Reader TTS

ABSTRACT SNAREs (soluble _N-_ethylmaleimide-sensitive factor attachment protein receptors) and Rab-GTPases, together with their cofactors, mediate the attachment step in the membrane fusion

of vesicles. But how bilayer mixing—the subsequent core process of fusion—is catalysed remains unclear. Ca2+/calmodulin controls this terminal process in many intracellular fusion events.

Here we identify V0, the membrane-integral sector of the vacuolar H+-ATPase, as a target of calmodulin on yeast vacuoles. Between docking and bilayer fusion, V0 sectors from opposing

membranes form complexes. V0 _trans_-complex formation occurs downstream from _trans_-SNARE pairing, and depends on both the Rab-GTPase Ypt7 and calmodulin. The maintenance of existing

complexes and completion of fusion are independent of _trans_-SNARE pairs. Reconstituted proteolipids form sealed channels, which can expand to form aqueous pores in a

Ca2+/calmodulin-dependent fashion. V0 _trans_-complexes may therefore form a continuous, proteolipid-lined channel at the fusion site. We propose that radial expansion of such a protein pore

may be a mechanism for intracellular membrane fusion. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS OPTIONS

Access through your institution Subscribe to this journal Receive 51 print issues and online access $199.00 per year only $3.90 per issue Learn more Buy this article * Purchase on

SpringerLink * Instant access to full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about

institutional subscriptions * Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS STRUCTURAL BASIS FOR VPS34 KINASE ACTIVATION BY RAB1 AND RAB5 ON MEMBRANES

Article Open access 10 March 2021 NSF/ΑSNAP2-MEDIATED _CIS_-SNARE COMPLEX DISASSEMBLY PRECEDES VESICLE FUSION IN _ARABIDOPSIS_ CYTOKINESIS Article 01 June 2023 RAB GTPASES AND

PHOSPHOINOSITIDES FINE-TUNE SNARES DEPENDENT TARGETING SPECIFICITY OF INTRACELLULAR VESICLE TRAFFIC Article Open access 20 March 2024 REFERENCES * Jahn, R. & Südhof, T. C. Membrane

fusion and exocytosis. _Annu. Rev. Biochem._ 68, 863–911. (1999). Article  CAS  Google Scholar  * Pfeffer, S. R. Transport vesicle targeting: tethers before SNAREs. _Nature Cell Biol._ 1,

17–19 (1999) Article  Google Scholar  * Mayer, A. Membrane fusion: SNAREs only? _Curr. Opin. Cell Biol._ 11, 447–452 (1999). Article  CAS  Google Scholar  * Lindau, M. & Almers, W.

Structure and function of fusion pores in exocytosis and ectoplasmic membrane fusion. _Curr. Opin. Cell Biol._ 7, 509–517 (1995). Article  CAS  Google Scholar  * Chernomordik, L. V. &

Zimmerberg, J. Bending membranes to the task: structural intermediates in bilayer fusion. _Curr. Opin. Struct. Biol._ 5, 541–547 (1995). Article  CAS  Google Scholar  * Hanson, P. I. et al.

Structure & conformational changes in NSF and its membrane receptor complexes visualized by quick-freeze/deep-etch electron microscopy. _Cell_ 90, 523–535 (1997). CAS  Google Scholar  *

Skehel, J. J. & Wiley, D. C. Coiled coils in both intracellular vesicle and viral membrane fusion. _Cell_ 95, 871–874 (1998). Article  CAS  Google Scholar  * Weber, T. et al. SNAREpins:

