Limits...
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.

Show MeSH

Related in: MedlinePlus

i-SNAREs sharpen the specificity of membrane fusion in the reconstituted Golgi- mimetic system. The Golgi-mimetic mixture of SNAREs was reconstituted into acceptor liposomes as described in the Materials and methods. Stoichiometry of SNAREs in the CGN, five Golgi cisternae (C1–C5), and the TGN was determined by quantitative immuno-EM in mammalian cells. The percentage of total immunogold particles for individual SNAREs in each cisternae (see Table I in Volchuk et al., 2004) was normalized to relative molar amounts of individual SNAREs in whole cells determined by quantitative immunoprecipitation (see Fig. 7 in Volchuk et al., 2004). Syntaxin 5 was used as a standard for normalization. (A and B) Acceptor liposomes mimicking the SNARE composition of individual cisternae, but lacking v-SNAREs Bet1 and Sft1, respectively, were generated by using the molar ratios of SNAREs in every compartment. In addition, individual i-SNAREs were omitted from the reconstitution (shown as −[i-SNARE]). Acceptor liposomes were mixed with either (A) Bet1- or (B) Sft1-containing donor liposomes, and fusion was monitored as described in the Materials and methods. The specific compositions of Golgi-mimetic liposomes are as follows: (A) Complete (Sed5, Bos1, Sec22, Gos1, Ykt6, Sft1); −[i-Gos1] (Sed5, Bos1, Sec22, Ykt6, Sft1); −[i-Sft1] (Sed5, Bos1, Sec22, Gos1, Ykt6); and cis t-SNARE only (Sed5, Sec22, Bos1). (B) Complete (Sed5, Bos1, Sec22, Bet1, Gos1, Ykt6); −[i-Bos1] (Sed5, Sec22, Bet1, Gos1, Ykt6); −[i-Bos1, i-Bet1] (Sed5, Sec22, Gos1, Ykt6); and trans t-SNARE only (Sed5, Ykt6, Gos1).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171956&req=5

fig7: i-SNAREs sharpen the specificity of membrane fusion in the reconstituted Golgi- mimetic system. The Golgi-mimetic mixture of SNAREs was reconstituted into acceptor liposomes as described in the Materials and methods. Stoichiometry of SNAREs in the CGN, five Golgi cisternae (C1–C5), and the TGN was determined by quantitative immuno-EM in mammalian cells. The percentage of total immunogold particles for individual SNAREs in each cisternae (see Table I in Volchuk et al., 2004) was normalized to relative molar amounts of individual SNAREs in whole cells determined by quantitative immunoprecipitation (see Fig. 7 in Volchuk et al., 2004). Syntaxin 5 was used as a standard for normalization. (A and B) Acceptor liposomes mimicking the SNARE composition of individual cisternae, but lacking v-SNAREs Bet1 and Sft1, respectively, were generated by using the molar ratios of SNAREs in every compartment. In addition, individual i-SNAREs were omitted from the reconstitution (shown as −[i-SNARE]). Acceptor liposomes were mixed with either (A) Bet1- or (B) Sft1-containing donor liposomes, and fusion was monitored as described in the Materials and methods. The specific compositions of Golgi-mimetic liposomes are as follows: (A) Complete (Sed5, Bos1, Sec22, Gos1, Ykt6, Sft1); −[i-Gos1] (Sed5, Bos1, Sec22, Ykt6, Sft1); −[i-Sft1] (Sed5, Bos1, Sec22, Gos1, Ykt6); and cis t-SNARE only (Sed5, Sec22, Bos1). (B) Complete (Sed5, Bos1, Sec22, Bet1, Gos1, Ykt6); −[i-Bos1] (Sed5, Sec22, Bet1, Gos1, Ykt6); −[i-Bos1, i-Bet1] (Sed5, Sec22, Gos1, Ykt6); and trans t-SNARE only (Sed5, Ykt6, Gos1).

