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i-SNAREs: inhibitory SNAREs that fine-tune the specificity of membrane fusion.

Varlamov O, Volchuk A, Rahimian V, Doege CA, Paumet F, Eng WS, Arango N, Parlati F, Ravazzola M, Orci L, Söllner TH, Rothman JE - J. Cell Biol. (2003)

Bottom Line: A new functional class of SNAREs, designated inhibitory SNAREs (i-SNAREs), is described here.A striking pattern emerges in which certain subunits of the cis-Golgi SNAREpin function as i-SNAREs that inhibit fusion mediated by the trans-Golgi SNAREpin, and vice versa.Although the opposing distributions of the cis- and trans-Golgi SNAREs themselves could provide for a countercurrent fusion pattern in the Golgi stack, the gradients involved would be strongly sharpened by the complementary countercurrent distributions of the i-SNAREs.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biochemistry and Biophysics, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., Box 251, New York, NY 10021, USA.

ABSTRACT
A new functional class of SNAREs, designated inhibitory SNAREs (i-SNAREs), is described here. An i-SNARE inhibits fusion by substituting for or binding to a subunit of a fusogenic SNAREpin to form a nonfusogenic complex. Golgi-localized SNAREs were tested for i-SNARE activity by adding them as a fifth SNARE together with four other SNAREs that mediate Golgi fusion reactions. A striking pattern emerges in which certain subunits of the cis-Golgi SNAREpin function as i-SNAREs that inhibit fusion mediated by the trans-Golgi SNAREpin, and vice versa. Although the opposing distributions of the cis- and trans-Golgi SNAREs themselves could provide for a countercurrent fusion pattern in the Golgi stack, the gradients involved would be strongly sharpened by the complementary countercurrent distributions of the i-SNAREs.

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i-SNAREs compete with the tcis light chain Bos1, resulting in inactive pseudo t-SNARE. (A–C) We coreconstituted stoichiometric amounts of the tcis SNAREs (Sed5/Sec22, Bos1) and i-SNAREs into acceptor liposomes. Where indicated, an excess of one of the t-SNARE subunits was added. To achieve a substantial inhibition, the i-SNARE Sft1 was added at a twofold molar excess. Fusion activity of modified acceptor liposomes with donor vcis Bet1 liposomes is plotted as a percentage of control fusion (control liposomes contain only t-SNAREs). The proposed mechanism of action of the i-SNARE is illustrated at the top of each graph. The inhibitors of the cis-Golgi complex may compete with the cognate light chain Bos1, generating fusion-incompetent pseudo t-SNARE.
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fig4: i-SNAREs compete with the tcis light chain Bos1, resulting in inactive pseudo t-SNARE. (A–C) We coreconstituted stoichiometric amounts of the tcis SNAREs (Sed5/Sec22, Bos1) and i-SNAREs into acceptor liposomes. Where indicated, an excess of one of the t-SNARE subunits was added. To achieve a substantial inhibition, the i-SNARE Sft1 was added at a twofold molar excess. Fusion activity of modified acceptor liposomes with donor vcis Bet1 liposomes is plotted as a percentage of control fusion (control liposomes contain only t-SNAREs). The proposed mechanism of action of the i-SNARE is illustrated at the top of each graph. The inhibitors of the cis-Golgi complex may compete with the cognate light chain Bos1, generating fusion-incompetent pseudo t-SNARE.

Mentions: Fig. 4 shows the results of this analysis for the cis-Golgi fusion reaction and its i-SNAREs (Gos1, Tlg1, and Sft1). Inhibition was effectively reversed in all cases by an excess of the tcis light chain Bos1, but was not reversed by an excess of the other light chain (Sec22) or by an excess of the heavy chain (Sed5). This result establishes that all three i-SNAREs operate in the cis-Golgi fusion reaction by the same mechanism—they compete with Bos1 to form pseudo t-SNAREs (Sed5/Sec22,Gos1; Sed5/Sec22,Tlg1; Sed5/Sec22,Sft1). The tetrameric complex predicted by this mechanism (Sed5–Sec22–Gos1–Bet1) and the trimeric complex (Sed5–Sec22–Sft1) have been previously reported to form with cytoplasmic domains in solution by Tsui et al. (2001). All three of these pseudo t-SNAREs have been found to be nonfusogenic with v-SNAREs tested (Parlati et al., 2002). Although the trimeric i-SNARE–containing complexes Sed5/Sec22,Tlg1 and Sed5/Sec22,Sft1 form in solution, they are very labile, and thus may form only transiently in vivo (unpublished data). The quaternary complex Sed5–Sec22–Gos1–Bet1 forms in solution with high efficiency, similar to the cognate cis-Golgi complex Sed5–Sec22–Bos1–Bet1 (Tsui et al., 2001; unpublished data). To test whether this i-SNARE–containing complex represents the “dead-end” bi-product of SNARE pairing, we examined the effect of NSF on its stability. Both Sed5–Sec22–Bos1–Bet1 and Sed5–Sec22–Gos1–Bet1 are disrupted in the presence of NSF and α-SNAP, suggesting that both the cognate and the i-SNARE quaternary complexes are the substrates for NSF (unpublished data).


i-SNAREs: inhibitory SNAREs that fine-tune the specificity of membrane fusion.