Minimal machinery for membrane fusion. _Cell_ 92, 759–772 (1998). CAS  Google Scholar  * Otter-Nilsson, M. et al. Cytosolic ATPases, p97 and NSF, are sufficient to mediate rapid membrane

fusion. _EMBO J._ 18, 2074–2083 (1999). Article  CAS  Google Scholar  * Brügger, B. et al. Putative fusogenic activity of NSF is restricted to a lipid mixture whose coalescence is also

triggered by other factors. _EMBO J._ 19, 1272–1278 (2000). Article  Google Scholar  * Coorssen, J. R., Blank, P. S., Tahara, M. & Zimmerberg, J. Biochemical and functional studies of

cortical vesicle fusion: The SNARE complex and Ca2+ sensitivity. _J. Cell Biol._ 143, 1845–1857 (1998). Article  CAS  Google Scholar  * Chen, Y. A. et al. SNARE complex formation is

triggered by Ca2+ and drives membrane fusion. _Cell_ 97, 165–174 (1999). Article  CAS  Google Scholar  * Xu, T. et al. Inhibition of SNARE complex assembly differentially affects kinetic

components of exocytosis. _Cell_ 99, 713–722 (1999). Article  CAS  Google Scholar  * Ungermann, C., Sato, K. & Wickner, W. Defining the functions of _trans_-SNARE pairs. _Nature_ 396,

543–548 (1998). Article  ADS  CAS  Google Scholar  * Ungermann, C., Wickner, W. & Xu, Z. Y. Vacuole acidification is required for _trans_-SNARE pairing, LMA1 release, and homotypic

fusion. _Proc. Natl Acad. Sci. USA_ 96, 11194–11199 (1999). Article  ADS  CAS  Google Scholar  * Peters, C. & Mayer, A. Ca2+/calmodulin signals the completion of docking and triggers a

late step of vacuole fusion. _Nature_ 396, 575–580 (1998). Article  ADS  CAS  Google Scholar  * Peters, C. et al. Control of the terminal step of membrane fusion by protein phosphatase 1.

_Science_ 285, 1084–1087 (1999). Article  CAS  Google Scholar  * Pryor, P. R. et al. The role of intraorganellar Ca2+ in late endosome–lysosome heterotypic fusion and in the reformation of

lysosomes from hybrid organelles. _J. Cell Biol._ 149, 1053–1062 (2000). Article  CAS  Google Scholar  * Colombo, M. I., Beron, W. & Stahl, P. D. Calmodulin regulates endosome fusion.

_J. Biol. Chem._ 272, 7707–7712 (1997). Article  CAS  Google Scholar  * Holroyd, C., Kistner, U., Annaert, W. & Jahn, R. Fusion of endosomes involved in synaptic vesicle recycling. _Mol.

Biol. Cell_ 10, 3035–3044 (1999). Article  CAS  Google Scholar  * Porat, A. & Elazar, Z. Regulation of intra-Golgi membrane transport by calcium. _J. Biol. Chem._ 275, 29233–29237

(2000). Article  CAS  Google Scholar  * Mayer, A. & Wickner, W. Docking of yeast vacuoles is catalysed by the ras-like GTPase Ypt7p after symmetric priming by Sec18p (NSF). _J. Cell

Biol._ 136, 307–317 (1997). Article  CAS  Google Scholar  * Ungermann, C., Nichols, B. J., Pelham, H. R. & Wickner, W. A vacuolar v-t-SNARE complex, the predominant form in vivo and on

isolated vacuoles, is disassembled and activated for docking and fusion. _J. Cell Biol._ 140, 61–69 (1998). Article  CAS  Google Scholar  * Hartinger, J., Stenius, K., Högemann, D. &

Jahn, R. 16-BAC/SDS-PAGE: a two-dimensional gel electrophoresis system suitable for the separation of integral membrane proteins. _Anal. Biochem._ 240, 126–133 (1996). Article  CAS  Google

Scholar  * Cohen, A., Perzov, N., Nelson, H. & Nelson, N. A novel family of yeast chaperons involved in the distribution of V-ATPase and other membrane proteins. _J. Biol. Chem._ 274,

26885–26893 (1999). Article  CAS  Google Scholar  * Stevens, T. H. & Forgac, M. Structure, function and regulation of the vacuolar ATPase. _Annu. Rev. Cell Dev. Biol._ 13, 779–808

