Claudio G. Giraudo

Alternative Zippering as an On-Off Switch for SNARE-Mediated Fusion

Membrane fusion between vesicles and target membranes involves the zippering of a four-helix bundle generated by constituent helices derived from target– and vesicle–soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs). In neurons, the protein complexin clamps otherwise spontaneous fusion by SNARE proteins, allowing neurotransmitters and other mediators to be secreted when and where they are needed as this clamp is released. The membrane-proximal accessory helix of complexin is necessary for clamping, but its mechanism of action is unknown. Here, we present experiments using a reconstituted fusion system that suggest a simple model in which the complexin accessory helix forms an alternative four-helix bundle with the target-SNARE near the membrane, preventing the vesicle-SNARE from completing its zippering.

SNAREs can promote complete fusion and hemifusion as alternative outcomes

Using a cell fusion assay, we show here that in addition to complete fusion SNAREs also promote hemifusion as an alternative outcome. Approximately 65% of events resulted in full fusion, and the remaining 35% in hemifusion; of those, approximately two thirds were permanent and approximately one third were reversible. We predict that this relatively close balance among outcomes could be tipped by binding of regulatory proteins to the SNAREs, allowing for dynamic physiological regulation between full fusion and reversible kiss-and-run–like events.

Ganglioside Glycosyltransferases Organize in Distinct Multienzyme Complexes in CHO-K1 Cells

The synthesis of gangliosides is compartmentalized in the Golgi complex. In most cells, glycosylation of LacCer, GM3, and GD3 to form higher order species (GA2, GM2, GD2, GM1, GD1b) is displaced toward the most distal aspects of the Golgi and the trans-Golgi network, where the involved transferases (GalNAcT and GalT2) form physical and functional associations. Glycosylation of the simple species LacCer, GM3, and GD3, on the other hand, is displaced toward more proximal Golgi compartments, and we investigate here whether the involved transferases (GalT1, SialT1, and SialT2) share the property of forming physical associations. Co-immunoprecipitation experiments from membranes of CHO-K1 cells expressing epitope-tagged versions of these enzymes indicate that GalT1, SialT1, and SialT2 associate physically in a SialT1-dependent manner and that their N-terminal domains participate in these interactions. Microscopic fluorescence resonance energy transfer and fluorescence recovery after photobleaching in living cells confirmed the interactions, and in addition to showing a Golgi apparatus localization of the complexes, mapped their formation to the endoplasmic reticulum. Neither co-immunoprecipitation nor fluorescence resonance energy transfer detected interactions between either GalT2 or GalNAcT and GalT1 or SialT1 or SialT2. These results, and triple color imaging of Golgi-derived microvesicles in nocodazole-treated cells, suggest that ganglioside synthesis is organized in distinct units each formed by associations of particular glycosyltransferases, which concentrate in different sub-Golgi compartments.

Complexin activates and clamps SNAREpins by a common mechanism involving an intermediate energetic state

The core mechanism of intracellular vesicle fusion consists of SNAREpin zippering between vesicular and target membranes. Recent studies indicate that the same SNARE-binding protein, complexin (CPX), can act either as a facilitator or as an inhibitor of membrane fusion, constituting a controversial dilemma. Here we take energetic measurements with the surface force apparatus that reveal that CPX acts sequentially on assembling SNAREpins, first facilitating zippering by nearly doubling the distance at which v- and t-SNAREs can engage and then clamping them into a half-zippered fusion-incompetent state. Specifically, we find that the central helix of CPX allows SNAREs to form this intermediate energetic state at 9–15 nm but not when the bilayers are closer than 9 nm. Stabilizing the activated-clamped state at separations of less than 9 nm requires the accessory helix of CPX, which prevents membrane-proximal assembly of SNAREpins.

Distinct Domains of Complexins Bind SNARE Complexes and Clamp Fusion in Vitro

In regulated exocytosis, the core membrane fusion machinery proteins, the SNARE proteins, are assisted by a group of regulatory factors in order to couple membrane fusion to an increase of intracellular calcium ion (Ca2+) concentration. Complexin-I and synaptotagmin-I have been shown to be key elements for this tightly regulated process. Many studies suggest that complexin-I can arrest the fusion reaction and that synaptotagmin-I can release the complexin-I blockage in a calcium-dependent manner. Although the actual molecular mechanism by which they exert their function is still unknown, recent in vivo experiments postulate that domains of complexin-I produce different effects on neurotransmitter release. Herein, by using an in vitro flipped SNARE cell fusion assay, we have identified and characterized the minimal functional domains of complexin-I necessary to couple calcium and synaptotagmin-I to membrane fusion. Moreover, we provide evidence that other isoforms of complexin, complexin-II, -III, and -IV, can also be functionally coupled to synaptotagmin-I and calcium. These correspond closely to results from in vivo experiments, providing further validation of the physiological relevance of the flipped SNARE system.

