Portal de Periódicos da CAPES

Concerted Auto-regulation in Yeast Endosomal t-SNAREs

2005; Elsevier BV; Volume: 280; Issue: 22 Linguagem: Inglês

10.1074/jbc.m500841200

1083-351X

Fabienne Paumet, Vahid Rahimian, Maurizio Di Liberto, James E. Rothman,

Calcium signaling and nucleotide metabolism

In yeast, the assembly of the target (t)-SNAREs [Tlg2p/Tlg1p,Vti1p] and [Pep12p/Tlg1p,Vti1p] with the vesicular (v)-SNARE Snc2p promotes endocytic fusion. Here, selected mutations and truncations of SNARE proteins were tested in an in vitro fusion assay to identify potential regulatory regions in these proteins, and two distinct regions were found. The first is represented by the combined effect of the three t-SNARE N-terminal regions and the second is located within the Tlg1p SNARE motif. These internal controls provide a potential mechanism to enable SNARE-dependent fusion to be regulated. In yeast, the assembly of the target (t)-SNAREs [Tlg2p/Tlg1p,Vti1p] and [Pep12p/Tlg1p,Vti1p] with the vesicular (v)-SNARE Snc2p promotes endocytic fusion. Here, selected mutations and truncations of SNARE proteins were tested in an in vitro fusion assay to identify potential regulatory regions in these proteins, and two distinct regions were found. The first is represented by the combined effect of the three t-SNARE N-terminal regions and the second is located within the Tlg1p SNARE motif. These internal controls provide a potential mechanism to enable SNARE-dependent fusion to be regulated. The core mechanism of SNARE 1The abbreviations used are: SNARE, soluble NSF attachment protein receptor; t-SNARE, target-SNARE; v-SNARE, vesicular-SNARE; SNAP, soluble NSF attachment protein; NSF, N-ethylmaleimide-sensitive factor; GST, glutathione S-transferase; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride.1The abbreviations used are: SNARE, soluble NSF attachment protein receptor; t-SNARE, target-SNARE; v-SNARE, vesicular-SNARE; SNAP, soluble NSF attachment protein; NSF, N-ethylmaleimide-sensitive factor; GST, glutathione S-transferase; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride. (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor)-mediated fusion is strikingly simple: a target-SNARE (t-SNARE) generates fusion only with its cognate vesicular-SNARE (v-SNARE) (1Scales S.J. Chen Y.A. Yoo B.Y. Patel S.M. Doung Y.C. Scheller R.H. Neuron. 2000; 26: 457-464Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 2McNew J.A. Parlati F. Fukuda R. Johnston R.J. Paz K. Paumet F. Sollner T.H. Rothman J.E. Nature. 2000; 407: 153-159Crossref PubMed Scopus (522) Google Scholar, 3Parlati F. Varlamov O. Paz K. McNew J.A. Hurtado D. Sollner T. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 5424-5429Crossref PubMed Scopus (144) Google Scholar, 4Paumet F. Brugger B. Parlati F. McNew J.A. Sollner T.H. Rothman J.E. J. Cell Biol. 2001; 155: 961-968Crossref PubMed Scopus (55) Google Scholar, 5Paumet F. Rahimian V. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3376-3380Crossref PubMed Scopus (64) Google Scholar). The cognate t- and v-SNARE pair forms a stable helical complex composed of four helices (three from the t-SNARE and one from the v-SNARE). Each of the four helices comes from a different subfamily: the Syntaxins, the Bet1 group, the Bos1 group, and the R-SNAREs (6Pelham H. Trends Cell Biol. 2001; 11: 99-101Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Upon assembly, all membrane anchors of the helical bundle are at one end and the N-terminal domains at the other end (7Sutton R. Fasshauer D. Jahn R. Brunger A. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1878) Google Scholar, 8Antonin W. Fasshauer D. Becker S. Jahn R. Schneider T.R. Nat. Struct. Biol. 2002; 9: 107-111Crossref PubMed Scopus (204) Google Scholar). The largely unstructured monomers undergo a conformational change during SNARE assembly, which releases enough free energy to overcome the repulsive forces of the opposed membranes, and leads to lipid mixing (9Weber T. Zemelman B. McNew J. Westermann B. Gmachl M. Parlati F. Söllner T. Rothman T. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (1986) Google Scholar). After fusion, the ATPase NSF and α-SNAP disassemble SNARE complexes, and monomers are recycled (10Sollner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1562) Google Scholar). The finding that SNAREs constitute the core fusion machinery and are sufficient for membrane fusion raises the question of how they are regulated. Many SNAREs, in particular the Syntaxin heavy chains, possess N-terminal extensions, which can independently fold and are able to modulate SNARE activity. The neuronal Syntaxin1, as well as Sso1p (yeast homolog of Syntaxin1) and Syntaxin7 (late endocytic mammalian Syntaxin), adopt a “closed” conformation. In this case, their N-terminal domain, structured in a three-helix bundle, is able to interact with the SNARE motif, generating the so-called closed conformation. This interaction blocks the binding of the light chains and inhibits the t-SNARE complex formation (11Fernandez I. Ubach J. Dulubova I. Zhang X. Sudhof T.C. Rizo J. Cell. 1998; 94: 841-849Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 12Dulubova I. Sugita S. Hill S. Hosaka M. Fernandez I. Sudhof T.C. Rizo J. EMBO J. 1999; 18: 4372-4382Crossref PubMed Scopus (541) Google Scholar, 13Nicholson K.L. Munson M. Miller R.B. Filip T.J. Fairman R. Hughson F.M. Nat. Struct. Biol. 1998; 5: 793-802Crossref PubMed Scopus (172) Google Scholar, 14Munson M. Chen X. Cocina A.E. Schultz S.M. Hughson F.M. Nat. Struct. Biol. 2000; 7: 894-902Crossref PubMed Scopus (132) Google Scholar, 15Antonin W. Dulubova I. Arac D. Pabst S. Plitzner J. Rizo J. Jahn R. J. Biol. Chem. 2002; 277: 36449-36456Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). The removal of the Syntaxin1 N-terminal domain results in an increase of fusion (16Parlati F. Weber T. McNew J.A. Westermann B. Sollner T.H. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12565-12570Crossref PubMed Scopus (213) Google Scholar). The Syntaxin Vam3p on the other hand, uses a different mechanism. In the yeast vacuole, the three-helix bundle of Vam3p-N-terminal domain does not interact intramolecularly with the SNARE motif (17Dulubova I. Yamaguchi T. Wang Y. Sudhof T.C. Rizo J. Nat. Struct. Biol. 2001; 8: 258-264Crossref PubMed Scopus (125) Google Scholar). Nonetheless, the removal of this N-terminal domain influences the SNARE complex assembly (18Laage R. Ungermann C. Mol. Biol. Cell. 2001; 12: 3375-3385Crossref PubMed Scopus (42) Google Scholar). Despite the fact that the mechanisms differ (closed or opened Syntaxins), all heavy chain N-terminal domains tested act by reducing t-SNARE assembly (11Fernandez I. Ubach J. Dulubova I. Zhang X. Sudhof T.C. Rizo J. Cell. 1998; 94: 841-849Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar, 12Dulubova I. Sugita S. Hill S. Hosaka M. Fernandez I. Sudhof T.C. Rizo J. EMBO J. 1999; 18: 4372-4382Crossref PubMed Scopus (541) Google Scholar, 13Nicholson K.L. Munson M. Miller R.B. Filip T.J. Fairman R. Hughson F.M. Nat. Struct. Biol. 1998; 5: 793-802Crossref PubMed Scopus (172) Google Scholar, 14Munson M. Chen X. Cocina A.E. Schultz S.M. Hughson F.M. Nat. Struct. Biol. 2000; 7: 894-902Crossref PubMed Scopus (132) Google Scholar, 15Antonin W. Dulubova I. Arac D. Pabst S. Plitzner J. Rizo J. Jahn R. J. Biol. Chem. 2002; 277: 36449-36456Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar,18Laage R. Ungermann C. Mol. Biol. Cell. 2001; 12: 3375-3385Crossref PubMed Scopus (42) Google Scholar). The light chains also have N-terminal domains, but their role is less clear. Neither appears to adopt a closed intra-molecular conformation nor does their N-terminal domain seem to influence the rate of the t-SNARE complex formation (15Antonin W. Dulubova I. Arac D. Pabst S. Plitzner J. Rizo J. Jahn R. J. Biol. Chem. 2002; 277: 36449-36456Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 19Misura K.M.S. Bock J.B. Gonzalez Jr., L.C. Scheller R.H. Weis W.