(1997). Article  CAS  Google Scholar  * Kane, P. M. & Parra, K. Assembly and regulation of the yeast vacuolar H+-ATPase. _J. Exp. Biol._ 203, 81–87 (2000). CAS  PubMed  Google Scholar  *

Dunant, Y. & Israël, M. In vitro reconstitution of neurotransmitter release. _Neurochem. Res._ 23, 709–718 (1998). Article  CAS  Google Scholar  * Israël, M., Morel, N. & Lesbats,

B. Evidence for an association of the 15-kDa proteolipid of mediatophore with a 14-kDa polypeptide. _J. Neurochem._ 57, 2047–2053 (1991). Article  Google Scholar  * Israël, M., Meunier, F.

M., Morel, N. & Lesbats, B. Calcium-induced desensitization of acetylcholine release from synaptosomes or proteoliposomes equipped with mediatophore, a presynaptic membrane protein. _J.

Neurochem._ 49, 975–982 (1987). Article  Google Scholar  * Mayer, A., Wickner, W. & Haas, A. Sec18p (NSF) driven release of Sec17p (alpha-SNAP) can precede docking and fusion of yeast

vacuoles. _Cell_ 85, 83–94 (1996). Article  CAS  Google Scholar  * Finbow, M. E. & Harrison, M. A. The vacuolar ATPase: a universal proton pump of eukaryotes. _Biochem. J._ 324, 697–712

(1997). Article  CAS  Google Scholar  * Weber, T. et al. SNAREpins are functionally resistant to disruption by NSF and alpha-SNAP. _J. Cell Biol._ 149, 1063–1072 (2000). Article  CAS  Google

Scholar  * Umemoto, N., Yoshihisa, T., Hirata, R. & Anraku, Y. Roles of the Vma3 gene product, subunit c of the vacuolar membrane H+-ATPase on vacuolar acidification and protein

transport. _J. Biol. Chem._ 265, 18447–18453 (1990). CAS  PubMed  Google Scholar  * Klionsky, D. J., Nelson, H. & Nelson, N. Compartment acidification is required for efficient sorting

of proteins to the vacuole in _Saccharomyces cerevisiae_. _J. Biol. Chem._ 267, 3416–3422 (1992). CAS  PubMed  Google Scholar  * Morano, K. A. & Klionsky, D. J. Differential effects of

compartment deacidification on the targeting of membrane and soluble proteins to the vacuole in yeast. _J. Cell Sci._ 107, 2813–2824 (1994). CAS  PubMed  Google Scholar  * Yamashiro, C. T.

et al. Role of vacuolar acidification in protein sorting and zymogen activation: a genetic analysis of the yeast vacuolar proton translocating ATPase. _Mol. Cell. Biol._ 10, 3737–3749

(1990). Article  CAS  Google Scholar  * Kibble, V. A. & Burgoyne, R. D. Calmodulin increases the initial rate of exocytosis in adrenal chromaffin cells. _Eur. J. Physiol._ 431, 464–466

(1996). Article  CAS  Google Scholar  * Chen, Y., Duvvuri, V., Schulman, H. & Scheller, R. H. Calmodulin and protein kinase C increase Ca2+-stimulated secretion by modulating

membrane-attached exocytic machinery. _J. Biol. Chem._ 274, 26469–26476 (1999). Article  CAS  Google Scholar  * Bennett, M. K., Calakos, N., Kreiner, T. & Scheller, R. H. Synaptic

vesicle membrane proteins interact to form a multimeric complex. _J. Cell Biol._ 116, 761–775 (1992). Article  CAS  Google Scholar  * Galli, T., McPherson, P. S. & De Camilli, P. The V0

sector of the V-ATPase, synaptobrevin, and synaptophysin are associated on synaptic vesicles in a Triton X-100-resistant, freeze-thawing sensitive complex. _J. Biol. Chem._ 271, 2193–2198

(1996). Article  CAS  Google Scholar  * Harvey, W. R. & Wieczorek, H. Animal plasma membrane energization by chemiosmotic H+ V-ATPases. _J. Exp. Biol._ 200, 203–216 (1997). CAS  PubMed 

Google Scholar  * Conchon, S., Cao, X., Barlowe, C. & Pelham, H. R. B. Got1p and Sft2p: membrane proteins involved in traffic to the Golgi complex. _EMBO J._ 18, 3934–3946 (1999).