Cytoplasmic Tails of SialT2 and GalNAcT Impose Their Respective Proximal and Distal Golgi Localization

Complex glycolipid synthesis is catalyzed by different glycosyltransferases resident of the Golgi complex. Most of them are type II membrane proteins comprising a lumenal, C‐terminal domain linked to an N‐terminal domain (Ntd) constituted by a short cytoplasmic tail (ct), a transmembrane, and a lumenal stem regions. They concentrate selectively in different sub‐Golgi compartments, in an overlapped manner, acting in succession in the addition of sugars to acceptor glycolipids. The Ntds are sufficient to localize glycosyltransferases in the Golgi complex, but it is not clear whether they also confer selective concentration in sub‐Golgi compartments. Here, we studied whether the Ntd of SialT2, localized in the proximal Golgi, and the one of GalNAcT, a trans/TGN Golgi‐concentrated enzyme, concentrate reporter proteins in the corresponding sub‐Golgi compartment. The sub‐Golgi concentration of the Ntds fused to spectral variants of the GFP was determined in CHO‐K1 cells from their behavior upon addition of brefeldin A. Fluorescence microscopy and subcellular fractionation showed that the SialT2 Ntd concentrates in a proximal sub‐Golgi compartment – and that of GalNAcT in TGN elements. Exchanging the transmembrane region and the cts of SialT2 and GalNAcT indicates that information for proximal or distal Golgi concentration is associated with the cts.

Dual roles of Munc18-1 rely on distinct binding modes of the central cavity with Stx1A and SNARE complex

The Munc18 central cavity plays a major role in trafficking syntaxin 1 (Stx 1) to the plasma membrane and in activating SNARE-mediated membrane fusion. This paper provides critical insight into the mechanisms of how the Stx1A H3 domain can compete with the SNARE complex for binding the Munc18 central cavity, first inhibiting, and later assisting, SNARE-complex assembly.

Identification of a Site in Sar1 Involved in the Interaction with the Cytoplasmic Tail of Glycolipid Glycosyltransferases

Glycolipid glycosyltransferases (GGT) are transported from the endoplasmic reticulum (ER) to the Golgi, their site of residence, via COPII vesicles. An interaction of a (R/K)X(R/K) motif at their cytoplasmic tail (CT) with Sar1 is critical for the selective concentration in the transport vesicles. In this work using computational docking, we identify three putative binding pockets in Sar1 (sites A, B, and C) involved in the interaction with the (R/K)X(R/K) motif. Sar1 mutants with alanine replacement of amino acids in site A were tested in vitro and in cells. In vitro, mutant versions showed a reduced ability to bind immobilized peptides with the CT sequence of GalT2. In cells, Sar1 mutants (Sar1D198A) specifically affect the exiting of GGT from the ER, resulting in an ER/Golgi concentration ratio favoring the ER. Neither the typical Golgi localization of GM130 nor the exiting and transport of the G protein of the vesicular stomatitis virus were affected. The protein kinase inhibitor H89 produced accumulation of Sec23, Sar1, and GalT2 at the ER exit sites; Sar1D189A also accumulated at these sites, but in this case GalT2 remained disperse along ER membranes. The results indicate that amino acids in site A of Sar1 are involved in the interaction with the CT of GGT for concentration at ER exiting sites.

SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity

Significance Vital physiological processes—such as the cytotoxic immune response—require the coordinated action of the atypical fusion protein Syntaxin 11 (STX11) and the Sec/Munc protein Munc18-2 for releasing effector proteins housed in membrane-enclosed secretory granules. Human mutations in STX11 and Munc18-2 genes lead to severe immunodeficiency and hemostasis disorders. However it is still unclear how STX11, a lipid-anchored SNARE, and Munc18-2 mediate membrane fusion. By using an in vitro fusion assay, we found that STX11 mainly mediates lipid mixing when combined with human lymphocyte-interacting SNAREs. Remarkably, Munc18-2 induces association among these SNAREs and facilitates the transition from a hemifusion-like state to complete fusion. Our findings support a model in which SM proteins play a direct role in membrane merging. The atypical lipid-anchored Syntaxin 11 (STX11) and its binding partner, the Sec/Munc (SM) protein Munc18-2, facilitate cytolytic granule release by cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells. Patients carrying mutations in these genes develop familial hemophagocytic lymphohistiocytosis, a primary immunodeficiency characterized by impaired lytic granule exocytosis. However, whether a SNARE such as STX11, which lacks a transmembrane domain, can support membrane fusion in vivo is uncertain, as is the precise role of Munc18-2 during lytic granule exocytosis. Here, using a reconstituted “flipped” cell–cell fusion assay, we show that lipid-anchored STX11 and its cognate SNARE proteins mainly support exchange of lipids but not cytoplasmic content between cells, resembling hemifusion. Strikingly, complete fusion is stimulated by addition of wild-type Munc18-2 to the assay, but not of Munc18-2 mutants with abnormal STX11 binding. Our data reveal that Munc18-2 is not just a chaperone of STX11 but also directly contributes to complete membrane merging by promoting SNARE complex assembly. These results further support the concept that SM proteins in general are part of the core fusion machinery. This fusion mechanism likely contributes to other cell-type–specific exocytic processes such as platelet secretion.