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The Carboxy-Terminal Domain of Complexin I Stimulates Liposome Fusion

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The Carboxy-Terminal Domain of Complexin I Stimulates Liposome Fusion The carboxy-terminal domain of complexin I stimulates liposome fusion Jo¨ rg Malsam1, Florian Seiler1, Yvette Schollmeier, Patricia Rusu, Jean Michel Krause, and Thomas H. So¨ llner2 Heidelberg University Biochemistry Center, 69120 Heidelberg, Germany Communicated by James E. Rothman, Columbia University Medical Center, New York, NY, December 17, 2008 (received for review August 8, 2008) Regulated exocytosis requires tight coupling of the membrane CpxI/II double- and CpxI/II/III triple-knockout mice (15). Other fusion machinery to a triggering signal and a fast response time. studies, including complexin overexpression, in vitro liposome Complexins are part of this regulation and, together with synap- fusion assays, and cell-cell fusion experiments, indicate that totagmins, control calcium-dependent exocytosis. Stimulatory and complexins can also function as fusion clamps (16–19). The inhibitory functions have been reported for complexins. To test if clamp is released by synaptotagmin 1 in a calcium-dependent complexins directly affect membrane fusion, we analyzed the 4 manner. Recent analysis revealed that distinct domains of com- known mammalian complexin isoforms in a reconstituted fusion plexins differently control neurotransmitter release and mem- assay. In contrast to complexin III (CpxIII) and CpxIV, CpxI and CpxII brane fusion. The amino-terminal 26 amino acids of CpxI stimulated soluble N-ethylmaleimide-sensitive factor attachment contain a stimulatory function followed by an inhibitory ␣-helix protein receptor (SNARE)-pin assembly and membrane fusion. This (amino acids 29–48) and the SNARE binding helix (amino acids stimulatory effect required a preincubation at low temperature 48–70) (20, 21). Although low-affinity binding of CpxI to and was specific for neuronal t-SNAREs. Stimulation of membrane neuronal t-SNAREs has been reported, high-affinity binding fusion was lost when the carboxy-terminal domain of CpxI was requires v-/t-SNARE complexes (22–25). The crystal structure deleted or serine 115, a putative phosphorylation site, was mu- of CpxI, together with the assembled cis v-/t-SNARE core tated. Transfer of the carboxy-terminal domain of CpxI to CpxIII complex, shows that CpxI binds in an antiparallel manner to a resulted in a stimulatory CpxIII-I chimera. Thus, the carboxy- groove at the surface of the 4-helix SNARE bundle, which is terminal domains of CpxI and CpxII promote the fusion of high- formed by Syx1 and VAMP2 (26, 27). Thus, complexins are curvature liposomes. perfectly positioned to control SNAREpin assembly directly, and it has been suggested that complexins stabilize SNAREpin exocytosis ͉ SNARE intermediates (26). In addition, clusters of conserved amino acids are present in the amino- and carboxy-terminal regions that embrane fusion is initiated by the pairing of compartment- flank the SNARE complex-binding helix of complexins. It is Mspecific v-SNAREs and t-SNAREs between opposite largely unknown to what degree and how these amino- and membranes, resulting in the formation of so-called ‘‘trans- carboxy-terminal regions control membrane fusion. Thus, to SNARE’’ complexes or SNAREpins (1). At the neuronal syn- shed light on the function of complexins in SNARE complex apse, the v-SNARE VAMP2/synaptobrevin2 is located on syn- assembly and membrane fusion, we compared the molecular aptic vesicles and binds the t-SNARE, consisting of syntaxin 1 function of the 4 mammalian complexin isoforms in an in vitro (Syx1) and SNAP-25, at the presynaptic plasma membrane (2). liposome fusion assay. Remarkably, we observed a complexin The assembly of the SNAREpin starts at the membrane distal isoform-specific stimulation of membrane fusion that requires end of the SNARE motifs and proceeds in a membrane proximal the carboxy-terminus of complexins. The removal of the stim- direction bringing the lipid bilayers in close proximity (3–5). This ulatory carboxy-terminus of CpxI or point mutations in S115 process occurs in a stepwise manner, and regulatory proteins abolish the stimulation. The transfer of the stimulatory CpxI likely stabilize intermediates along the assembly pathway. In- carboxy-terminus to CpxIII results in a stimulatory chimera. deed, synaptic vesicles docked at the active zone appear to be Results linked to the plasma membrane by such partially assembled CpxI and CpxII Stimulate Membrane Fusion. To analyze Cpx func- SNAREpins, which ensure membrane fusion within a submilli- tion, we used an in vitro fusion assay that consists of liposomes second response time (6). The physiological trigger for synaptic containing reconstituted SNAREs. When v-SNARE liposomes vesicle fusion is a local increase in the calcium concentration, with a quenched pair of N-(7-nitro-2,1,3-benzoxadiaziole-4-yl) caused by the opening of calcium channels (7, 8). In the presence (NBD)-labeled and rhodamine-labeled lipids fuse with unla- of calcium, the calcium sensor synaptotagmin 1 localized on beled t-SNARE liposomes, the NBD fluorescence increases (1, synaptic vesicles interacts with t-SNAREs and negatively 28). VAMP2 and preassembled full-length Syx1/SNAP-25 com- charged phospholipids, leading to fusion pore opening (9). plexes were reconstituted into liposomes at a protein-to-lipid In addition to synaptotagmin 1, complexins (also called syn- ratio of about 1:200, which corresponds approximately to the aphins) participate in calcium-dependent exocytosis (10–12). VAMP2/lipid ratio found in purified synaptic vesicles (29). Complexins are small cytoplasmic proteins that contain a central ␣-helix, which binds distinct SNAREs. In Drosophila, only 1 complexin orthologue has been identified, and its inactivation Author contributions: J.M., F.S., and T.H.S. designed research; J.M., F.S., Y.S., and P.R. results in a dramatic increase in the spontaneous fusion of performed research; J.M.K. contributed new reagents/analytical tools; J.M., F.S., and T.H.S. synaptic vesicles at neuromuscular junctions (13). In vertebrates, analyzed data; and J.M., F.S., and T.H.S. wrote the paper. 4 complexin isoforms are known (14). Complexin I (CpxI) and The authors declare no conflict of interest. CpxII are highly homologous to each other and more distantly Freely available online through the PNAS open access option. related to CpxIII and CpxIV, which contain CAAX boxes at 1J.M. and F.S. contributed equally to this work. their carboxy-termini, anchoring these isoforms to membranes. 2To whom correspondence should be addressed. E-mail: thomas.soellner@bzh. CpxI/II double- and CpxI/II/III triple-knockout mice die at birth uni-heidelberg.de. 2ϩ and are impaired in synaptic vesicle exocytosis at the Ca - This article contains supporting information online at www.pnas.org/cgi/content/full/ dependent step (12, 15). In contrast to the complexin knockout 0812813106/DCSupplemental. NEUROSCIENCE in Drosophila, nonsynchronous release is slightly reduced in © 2009 by The National Academy of Sciences of the USA www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812813106 PNAS ͉ February 10, 2009 ͉ vol. 106 ͉ no. 6 ͉ 2001–2006 Downloaded by guest on September 25, 2021 amounts, aliquots were analyzed postfusion by SDS/PAGE and Coomassie Blue staining (Fig. S2). The stimulatory effect was strictly dependent on the preincubation of CpxI with v-SNARE and t-SNARE liposomes at low temperature (Fig. S3), which usually blocks membrane fusion in the in vitro assay (1). Re- markably, in the presence of CpxI and CpxII, a significant fraction of liposomes already fused at low temperature and during the warming up phase (see elevated initial signal in Fig. 1A and Fig. S1). Separate preincubations of CpxI with either v-SNARE or t-SNARE liposomes did not stimulate membrane fusion, indicating that CpxI acts on SNAREpins (data not shown). Fusion stimulation by CpxI and CpxII was dose depen- dent (Fig. 1B). Plotting the NBD fluorescence signal 1 min after sample warming up against the complexin amount showed that half-maximal stimulation was reached when CpxI (5 ␮M) and the surface-exposed t-SNARE reached a ratio of Ϸ1:1 (Fig. 1B). CpxIII and Drosophila complexin showed only minor effects on the fusion signal at the indicated concentrations (Fig. 1A). CpxIV inhibited membrane fusion to some extent, consistent with the findings of a previous publication (16). CpxIII had no effect on liposome fusion at the indicated concentration (Fig. 1A) but inhibited fusion when the amounts were increased (Fig. S4), which is consistent with a lower affinity of CpxIII for SNAREs (14). We also tested Drosophila complexin in combi- nation with the mammalian SNAREs and the Drosophila SNARE orthologues in reconstituted liposomes. Similar to the human CpxIII, the Drosophila complexin inhibited the fusion reaction when used at high concentrations (data not shown). Remarkably, the inhibitory effect of CpxIV occurred indepen- dent of the low temperature preincubation (Fig. S3). Thus, the fusion stimulation is specific to complexin isoform and stage. CpxI Stimulates SNAREpin Assembly. Because CpxI stimulates the initial phase of the fusion reaction, it could accelerate SNARE- pin assembly. To test this hypothesis, it is necessary to detect SNAREpins. Preincubation of v- and t-SNARE liposomes at low Fig. 1. Complexin isoforms differentially stimulate/inhibit membrane fu- temperature prevents membrane fusion to a large degree but sion. (A) Effect of complexin isoforms on liposome fusion kinetics. v-SNARE allows the formation of SNAREpins (1). VAMP2 on the lipo- (VAMP2) liposomes labeled with rhodamine and NBD lipids were incubated in some surface, which is not incorporated into SNAREpins, can be the absence or presence of the indicated complexin isoforms with unlabeled t-SNARE (Syx1/SNAP-25) liposomes for1honice. With the exception of removed by the cleavage with tetanus toxin, whereas VAMP2 Drosophila complexin (dm Cpx), all other complexin isoforms were human assembled into SNARE complexes is protected (6). However, orthologues (CpxI–CpxIV). After warming up of the reaction to 37 °C, the VAMP2 is also trapped in the lumen of the liposomes, and thus increase in NBD fluorescence was monitored for 30 min at 37 °C.
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