Article  CAS  Google Scholar  * Zimmerberg, J., Vogel, S. & Chernomordik, L. V. Mechanisms of membrane fusion. _Annu. Rev. Biophys. Biomol. Struct._ 22, 433–466 (1993). Article  CAS 

Google Scholar  * Albillos, A. et al. The exocytotic event in chromaffin cells revealed by patch amperometry. _Nature_ 389, 509–512 (1997). Article  ADS  CAS  Google Scholar  * Henkel, A.

& Betz, W. J. Staurosporine blocks evoked release of FM1-43 but not acetylcholine from frog motor nerve terminals. _J. Neurosci._ 15, 8246–8258 (1995). Article  CAS  Google Scholar  *

Scepek, S., Coorssen, J. R. & Lindau, M. Fusion pore expansion in horse eosinophils is modulated by Ca2+ and protein kinase C via distinct mechanisms. _EMBO J._ 17, 4340–4345 (1998).

Article  CAS  Google Scholar  * Plattner, H. & Knoll, G. in _Signal Transduction During Biomembrane Fusion_ (ed. D. H. O'Day) 19–46 (Academic, San Diego, 1993). Google Scholar  *

Garcia-Segura, L. M., Muller, D. & Dunant, Y. Increase in the number of presynaptic large intramembrane particles during synaptic transmission at the Torpedo nerve–electroplaque

junction. _Neuroscience_ 19, 63–78 (1986). Article  CAS  Google Scholar  * Chernomordik, L., Kozlov, M. M. & Zimmerberg, J. Lipids in biological membrane fusion. _J. Membr. Biol._ 146,

1–14 (1995). Article  CAS  Google Scholar  * Shevchenko, A. et al. Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels.

_Proc. Natl Acad. Sci. USA_ 93, 14440–14445 (1996). Article  ADS  CAS  Google Scholar  Download references ACKNOWLEDGEMENTS We thank D. Gallwitz, P. Kane, R. Piper and M. Harrison for

plasmids and strains; C. Baradoy for assistance; and the Boehringer Ingelheim Foundation and Deutsche Forschungsgemeinschaft (SFB446) for support. AUTHOR INFORMATION Author notes *

Christopher Peters, Martin J. Bayer, Jens S. Andersen and Matthias Mann: These authors contributed equally to this work AUTHORS AND AFFILIATIONS * Friedrich-Miescher-Laboratorium der

Max-Planck-Gesellschaft, Spemannstrasse 37-39, Tübingen, 72076, Germany Christopher Peters, Martin J. Bayer, Susanne Bühler & Andreas Mayer * Department of Molecular Biology, University

of Southern Denmark, Campusvej 55, Odense M, 5230, Denmark Jens S. Andersen & Matthias Mann Authors * Christopher Peters View author publications You can also search for this author

inPubMed Google Scholar * Martin J. Bayer View author publications You can also search for this author inPubMed Google Scholar * Susanne Bühler View author publications You can also search

for this author inPubMed Google Scholar * Jens S. Andersen View author publications You can also search for this author inPubMed Google Scholar * Matthias Mann View author publications You

can also search for this author inPubMed Google Scholar * Andreas Mayer View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR

Correspondence to Andreas Mayer. RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Peters, C., Bayer, M., Bühler, S. _et al._ _Trans_-complex formation by

proteolipid channels in the terminal phase of membrane fusion. _Nature_ 409, 581–588 (2001). https://doi.org/10.1038/35054500 Download citation * Received: 10 August 2000 * Accepted: 29

November 2000 * Issue Date: 01 February 2001 * DOI: https://doi.org/10.1038/35054500 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get

shareable link Sorry, a shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative