Thomas H. Söllner

SNAREpins: minimal machinery for membrane fusion.

Recombinant v- and t-SNARE proteins reconstituted into separate lipid bilayer vesicles assemble into SNAREpins-SNARE complexes linking two membranes. This leads to spontaneous fusion of the docked membranes at physiological temperature. Docked unfused intermediates can accumulate at lower temperatures and can fuse when brought to physiological temperature. A supply of unassembled v- and t-SNAREs is needed for these intermediates to form, but not for the fusion that follows. These data imply that SNAREpins are the minimal machinery for cellular membrane fusion.

Compartmental specificity of cellular membrane fusion encoded in SNARE proteins

Membrane-enveloped vesicles travel among the compartments of the cytoplasm of eukaryotic cells, delivering their specific cargo to programmed locations by membrane fusion. The pairing of vesicle v-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) with target membrane t-SNAREs has a central role in intracellular membrane fusion. We have tested all of the potential v-SNAREs encoded in the yeast genome for their capacity to trigger fusion by partnering with t-SNAREs that mark the Golgi, the vacuole and the plasma membrane. Here we find that, to a marked degree, the pattern of membrane flow in the cell is encoded and recapitulated by its isolated SNARE proteins, as predicted by the SNARE hypothesis.

A rab protein is required for the assembly of SNARE complexes in the docking of transport vesicles

Rab proteins are generally required for transport vesicle docking. We have exploited yeast secretion mutants to demonstrate that a rab protein is required for v-SNAREs and t-SNAREs to assemble. The absence of the rab protein in the docking complex suggests that, in a broad sense, rab proteins participate in a reaction catalyzing SNARE complex assembly. In so doing, rab proteins could help impart an additional layer of specificity to vesicle docking. This mechanism likely involves the Sec1 homolog Sly1, which we identified in isolated docking complexes. We also report the identification of a novel v-SNARE (Ykt6p) component of the yeast ER-Golgi docking complex that has a CAAX box and is predicted to be lipid anchored. The surprising finding that docking complexes can contain many distinct species of SNAREs (Sed5p, Bos1p, Sec22p, Ykt6p, and likely Bet1p, p28, and p14) suggests that multimeric interactions are features of the fusion machinery, and may also improve the fidelity of vesicle targeting.

Bidirectional Transport by Distinct Populations of COPI-Coated Vesicles

Electron microscope immunocytochemistry reveals that both anterograde-directed (proinsulin and VSV G protein) and retrograde-directed (the KDEL receptor) cargo are present in COPI-coated vesicles budding from every level of the Golgi stack in whole cells; however, they comprise two distinct populations that together can account for at least 80% of the vesicles budding from Golgi cisternae. Segregation of anterograde- from retrograde-directed cargo into distinct sets of COPI-coated vesicles is faithfully reproduced in the cell-free Golgi transport system, in which VSV G protein and KDEL receptor are packaged into separable vesicles, even when budding is driven by highly purified coatomer and a recombinant ARF protein.

COUPLING OF COAT ASSEMBLY AND VESICLE BUDDING TO PACKAGING OF PUTATIVE CARGO RECEPTORS

COPI-coated vesicle budding from lipid bilayers whose composition resembles mammalian Golgi membranes requires coatomer, ARF, GTP, and cytoplasmic tails of putative cargo receptors (p24 family proteins) or membrane cargo proteins (containing the KKXX retrieval signal) emanating from the bilayer surface. Liposome-derived COPI-coated vesicles are similar to their native counterparts with respect to diameter, buoyant density, morphology, and the requirement for an elevated temperature for budding. These results suggest that a bivalent interaction of coatomer with membrane-bound ARF[GTP] and with the cytoplasmic tails of cargo or putative cargo receptors is the molecular basis of COPI coat assembly and provide a simple mechanism to couple uptake of cargo to transport vesicle formation.