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9184-9189Crossref PubMed Scopus (46) Google Scholar, 20Gonzalez Jr., L.C. Weis W.I. Scheller R.H. J. Biol. Chem. 2001; 276: 24203-24211Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Previously, we characterized two yeast endocytic complexes, the early endosome/trans-Golgi network complex [Tlg2p/Tlg1p,Vti1p] and the late endocytic complex [Pep12p/Tlg1p,Vti1p], both fusing with Snc2p v-SNARE although very slowly in vitro (4Paumet F. Brugger B. Parlati F. McNew J.A. Sollner T.H. Rothman J.E. J. Cell Biol. 2001; 155: 961-968Crossref PubMed Scopus (55) Google Scholar, 5Paumet F. Rahimian V. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3376-3380Crossref PubMed Scopus (64) Google Scholar). In the present study, using these SNARE complexes, we determined the influence of their N-terminal domains on fusion. Furthermore, we investigated whether the SNARE motif itself influences the kinetics of liposome fusion. Peptides—Snc2-C-pept (GERLTSIEDKADNLAISAQGFKRGANRVRKQMWWKD) (4Paumet F. Brugger B. Parlati F. McNew J.A. Sollner T.H. Rothman J.E. J. Cell Biol. 2001; 155: 961-968Crossref PubMed Scopus (55) Google Scholar) was synthesized by the Microchemistry Core Facility of the Memorial Sloan Kettering Cancer Institute (New York) and VAMP8-C-pept (GENLEHLRNKTEDLEATSEHFKTTSQKVARKFWWKN) by SynPep Corp. (Dublin, CA). Both peptides were dissolved in 10 mm HCl and then diluted in a reconstitution buffer (25 mm Hepes-KOH pH 7.4, 100 mm KCl, 10% glycerol) to a concentration of 3 mg/ml. Plasmid Constructs—The coding sequences of the mammalian SNAREs were cloned as following: hSyntaxin7 was amplified by PCR using the primers FO50 (GGGCATATCCATATGTCTTACACTCCAGGAGTTGGT) and FO51 (GCGAATTCTCAGTGGTTCAATCCCCAAA), hSyntaxin8 using FO61 (GGGCATATCCATATGGCCCCGGACCCCTGGTTC) and FO62 (GCGAATTCTCAGTTGGTCGGCCAGACTGC), mVti1b using FO58 (GGGCATATCCATATGGCCGCCTCCGCCGCCTCC) and FO59 (GCGAATTCTCAATGGTGTCGAAAGAATTT), and hVAMP8 using FO108 (GGGCATATCCATATGGAGGAAGCCAGTGAAGGT) and FO109 (TTTGAATTCTTAAGAGAAGGCACCAGTGGCAAAGAGCACAATGAAGAGGATGATGATAAAAAC). The cytosolic form of hVAMP8 (hVAMP8-ΔTMD) was amplified with FO108 and FO109 (GCGAATTCTCACTTCACGTTCTTCCACCAGAA). All PCR products were digested with NdeI and EcoRI and ligated in myc-pGEX-2T (modified from Amersham Biosciences) resulting, respectively, in FD132 (GST-myc-hSyn7), FD134 (GST-myc-hSyn8), FD120 (GST-myc-mVti1b), FD136 (GST-myc-hVAMP8), and FD196 (GST-hVAMP8-ΔTMD). Thrombin sites (indicated with an asterisk) were inserted in Tlg2p with the primers FO3 (GGGCCTCGATATTGAAGACCTGGTACCAAGAACGTTGCAGAGACAGC) and FO4 (GCTGTCTCTGCAACGTTCTTGGTACCAGGTCTTCAATATCGAGGCCC) replacing the amino acids YSKR in positions 191–194 with LVPR and resulting in Tlg2* and in Tlg1p in positions 114–117 (114LNTS) with the primers FO14 (CGGTGGAAAATTCAACACTGGTACCCCGGATGGCTGAGAACAATGATGG) and FO15 (CCATCATTGTTCTCAGCCATCCGGGGTACCAGTGTTGAATTTTCCACCG) resulting in Tlg1*. The thrombin site in Vti1p was inserted with the primers FO97 (ATTGACGATGACCAAAGGCTGGTACCGAGGAGCAACCATGCAATCTTA) and FO98 (TAAGATTGCATGGTTGCTCCTCGGTACCAGCCTTTGGTCATCGTCAAT), replacing the amino acids QQLL in positions 118–121 and resulting in Vti1*. Finally, the thrombin site in Pep12p was inserted with the primers FO99 (AAGATTTTGCTGATAAGCACGCGGTACTAGTTTTATCGGGTCTCTCTC) and FO102 (GAGAGAGACCCGATAAAACTAGTACCGCGTGCTTATCAGCAAAATCTT), replacing the amino acids NEEF in positions 188–191 and resulting in Pep12*. The DNA products of Tlg2*, Tlg1*, and Pep12* were digested by NdeI/BamH1 and Vti1* by EcoRI/BamHI. All of them were cloned in pET28a vector resulting in FD97 for His-Tlg2*, FD100 for His-Tlg1*, FD184 for His-Vti1*, and FD197 for His-Pep12*. The chimeric forms of the Tlg1/Syn8 SNARE motif were generated as follows: each fragment was amplified by PCR with FO38 (GGGAATTCCATATGGCTGAGAACAATGATGGT) and FO76 (CCGGAATTCATCCCCCATTGTTTGAGC) to generate N-Tlg1, FO77 (CCGGAATTCGAGAACCAGGGACAATTG) and FO78 (ATAAGAATGCGGCCGCTCAAGCAATGAATGCCAA) to generate C-Tlg1, FO63 (GGGCATATCCATATGTTGGGTTTTGATGAAATCCGG) and FO79 (CCGGAATTCATTCCCAATTTCCTGCCC) to generate N-Syn8, and FO80 (CCGGAATTCGATGAACAAAATGAGATA) and FO81 (ATAAGAATGCGGCCGCTCAGTTGGTCGGCCAGAC) to generate C-Syn8. These fragments were thus digested with NdeI/EcoRI or NdeI/NotI and ligated: N-Tlg1 with C-Syn8 to generate T1-S8, N-Tlg1 with C-Tlg1 to generate T1-T1, N-Syn8 with C-Tlg1 to generate S8-T1, and N-Syn8 with C-Syn8 to generate S8-S8. All of them were cloned in pGEX-2T resulting in FD161 for T1-T1, FD162 for T1-S8, FD163 for S8-T1, and FD164 for S8-S8. The coding sequence of Vti1p was obtained as described (21Fukuda R. McNew J.A. Weber T. Parlati F. Engel T. Nickel W. Rothman J.E. Sollner T.H. Nature. 2000; 407: 198-202Crossref PubMed Scopus (188) Google Scholar); Snc2p and the cytosolic domain of Snc2p as described (2McNew J.A. Parlati F. Fukuda R. Johnston R.J. Paz K. Paumet F. Sollner T.H. Rothman J.E. Nature. 2000; 407: 153-159Crossref PubMed Scopus (522) Google Scholar); Pep12p, ΔPep12p (deletion of the N-terminal domain), and ΔVti1p (deletion of the N-terminal domain) as described (5Paumet F. Rahimian V. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3376-3380Crossref PubMed Scopus (64) Google Scholar); and Δ36-Tlg2p (called in this paper Tlg2p for clarity) and Tlg1p as described (4Paumet F. Brugger B. Parlati F. McNew J.A. Sollner T.H. Rothman J.E. J. Cell Biol. 2001; 155: 961-968Crossref PubMed Scopus (55) Google Scholar). All plasmids were propagated in DH5α strain (Invitrogen). Protein Expression and Purification—Snc2p-His6, GST-Snc2p cytosolic domain, His6-Vti1p, His6-Tlg2p, His6-Tlg1p, GST-Pep12p, GST-ΔPep12p, and GST-ΔVti1p were prepared as described (2McNew J.A. Parlati F. Fukuda R. Johnston R.J. Paz K. Paumet F. Sollner T.H. Rothman J.E. Nature. 2000; 407: 153-159Crossref PubMed Scopus (522) Google Scholar, 4Paumet F. Brugger B. Parlati F. McNew J.A. Sollner T.H. Rothman J.E. J. Cell Biol. 2001; 155: 961-968Crossref PubMed Scopus (55) Google Scholar, 5Paumet F. Rahimian V. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3376-3380Crossref PubMed Scopus (64) Google Scholar, 21Fukuda R. McNew J.A. Weber T. Parlati F. Engel T. Nickel W. Rothman J.E. Sollner T.H. Nature. 2000; 407: 198-202Crossref PubMed Scopus (188) Google Scholar, 22McNew J.A. Coe J.G. Sogaard M. Zemelman B.V. Wimmer C. Hong W. Sollner T.H. FEBS Lett. 1998; 435: 89-95Crossref PubMed Scopus (50) Google Scholar). For GST-Syn7, GST-Syn8, GST-Vti1b, GST-VAMP8, and GST-ΔVAMP8; GST-T1-T1, GST-S8-S8, GST-T1-S8 and GST-S8-T1; and His6-Tlg2*, His6-Tlg1*, His6-Vti1*, and His6-Pep12*, plasmids used for protein expression were transformed into the Escherichia coli strain BL21star (DE3) (Invitrogen). Transformed cells were grown at 37 °C to an absorbance at 600 nm of 0.8, and protein expression was induced with 0.2 mm isopropyl 1-thio-β-d-galactopyranoside (Roche Applied Science) for 2 h at 37 °C. Cells were collected by centrifugation and lysed in buffer A, and the lysate was clarified by centrifugation (4Paumet F. Brugger B. Parlati F. McNew J.A. Sollner T.H. Rothman J.E. J. Cell Biol. 2001; 155: 961-968Crossref PubMed Scopus (55) Google Scholar). Supernatants containing His-tagged proteins were bound to nickel-nitrilotriacetic acid-agarose (Qiagen) and washed with buffer B containing 100 mm imidazole, and proteins were eluted with a 100 mm to 1 m imidazole gradient (in buffer B) (4Paumet F. Brugger B. Parlati F. McNew J.A. Sollner T.H. Rothman J.E. J. Cell Biol. 2001; 155: 961-968Crossref PubMed Scopus (55) Google Scholar). Supernatants containing GST-tagged protein were bound to glutathione-agarose beads (Sigma) and washed with buffer B, and proteins were eluted with 20 mm glutathione reduced (Roche Applied Science) in buffer B. GST-proteins were cleaved from GST on liposomes with 0.05 unit/μl thrombin (Sigma). SNARE Reconstitution into Liposomes—All t-SNAREs were reconstituted from individual proteins as described (9Weber T. Zemelman B. McNew J. Westermann B. Gmachl M. Parlati F. Söllner T. Rothman T. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (1986) Google Scholar). The typical lipid recovery efficiency in the recovered Nycodenz fraction was about 50% for acceptor liposomes and about 30% for donor liposomes. Fusion Assay—The lipid mixing assay was conducted as described (9Weber T. Zemelman B. McNew J. Westermann B. Gmachl M. Parlati F. Söllner T. Rothman T. Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (1986) Google Scholar, 16Parlati F. Weber T. McNew J.A. Westermann B. Sollner T.H. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12565-12570Crossref PubMed Scopus (213) Google Scholar). For some experiments, 3.0 nmol of Snc2-C-pept (4Paumet F. Brugger B. Parlati F. McNew J.A. Sollner T.H. Rothman J.E. J. Cell Biol. 2001; 155: 961-968Crossref PubMed Scopus (55) Google Scholar) or VAMP8-C-pept, or 6 nmol of cytosolic domain of Snc2p or cyt-VAMP8, were added as indicated in figure legends. The data were converted to rounds of fusion as described (16Parlati F. Weber T. McNew J.A. Westermann B. Sollner T.H. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12565-12570Crossref PubMed Scopus (213) Google Scholar). Note that the small decrease observed during the first 10 min of each fusion reaction is due to the temperature equilibration of the fluorophore. The N-terminal Domains of the t-SNARE Constitute a Potential Regulatory Switch—Both yeast endocytic t-SNAREs, [Tlg2p/Tlg1p,Vti1p] and [Pep12p/Tlg1p,Vti1p], must be activated to promote fusion of liposomes (4Paumet F. Brugger B. Parlati F. McNew J.A. Sollner T.H. Rothman J.E. J. Cell Biol. 2001; 155: 961-968Crossref PubMed Scopus (55) Google Scholar, 5Paumet F. Rahimian V. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 3376-3380Crossref PubMed Scopus (64) Google Scholar). This distinguishes them from all other fusogenic SNAREs tested. In vitro, this activation is provided artificially by a peptide (Snc2-C-pept) corresponding to the C-terminal part of the v-SNARE helical motif (4Paumet F. Brugger B. Parlati F. McNew J.A. Sollner T.H. Rothman J.E. J. Cell Biol. 2001; 155: 961-968Crossref PubMed Scopus (55) Google Scholar). It has been demonstrated that such peptides are able to bind and restructure t-SNARE complexes (23Melia T.J. Weber T. McNew J.A. Fisher L.E. Johnston R.J. Parlati F. Mahal L.K. Sollner T. Rothman J.E. J. Cell Biol. 2002; 158: 929-940Crossref PubMed Scopus (166) Google Scholar). The heavy chain, as well as both light chains of each endocytic t-SNARE, contributes a large N-terminal domain. These N-terminal extensions are neither homologous to each other nor to other SNAREs. To study their influence on fusion, we generated t-SNARE constructs in which one or more of these N-terminal domains could be removed by thrombin cleavage. First, the individual role of each t-SNARE N-terminal extension was investigated by including only one cleavable t-SNARE subunit per t-SNARE complex. We reconstituted in acceptor liposomes all possible combinations of both endocytic complexes: [Tlg2*/Tlg1,Vti1], [Tlg2/Tlg1*,Vti1], and [Tlg2/Tlg1,Vti1*] or [Pep12*/Tlg1,Vti1], [Pep12/Tlg1*,Vti1], and [Pep12/Tlg1,Vti1*] (asterisk identifies the thrombin-cleavable protein). All combinations were systematically tested for fusion with Snc2p donor liposomes, in the presence or in the absence of the Snc2-C-pept, both before (black curves) or after (red curves) thrombin cleavage (Fig. 1). As expected, thrombin itself does not affect fusion (Fig. 1A). We observed that the presence of the N-terminal domain of both Syntaxins Tlg2p-(36–191) and Pep12p-(1–188) reduced the fusion rate (Fig. 1B). The results observed for both endocytic complexes are consistent with those previously obtained for Syntaxin 1 (16Parlati F. Weber T. McNew J.A. Westermann B. Sollner T.H. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12565-12570Crossref PubMed Scopus (213) Google Scholar) and extend the potential regulatory role of the heavy chain N-terminal domain. Interestingly, the removal of the N-terminal domain of each light chain, Tlg1p-(1–117) and Vti1p-(1–118), increases the fusion kinetic as well (Fig. 1C). However, it is still necessary to activate the t-SNARE complexes with Snc2-C-pept to promote fusion. To investigate the interplay of the three N-terminal domains, we modulated the composition of the t-SNARE complex in the acceptor liposomes by including two or three cleavable proteins, thus generating: [Tlg2*/Tlg1*,Vti1], [Tlg2*/Tlg1,Vti1*], [Tlg2/Tlg1*,Vti1*], and [Tlg2*/Tlg1*,Vti1*] or [Pep12*/Tlg1*,Vti1], [Pep12*/Tlg1,Vti1*], [Pep12/Tlg1*,Vti1*], and [Pep12*/Tlg1*,Vti1*]. We observed that the kinetic control exerted by the N-terminal domains is additive (Fig. 2). The removal of two N-terminal domains out of three increases the fusion rate compared with the removal of only one (Fig. 2A). The most dramatic effect occurs when all three N-terminal domains are cleaved off (Fig. 2B) resulting in an average of 5-fold (for Tlg2 complex) or 2.5-fold (for Pep12 complex) increase in the extent of fusion. In conclusion, the combination of the t-SNARE N-terminal domains represents a potential molecular switch for controlling the kinetic of fusion. In Yeast, the Light Chain SNARE Motif Contributes to a Second Potential Regulatory Switch—Even when the inhibition created by the combined effect of the yeast t-SNARE N-terminal domains is removed, the rate of fusion in the absence of peptide activation is still very low (Fig. 2B). Thus an additional inhibitory element may be involved. To determine whether this second element is specific for yeast or is instead a general feature of endocytic SNAREs, we investigated the fusion capacity of a corresponding mammalian late endocytic complex (8Antonin W. Fasshauer D. Becker S. Jahn R. Schneider T.R. Nat. Struct. Biol. 2002; 9: 107-111Crossref PubMed Scopus (204) Google Scholar, 24Antonin W. Holroyd C. Fasshauer D. Pabst S. Von Mollard G.F. Jahn R. EMBO J. 2000; 19: 6453-6464Crossref PubMed Scopus (210) Google Scholar). We reconstituted the t-SNARE [Syntaxin7/Syntaxin8,Vti1b] into acceptor liposomes and the v-SNARE VAMP8 into donor liposomes and tested the fusion efficiency of this complex with or without VAMP8-C-pept. As shown in Fig. 3, these liposomes now fuse efficiently without any added activator, although the peptide still increases the fusion rate. Thus mammalian and yeast complexes have different inherent fusogenicities. Combined with the results presented above, we assumed that the additional inhibition of the yeast complexes arises from an element located in the SNARE motif. Mixing experiments were conducted to locate this switch. Both the yeast [Pep12p/Tlg1p,Vti1p] and the mammalian [Syntaxin7/Syntaxin8,Vti1b] t-SNAREs are necessary for fusion at the late endosome (25Becherer K.A. Rieder S.E. Emr S.D. Jones E.W. Mol. Biol. Cell. 1996; 7: 579-594Crossref PubMed Scopus (252) Google Scholar, 26Mullock B.M. Smith C.W. Ihrke G. Bright N.A. Lindsay M. Parkinson E.J. Brooks D.A. Parton R.G. James D.E. Luzio J.P. Piper R.C. Mol. Biol. Cell. 2000; 11: 3137-3153Crossref PubMed Scopus (125) Google Scholar, 27Wang H. Frelin L. Pevsner J. Gene (Amst.). 1997; 199: 39-48Crossref PubMed Scopus (49) Google Scholar). In addition, Syntaxin7 can complement yeast Pep12p mutants defective in fusion to the prevacuolar compartment (28Nakamura N. Yamamoto A. Wada Y. Futai M. J. Biol. Chem. 2000; 275: 6523-6529Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar), showing their homology. We tested whether various yeast/mammalian mixed complexes were fusogenic, the hypothesis being that if the yeast proteins carry a regulatory element in their sequence, replacing it with the mammalian homolog will release the negative regulation. We reconstituted the different t-SNARE yeast/mammal combinations into acceptor liposomes (Fig. 4) and tested their fusion capacity in the presence or absence of Snc2-C-pept. Exchanging