exocytotic or late endosomal SNAREs. We conclude that the specificity of SNARE pairing does not suffice to determine the specificity of organelle fusion.endocytosis ͉ exocytosis ͉ PC12 cells S oluble N-ethylmaleimide (NEM)-sensitive factor attachment receptor (SNARE) proteins are represented by a superfamily of small membrane-associated proteins that mediate membrane fusion in the secretory pathway. All SNAREs possess a SNARE motif that is usually flanked by a C-terminal transmembrane domain (for recent reviews, see refs.

Distinct Contributions of Vacuolar Qabc- and R-SNARE Proteins to Membrane Fusion Specificity

“…In contrast, the other conclusion provides the quite divergent concept that SNARE pairing is rather promiscuous and, thereby, largely incapable of regulating fusion specificity. Indeed, in vitro experiments with SNAREs in mammals showed that the endosomal and exocytic SNAREs readily form noncognate and mixed SNARE complexes (43,44) and cause promiscuous RPL fusion by non-physiological SNARE pairing (24). Despite the fact that we used the same approaches with the purified SNAREs and the RPL lipid mixing assay, our conclusions do not fully agree with either of these previous concepts.…”

“…Despite the fact that we used the same approaches with the purified SNAREs and the RPL lipid mixing assay, our conclusions do not fully agree with either of these previous concepts. This might be due to the differences in experimental details, including lipid compositions, lipid to protein ratios, and salt concentrations (22,24). Nevertheless, our findings establish that Q-SNAREs are the key components to mediate fusion specificity through the specific assembly of appropriate Qabc-SNARE complexes, whereas R-SNAREs have little contribution.…”

“…This RPL approach has been further employed to test whether SNAREs themselves ensure the specificity of membrane fusion (22)(23)(24). A high degree of specificity was observed by comprehensive analyses with yeast SNAREs (22), whereas RPLs bearing mammalian SNAREs in the endosomal and exocytic pathways can fuse rather promiscuously (24).…”

Synaptotagmin activates membrane fusion through a Ca2+-dependent trans interaction with phospholipids

“…The expression constructs for the neuronal SNAREs were the full-length syntaxin-1A (amino acid residues 1-288), its SNARE motif including the transmembrane region (183-288), a cysteine-free variant of SNAP-25A (1-206) and synaptobrevin-2 (full-length protein, 1-116; soluble portion, 1-96; C-terminal fragment of the soluble portion, 49-96) 30,39,45,46 . The expression constructs for endobrevin were the full-length protein (1-100) and its soluble portion (1-74) 46,47 . Full-length syntaxin-4 (1-298) 48 and a variant of SNAP-23 in which cysteine residues were replaced with serines were similarly cloned into the pET28a vector using the NdeI and XhoI restriction sites.…”

Sorting in early endosomes reveals connections to docking- and fusion-associated factors

“…One of the reasons for this gap in our knowledge is that, in contrast to fusion (3,4), it has been difficult to reconstitute sorting from endosomes in vitro. Compared to live cell approaches, in vitro assays allow direct biochemical access to the docking and fusion machineries (for instance, proteins can be depleted or perturbed using cell-impermeant inhibitors such as antibodies).…”