Mentions: To ascertain what effect, if any, i-SNAREs might have on the pattern of fusion mediated by the cis- and trans-Golgi SNAREpins, we used the liposome fusion assay to recreate the unique SNARE composition of the sequential compartments of the Golgi complex. We took advantage of the knowledge of the detailed cis/trans distributions and relative concentrations of the SNAREs in intermediate compartment/cis-Golgi network (CGN), the five Golgi cisternae (C1–C5), and the TGN established in a mammalian cell line (Volchuk et al., 2004). Because the mammalian SNAREs are orthologous to the yeast SNAREs, and in some cases have been shown to substitute for the yeast equivalents in vivo (McNew et al., 1997; Mollard and Stevens, 1998), we thought it would be reasonable to model the successive compartments of the mammalian Golgi by creating a series of liposomes of graded SNARE composition for fusion analyses, using the yeast orthologues of the mammalian SNAREs combined in the same proportions in which the mammalian proteins are present in successive Golgi compartments in the cell. The degree of similarity between yeast and mammalian SNAREs has been further validated by replacing yeast i-SNAREs with the mammalian orthologues (Fig. 6). Strikingly, the mammalian orthologue of Tlg1 (syntaxin 6) and the mammalian orthologue of Sft1 (GS15) inhibit fusion mediated by yeast tcis + vcis (Fig. 6, B and C) to a similar extent as their yeast counterparts. Similarly, the mammalian orthologue of Bet1, rBet1, and syntaxin 6 inhibit fusion mediated by yeast ttrans + vtrans (Fig. 6, E and F) even more potently than their yeast counterparts. Furthermore, both rBet1 and GS15 are functionally active and mediate fusion with yeast tcis and ttrans, respectively (unpublished data). We conclude that yeast and mammalian SNAREs from the Golgi are functionally interchangeable in the fusion assay. Therefore, Sed5 and the six Sed5-interacting SNAREs required for Golgi function (Bos1, Sec22, Bet1, Gos1, Ykt6, and Sft1) were coreconstituted into liposomes at the molar ratios approximating those in the cis/trans Golgi compartments (see Materials and methods for details; Table I and Fig. 7 of Volchuk et al., 2004). The concentration of Sed5 was kept constant in all of the Golgi-mimetic liposomes. Each Golgi-mimetic “acceptor” liposome preparation (representing successively the CGN, C1–C5, and TGN) was then tested for the efficiency of its fusion with fluorescent probe–containing “donor” liposomes containing either the vcis (Bet1) or vtrans (Sft1).


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 sharpen the specificity of membrane fusion in the reconstituted Golgi- mimetic system. The Golgi-mimetic mixture of SNAREs was reconstituted into acceptor liposomes as described in the Materials and methods. Stoichiometry of SNAREs in the CGN, five Golgi cisternae (C1–C5), and the TGN was determined by quantitative immuno-EM in mammalian cells. The percentage of total immunogold particles for individual SNAREs in each cisternae (see Table I in Volchuk et al., 2004) was normalized to relative molar amounts of individual SNAREs in whole cells determined by quantitative immunoprecipitation (see Fig. 7 in Volchuk et al., 2004). Syntaxin 5 was used as a standard for normalization. (A and B) Acceptor liposomes mimicking the SNARE composition of individual cisternae, but lacking v-SNAREs Bet1 and Sft1, respectively, were generated by using the molar ratios of SNAREs in every compartment. In addition, individual i-SNAREs were omitted from the reconstitution (shown as −[i-SNARE]). Acceptor liposomes were mixed with either (A) Bet1- or (B) Sft1-containing donor liposomes, and fusion was monitored as described in the Materials and methods. The specific compositions of Golgi-mimetic liposomes are as follows: (A) Complete (Sed5, Bos1, Sec22, Gos1, Ykt6, Sft1); −[i-Gos1] (Sed5, Bos1, Sec22, Ykt6, Sft1); −[i-Sft1] (Sed5, Bos1, Sec22, Gos1, Ykt6); and cis t-SNARE only (Sed5, Sec22, Bos1). (B) Complete (Sed5, Bos1, Sec22, Bet1, Gos1, Ykt6); −[i-Bos1] (Sed5, Sec22, Bet1, Gos1, Ykt6); −[i-Bos1, i-Bet1] (Sed5, Sec22, Gos1, Ykt6); and trans t-SNARE only (Sed5, Ykt6, Gos1).
© Copyright Policy
Related In: Results  -  Collection