Tlg1p with its mammalian homolog Syntaxin8 released the blockage. In this situation, the complex [Pep12p/ Syn8,Vti1p] becomes fusogenic without activation with Snc2-C-pept. When replacing both yeast light chains Tlg1p and Vti1p with their mammalian counterparts Syn8 and Vti1b, the resulting t-SNARE [Pep12p/Syn8,Vti1b] is even more fusogenic, suggesting a synergistic effect of both light chains for fusion. We also observe that Snc2p (Fig. 4A) and VAMP8 (Fig. 4B) can functionally replace each other. Altogether, these results suggest that within a given cellular pathway, variations in SNARE autoregulation may be specifically encoded within the light chains of the t-SNARE. The Autoregulation of Tlg1p Is Encoded in the N-terminal Half of Its SNARE Motif—The SNARE motif is in average 60 amino acids long and is organized in ∼16 layers based on its helical structure with the zero layer determining the center of the SNARE motif (7Sutton R. Fasshauer D. Jahn R. Brunger A. Nature. 1998; 395: 347-353Crossref PubMed Scopus (1878) Google Scholar, 8Antonin W. Fasshauer D. Becker S. Jahn R. Schneider T.R. Nat. Struct. Biol. 2002; 9: 107-111Crossref PubMed Scopus (204) Google Scholar). To identify the region responsible for the slow kinetics in yeast, we created chimeric constructs between the Syntaxin8 and the Tlg1p SNARE motif. In these constructs, the Tlg1p N-terminal part of the SNARE motif (from layers –9 to 0) was joined to the Syntaxin8 C-terminal part of the SNARE motif (from layers 0 to +9) resulting in T1-S8 protein and vice versa (resulting in S8-T1 protein). Tlg1p (T1-T1) and Syntaxin8 controls (S8-S8) were constructed using the same cloning strategy. Each of these constructs is shown in Fig. 5A. These chimeras were expressed as GST-recombinant proteins and reconstituted into donor liposomes together with the truncated forms of Pep12p and Vti1p. While both controls (T1-T1 and S8-S8) retain their fusogenic properties (Fig. 5, B and C, respectively), the chimera T1-S8 requires activation by a peptide (VAMP8-C-pept) for fusion (Fig. 5D). The chimera S8-T1, on the other hand, does not require peptide activation for fusion (Fig. 5E). We note that when C-Syn8 replaces C-Tlg1p, Snc2-C-pept does not activate the complex anymore (Fig. 5D). This effect may have different causes: either Snc2-C-pept is simply unable to bind the C-terminal part of Syn8, or Snc2-C-pept can still bind but is unable to activate the complex, suggesting in this case that an adequate cognate structure on C terminus is necessary for releasing the N-terminal switch. At this point of the study, we are unable to discriminate between both possibilities. Altogether, this demonstrates that a second switch region is buried in the Tlg1p SNARE motif and is restricted to its N-terminal portion (between the layers –9 and 0). This study suggests that SNAREs possess multiple distinct autoregulatory switches situated in different regions of the t-SNARE complex. These elements can be combined in particular t-SNAREs (yeast versus mammalian endocytic complexes), providing the SNARE system with functional flexibility to answer to the specific needs of different transports pathways in different cells. The first switch is provided by the combination of the three t-SNARE N-terminal domains. While an autoregulatory role for syntaxin N-terminal domain is well known (16Parlati F. Weber T. McNew J.A. Westermann B. Sollner T.H. Rothman J.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12565-12570Crossref PubMed Scopus (213) Google Scholar) and now includes Tlg2p and Pep12p, the role of the light chain N-terminal domains has been unclear. The light chain N-terminal domains do not interact intramolecularly with the SNARE motif and have no influence on the t-SNARE complex formation in solution (15Antonin W. Dulubova I. Arac D. Pabst S. Plitzner J. Rizo J. Jahn R. J. Biol. Chem. 2002; 277: 36449-36456Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 19Misura K.M.S. Bock J.B. Gonzalez Jr., L.C. Scheller R.H. Weis W.I. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 9184-9189Crossref PubMed Scopus (46) Google Scholar). In this study, we found that the N-terminal domains of the yeast endocytic light chains can additively control the kinetics of fusion. A second potential switch is buried in the SNARE motif of the yeast light chain Tlg1p. This regulation seems to be absent from a mammalian homolog. Possibly, the tighter autoregulation in yeast relates to the multiple roles for the endocytic v-SNARE, Snc2p. Not only do both endocytic complexes share this v-SNARE with each other, they also share it with the exocytic complex [Sso1p/Sec9p] (29Gurunathan S. Chapman-Shimshoni D. Trajkovic S. Gerst J.E. Mol. Biol. Cell. 2000; 11: 3629-3643Crossref PubMed Scopus (56) Google Scholar). By contrast, in mammals there are several distinct VAMPs which are further functionally specialized (30Bock J.B. Matern H.T. Peden A.A. Scheller R.H. Nature. 2001; 409: 839-841Crossref PubMed Scopus (516) Google Scholar). It is interesting to note that although the C-peptide binds the C-terminal part of the SNARE motif, it releases a regulatory element located within the N-terminal half. t-SNARE complexes are partially assembled across the most N-terminal portions of the SNARE domain (23Melia T.J. Weber T. McNew J.A. Fisher L.E. Johnston R.J. Parlati F. Mahal L.K. Sollner T. Rothman J.E. J. Cell Biol. 2002; 158: 929-940Crossref PubMed Scopus (166) Google Scholar, 31Fiebig K.M. Rice L.M. Pollock R. Brunger A.T. Nat. Struct. Biol. 1999; 6: 117-123Crossref PubMed Scopus (232) Google Scholar). By using a peptide homologous to snc2-C-pept, our group showed previously that VAMP2-C-pept binds and structures the C-terminal part of the t-SNARE complex (called the tc-fusion switch), consequently increasing the fusion rate (23Melia T.J. Weber T. McNew J.A. Fisher L.E. Johnston R.J. Parlati F. Mahal L.K. Sollner T. Rothman J.E. J. Cell Biol. 2002; 158: 929-940Crossref PubMed Scopus (166) Google Scholar). In particular, Melia et al. (23Melia T.J. Weber T. McNew J.A. Fisher L.E. Johnston R.J. Parlati F. Mahal L.K. Sollner T. Rothman J.E. J. Cell Biol. 2002; 158: 929-940Crossref PubMed Scopus (166) Google Scholar) observed that when the v-SNARE C-peptide is bound, t-SNARE becomes as resistant to proteolysis as when the entire soluble VAMP2 is bound, suggesting that structuring the C-terminal half is sufficient to induce complete coiled-coil formation across the SNARE bundle. Although we cannot pin-point the site of regulation for the second potential switch in the endocytic complexes, it seems likely that the N-terminal region is refolded in response to Snc2-C-pept binding. When N-Syn8 replaces N-Tlg1p and therefore compensates the N-terminal switch, peptides still have an activation effect on fusion (Fig. 5E). In this case, the peptides are still acting on the tc-fusion switch. In conclusion there are multiple autoregulatory switches engulfed into t-SNAREs. These switches are presumably governed by additional cellular regulatory mechanisms, which could be protein or lipids. One candidate regulator for the yeast endocytic complex is the GARP (Golgi-associated retrograde proteins) multisubunit tethering complex. This complex, together with Vps52p and Vps53p, is required for retrograde transport to the late Golgi. It has been shown recently that two members of this complex, Vps51p and Vps54p, interact directly with the N-terminal domain of Tlg1p (32Siniossoglou S. Pelham H.R.B. EMBO J. 2001; 20: 5991-5998Crossref PubMed Scopus (162) Google Scholar, 33Conibear E. Cleck J.N. Stevens T.H. Mol. Biol. Cell. 2003; 14: 1610-1623Crossref PubMed Scopus (140) Google Scholar). An attractive model is that the binding of Vps51p/Vps54p to Tlg1p stabilizes the N-terminal region of the SNARE motif and releases the SNARE complex to promote fusion. We thank Thomas J. Melia, Peter Antinozzi, and Thalia Becker for their comments and help as well as for the critical reading of the manuscript.