Varlamov O, Volchuk A, Rahimian V, Doege CA, Paumet F, Eng WS, Arango N, Parlati F, Ravazzola M, Orci L, Söllner TH, Rothman JE - J. Cell Biol. (2003)

i-SNAREs compete with the tcis light chain Bos1, resulting in inactive pseudo t-SNARE. (A–C) We coreconstituted stoichiometric amounts of the tcis SNAREs (Sed5/Sec22, Bos1) and i-SNAREs into acceptor liposomes. Where indicated, an excess of one of the t-SNARE subunits was added. To achieve a substantial inhibition, the i-SNARE Sft1 was added at a twofold molar excess. Fusion activity of modified acceptor liposomes with donor vcis Bet1 liposomes is plotted as a percentage of control fusion (control liposomes contain only t-SNAREs). The proposed mechanism of action of the i-SNARE is illustrated at the top of each graph. The inhibitors of the cis-Golgi complex may compete with the cognate light chain Bos1, generating fusion-incompetent pseudo t-SNARE.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
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fig4: i-SNAREs compete with the tcis light chain Bos1, resulting in inactive pseudo t-SNARE. (A–C) We coreconstituted stoichiometric amounts of the tcis SNAREs (Sed5/Sec22, Bos1) and i-SNAREs into acceptor liposomes. Where indicated, an excess of one of the t-SNARE subunits was added. To achieve a substantial inhibition, the i-SNARE Sft1 was added at a twofold molar excess. Fusion activity of modified acceptor liposomes with donor vcis Bet1 liposomes is plotted as a percentage of control fusion (control liposomes contain only t-SNAREs). The proposed mechanism of action of the i-SNARE is illustrated at the top of each graph. The inhibitors of the cis-Golgi complex may compete with the cognate light chain Bos1, generating fusion-incompetent pseudo t-SNARE.
Mentions: Fig. 4 shows the results of this analysis for the cis-Golgi fusion reaction and its i-SNAREs (Gos1, Tlg1, and Sft1). Inhibition was effectively reversed in all cases by an excess of the tcis light chain Bos1, but was not reversed by an excess of the other light chain (Sec22) or by an excess of the heavy chain (Sed5). This result establishes that all three i-SNAREs operate in the cis-Golgi fusion reaction by the same mechanism—they compete with Bos1 to form pseudo t-SNAREs (Sed5/Sec22,Gos1; Sed5/Sec22,Tlg1; Sed5/Sec22,Sft1). The tetrameric complex predicted by this mechanism (Sed5–Sec22–Gos1–Bet1) and the trimeric complex (Sed5–Sec22–Sft1) have been previously reported to form with cytoplasmic domains in solution by Tsui et al. (2001). All three of these pseudo t-SNAREs have been found to be nonfusogenic with v-SNAREs tested (Parlati et al., 2002). Although the trimeric i-SNARE–containing complexes Sed5/Sec22,Tlg1 and Sed5/Sec22,Sft1 form in solution, they are very labile, and thus may form only transiently in vivo (unpublished data). The quaternary complex Sed5–Sec22–Gos1–Bet1 forms in solution with high efficiency, similar to the cognate cis-Golgi complex Sed5–Sec22–Bos1–Bet1 (Tsui et al., 2001; unpublished data). To test whether this i-SNARE–containing complex represents the “dead-end” bi-product of SNARE pairing, we examined the effect of NSF on its stability. Both Sed5–Sec22–Bos1–Bet1 and Sed5–Sec22–Gos1–Bet1 are disrupted in the presence of NSF and α-SNAP, suggesting that both the cognate and the i-SNARE quaternary complexes are the substrates for NSF (unpublished data).

Bottom Line: A new functional class of SNAREs, designated inhibitory SNAREs (i-SNAREs), is described here.A striking pattern emerges in which certain subunits of the cis-Golgi SNAREpin function as i-SNAREs that inhibit fusion mediated by the trans-Golgi SNAREpin, and vice versa.Although the opposing distributions of the cis- and trans-Golgi SNAREs themselves could provide for a countercurrent fusion pattern in the Golgi stack, the gradients involved would be strongly sharpened by the complementary countercurrent distributions of the i-SNAREs.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular Biochemistry and Biophysics, Memorial Sloan-Kettering Cancer Center, 1275 York Ave., Box 251, New York, NY 10021, USA.

ABSTRACT
A new functional class of SNAREs, designated inhibitory SNAREs (i-SNAREs), is described here. An i-SNARE inhibits fusion by substituting for or binding to a subunit of a fusogenic SNAREpin to form a nonfusogenic complex. Golgi-localized SNAREs were tested for i-SNARE activity by adding them as a fifth SNARE together with four other SNAREs that mediate Golgi fusion reactions. A striking pattern emerges in which certain subunits of the cis-Golgi SNAREpin function as i-SNAREs that inhibit fusion mediated by the trans-Golgi SNAREpin, and vice versa. Although the opposing distributions of the cis- and trans-Golgi SNAREs themselves could provide for a countercurrent fusion pattern in the Golgi stack, the gradients involved would be strongly sharpened by the complementary countercurrent distributions of the i-SNAREs.

Show MeSH
Related in: MedlinePlus