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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 for the cis-Golgi fusion reaction. (A) Increasing concentrations of candidate i-SNAREs were incorporated into acceptor liposomes containing the tcis complex. (B) Proteoliposomes were analyzed by SDS-PAGE and Coomassie blue staining. The position of each candidate i-SNARE is indicated by asterisks. The stoichiometry of proteins in liposomes was determined by densitometry. (C) The resulting acceptor liposomes were incubated with donor liposomes containing the v-SNARE Bet1, and relative fusion activities are plotted as a function of the molar ratio of the candidate i-SNARE to the t-SNARE.
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fig2: i-SNAREs for the cis-Golgi fusion reaction. (A) Increasing concentrations of candidate i-SNAREs were incorporated into acceptor liposomes containing the tcis complex. (B) Proteoliposomes were analyzed by SDS-PAGE and Coomassie blue staining. The position of each candidate i-SNARE is indicated by asterisks. The stoichiometry of proteins in liposomes was determined by densitometry. (C) The resulting acceptor liposomes were incubated with donor liposomes containing the v-SNARE Bet1, and relative fusion activities are plotted as a function of the molar ratio of the candidate i-SNARE to the t-SNARE.

Mentions: Both the cis- and trans-Golgi fusion reactions were tested against the potential i-SNAREs that are known to form complexes in solution with Sed5 (Tsui et al., 2001). The cis-Golgi fusion reaction (tcis = Sed5/Sec22, Bos1; vcis = Bet1) is strongly inhibited by the ttrans light chain Gos1 (K50 = 0.8) and the TGN/endosome-localized light chain Tlg1 (syntaxin 6; Holthuis et al., 1998; Bock et al., 2001; Paumet et al., 2001; K50 = 1.0), and is significantly inhibited by the vtrans Sft1 (K50 = 2.0; Fig. 2). Typically, a threefold molar excess of i-SNAREs inhibited 90% of the fusion activity. The ttrans light chain Ykt6, the Sed5-interacting SNARE Vti1 (localized both to the Golgi and to a prevacuolar compartment; Nichols and Pelham, 1998), and the yeast homologue of v-SNARE synaptobrevin Snc1 (Protopopov et al., 1993) had no significant effect (Fig. 2).


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 for the cis-Golgi fusion reaction. (A) Increasing concentrations of candidate i-SNAREs were incorporated into acceptor liposomes containing the tcis complex. (B) Proteoliposomes were analyzed by SDS-PAGE and Coomassie blue staining. The position of each candidate i-SNARE is indicated by asterisks. The stoichiometry of proteins in liposomes was determined by densitometry. (C) The resulting acceptor liposomes were incubated with donor liposomes containing the v-SNARE Bet1, and relative fusion activities are plotted as a function of the molar ratio of the candidate i-SNARE to the t-SNARE.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: i-SNAREs for the cis-Golgi fusion reaction. (A) Increasing concentrations of candidate i-SNAREs were incorporated into acceptor liposomes containing the tcis complex. (B) Proteoliposomes were analyzed by SDS-PAGE and Coomassie blue staining. The position of each candidate i-SNARE is indicated by asterisks. The stoichiometry of proteins in liposomes was determined by densitometry. (C) The resulting acceptor liposomes were incubated with donor liposomes containing the v-SNARE Bet1, and relative fusion activities are plotted as a function of the molar ratio of the candidate i-SNARE to the t-SNARE.
Mentions: Both the cis- and trans-Golgi fusion reactions were tested against the potential i-SNAREs that are known to form complexes in solution with Sed5 (Tsui et al., 2001). The cis-Golgi fusion reaction (tcis = Sed5/Sec22, Bos1; vcis = Bet1) is strongly inhibited by the ttrans light chain Gos1 (K50 = 0.8) and the TGN/endosome-localized light chain Tlg1 (syntaxin 6; Holthuis et al., 1998; Bock et al., 2001; Paumet et al., 2001; K50 = 1.0), and is significantly inhibited by the vtrans Sft1 (K50 = 2.0; Fig. 2). Typically, a threefold molar excess of i-SNAREs inhibited 90% of the fusion activity. The ttrans light chain Ykt6, the Sed5-interacting SNARE Vti1 (localized both to the Golgi and to a prevacuolar compartment; Nichols and Pelham, 1998), and the yeast homologue of v-SNARE synaptobrevin Snc1 (Protopopov et al., 1993) had no significant effect (Fig. 2).

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