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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 inhibit the trans-Golgi fusion reaction. We coreconstituted stoichiometric amounts of the ttrans SNAREs (Sed5/Ykt6, Gos1) 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 Tlg1 was added at a twofold molar excess (B). Fusion activity of modified acceptor liposomes with donor vtrans Sft1 liposomes is plotted as a percentage of control fusion (control liposomes contain only t-SNAREs).
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fig5: i-SNAREs inhibit the trans-Golgi fusion reaction. We coreconstituted stoichiometric amounts of the ttrans SNAREs (Sed5/Ykt6, Gos1) 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 Tlg1 was added at a twofold molar excess (B). Fusion activity of modified acceptor liposomes with donor vtrans Sft1 liposomes is plotted as a percentage of control fusion (control liposomes contain only t-SNAREs).

Mentions: When the trans-Golgi fusion reaction was tested in a functional competition test, no t-SNARE subunit added in excess is capable of reversing inhibition by the i-SNAREs Bet1 and Tlg1 (Fig. 5). This means either that this class of i-SNAREs acts as pseudo v-SNAREs (binding ttrans) or that i-SNAREs bind to the vtrans/ttrans complex, forming an inactive oligomer (the noncompetitive mechanism). The pseudo v-SNARE mechanism predicts that the i-SNAREs could compete with the cognate v-SNARE. Consistent with this, the i-SNARE Bet1 is known to form a stable complex with Sed5, Gos1, and Ykt6, which has been isolated from immunoprecipitates of animal cell extracts (Zhang and Hong, 2001). Out of all the i-SNAREs, Bet1 has the highest inhibitory potency toward the trans-Golgi fusion reaction (a stoichiometric amount of Bet1 inhibits fusion by 70%; Fig. 3). Binding experiments with the soluble recombinant proteins were inconclusive in further testing the possible pseudo v-SNARE mechanism. We observed no competition between the i-SNARE Bet1 and the v-SNARE Sft1 for binding to ttrans (unpublished data). We conclude that in the case of the cis-Golgi fusion reaction, the i-SNARE substitutes for a t-SNARE light chain Bos1 to form a nonfunctional pseudo t-SNARE. In the case of the trans-Golgi fusion reaction, the mechanism of the i-SNARE action is not established.


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 inhibit the trans-Golgi fusion reaction. We coreconstituted stoichiometric amounts of the ttrans SNAREs (Sed5/Ykt6, Gos1) 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 Tlg1 was added at a twofold molar excess (B). Fusion activity of modified acceptor liposomes with donor vtrans Sft1 liposomes is plotted as a percentage of control fusion (control liposomes contain only t-SNAREs).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171956&req=5

fig5: i-SNAREs inhibit the trans-Golgi fusion reaction. We coreconstituted stoichiometric amounts of the ttrans SNAREs (Sed5/Ykt6, Gos1) 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 Tlg1 was added at a twofold molar excess (B). Fusion activity of modified acceptor liposomes with donor vtrans Sft1 liposomes is plotted as a percentage of control fusion (control liposomes contain only t-SNAREs).
Mentions: When the trans-Golgi fusion reaction was tested in a functional competition test, no t-SNARE subunit added in excess is capable of reversing inhibition by the i-SNAREs Bet1 and Tlg1 (Fig. 5). This means either that this class of i-SNAREs acts as pseudo v-SNAREs (binding ttrans) or that i-SNAREs bind to the vtrans/ttrans complex, forming an inactive oligomer (the noncompetitive mechanism). The pseudo v-SNARE mechanism predicts that the i-SNAREs could compete with the cognate v-SNARE. Consistent with this, the i-SNARE Bet1 is known to form a stable complex with Sed5, Gos1, and Ykt6, which has been isolated from immunoprecipitates of animal cell extracts (Zhang and Hong, 2001). Out of all the i-SNAREs, Bet1 has the highest inhibitory potency toward the trans-Golgi fusion reaction (a stoichiometric amount of Bet1 inhibits fusion by 70%; Fig. 3). Binding experiments with the soluble recombinant proteins were inconclusive in further testing the possible pseudo v-SNARE mechanism. We observed no competition between the i-SNARE Bet1 and the v-SNARE Sft1 for binding to ttrans (unpublished data). We conclude that in the case of the cis-Golgi fusion reaction, the i-SNARE substitutes for a t-SNARE light chain Bos1 to form a nonfunctional pseudo t-SNARE. In the case of the trans-Golgi fusion reaction, the mechanism of the i-SNARE action is not established.

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