MOM19, an import receptor for mitochondrial precursor proteins

We have identified a 19 kd protein of the mitochondrial outer membrane (MOM19). Monospecific IgG and Fab fragments directed against MOM19 inhibit import of precursor proteins destined for the various mitochondrial subcompartments, including porin, cytochrome c1, Fe/S protein, F0 ATPase subunit 9, and F1 ATPase subunit beta. Inhibition occurs at the level of high affinity binding of precursors to mitochondria. Consistent with previous functional studies that suggested the existence of distinct import sites for ADP/ATP carrier and cytochrome c, we find that import of those precursors is not inhibited. We conclude that MOM19 is identical to, or closely associated with, a specific mitochondrial import receptor.

Topological restriction of SNARE-dependent membrane fusion.

To fuse transport vesicles with target membranes, proteins of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) complex must be located on both the vesicle (v-SNARE) and the target membrane (t-SNARE). In yeast, four integral membrane proteins, Sed5, Bos1, Sec22 and Bet1 (refs 2, 3,4,5,6), each probably contribute a single helix to form the SNARE complex that is needed for transport from endoplasmic reticulum to Golgi. This generates a four-helix bundle, which ultimately mediates the actual fusion event. Here we explore how the anchoring arrangement of the four helices affects their ability to mediate fusion. We reconstituted two populations of phospholipid bilayer vesicles, with the individual SNARE proteins distributed in all possible combinations between them. Of the eight non-redundant permutations of four subunits distributed over two vesicle populations, only one results in membrane fusion. Fusion only occurs when the v-SNARE Bet1 is on one membrane and the syntaxin heavy chain Sed5 and its two light chains, Bos1 and Sec22, are on the other membrane where they form a functional t-SNARE. Thus, each SNARE protein is topologically restricted by design to function either as a v-SNARE or as part of a t-SNARE complex.

A mitochondrial import receptor for the ADP/ATP carrier

We have identified a mitochondrial outer membrane protein of 72 kd (MOM72) that exhibits the properties of an import receptor for the ADP/ATP carrier (AAC), the most abundant mitochondrial protein. Monospecific antibodies and Fab fragments against MOM72 selectively inhibit import of AAC at the level of specific binding to the mitochondria. AAC bound to the mitochondrial surface is coprecipitated with antibodies against MOM72 after lysis of mitochondria with detergent. MOM72 thus has a complementary function to that of MOM19, which acts as an import receptor for the majority of mitochondrial proteins studied so far but not for the AAC. The import pathway of the precursor of MOM72 appears to involve MOM19 as receptor.

Multiple palmitoylation of synaptotagmin and the t-SNARE SNAP-25

Synaptotagmin, a likely calcium sensor for synaptic transmission, and SNAP‐25, a t‐SNARE of the presynaptic plasma membrane, are key proteins for the docking and fusion of synaptic and other vesicles. We report that both synaptotagmin and SNAP‐25 are palmitoylated with their fatty acids attached in a labile thioester‐type bond. A SNAP‐25 mutant with deleted palmitoylation sites is found exclusively in the cytosol after cell fractionation, whereas the palmitoylated form of SNAP‐25 is membrane‐bound, establishing that SNAP‐25 is membrane‐anchored via covalently linked palmitate.

Regulation of membrane fusion by the membrane-proximal coil of the t-SNARE during zippering of SNAREpins

We utilize structurally targeted peptides to identify a “tC fusion switch” inherent to the coil domains of the neuronal t-SNARE that pairs with the cognate v-SNARE. The tC fusion switch is located in the membrane-proximal portion of the t-SNARE and controls the rate at which the helical bundle that forms the SNAREpin can zip up to drive bilayer fusion. When the fusion switch is “off” (the intrinsic state of the t-SNARE), zippering of the helices from their membrane-distal ends is impeded and fusion is slow. When the tC fusion switch is “on,” fusion is much faster. The tC fusion switch can be thrown by a peptide that corresponds to the membrane-proximal half of the cognate v-SNARE, and binds reversibly to the cognate region of the t-SNARE. This structures the coil in the membrane-proximal domain of the t-SNARE and accelerates fusion, implying that the intrinsically unstable coil in that region is a natural impediment to the completion of zippering, and thus, fusion. Proteins that stabilize or destabilize one or the other state of the tC fusion switch would exert fine temporal control over the rate of fusion after SNAREs have already partly zippered up.