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

fig7: i-SNAREs sharpen the specificity of membrane fusion in the reconstituted Golgi- mimetic system. The Golgi-mimetic mixture of SNAREs was reconstituted into acceptor liposomes as described in the Materials and methods. Stoichiometry of SNAREs in the CGN, five Golgi cisternae (C1–C5), and the TGN was determined by quantitative immuno-EM in mammalian cells. The percentage of total immunogold particles for individual SNAREs in each cisternae (see Table I in Volchuk et al., 2004) was normalized to relative molar amounts of individual SNAREs in whole cells determined by quantitative immunoprecipitation (see Fig. 7 in Volchuk et al., 2004). Syntaxin 5 was used as a standard for normalization. (A and B) Acceptor liposomes mimicking the SNARE composition of individual cisternae, but lacking v-SNAREs Bet1 and Sft1, respectively, were generated by using the molar ratios of SNAREs in every compartment. In addition, individual i-SNAREs were omitted from the reconstitution (shown as −[i-SNARE]). Acceptor liposomes were mixed with either (A) Bet1- or (B) Sft1-containing donor liposomes, and fusion was monitored as described in the Materials and methods. The specific compositions of Golgi-mimetic liposomes are as follows: (A) Complete (Sed5, Bos1, Sec22, Gos1, Ykt6, Sft1); −[i-Gos1] (Sed5, Bos1, Sec22, Ykt6, Sft1); −[i-Sft1] (Sed5, Bos1, Sec22, Gos1, Ykt6); and cis t-SNARE only (Sed5, Sec22, Bos1). (B) Complete (Sed5, Bos1, Sec22, Bet1, Gos1, Ykt6); −[i-Bos1] (Sed5, Sec22, Bet1, Gos1, Ykt6); −[i-Bos1, i-Bet1] (Sed5, Sec22, Gos1, Ykt6); and trans t-SNARE only (Sed5, Ykt6, Gos1).
Mentions: To ascertain what effect, if any, i-SNAREs might have on the pattern of fusion mediated by the cis- and trans-Golgi SNAREpins, we used the liposome fusion assay to recreate the unique SNARE composition of the sequential compartments of the Golgi complex. We took advantage of the knowledge of the detailed cis/trans distributions and relative concentrations of the SNAREs in intermediate compartment/cis-Golgi network (CGN), the five Golgi cisternae (C1–C5), and the TGN established in a mammalian cell line (Volchuk et al., 2004). Because the mammalian SNAREs are orthologous to the yeast SNAREs, and in some cases have been shown to substitute for the yeast equivalents in vivo (McNew et al., 1997; Mollard and Stevens, 1998), we thought it would be reasonable to model the successive compartments of the mammalian Golgi by creating a series of liposomes of graded SNARE composition for fusion analyses, using the yeast orthologues of the mammalian SNAREs combined in the same proportions in which the mammalian proteins are present in successive Golgi compartments in the cell. The degree of similarity between yeast and mammalian SNAREs has been further validated by replacing yeast i-SNAREs with the mammalian orthologues (Fig. 6). Strikingly, the mammalian orthologue of Tlg1 (syntaxin 6) and the mammalian orthologue of Sft1 (GS15) inhibit fusion mediated by yeast tcis + vcis (Fig. 6, B and C) to a similar extent as their yeast counterparts. Similarly, the mammalian orthologue of Bet1, rBet1, and syntaxin 6 inhibit fusion mediated by yeast ttrans + vtrans (Fig. 6, E and F) even more potently than their yeast counterparts. Furthermore, both rBet1 and GS15 are functionally active and mediate fusion with yeast tcis and ttrans, respectively (unpublished data). We conclude that yeast and mammalian SNAREs from the Golgi are functionally interchangeable in the fusion assay. Therefore, Sed5 and the six Sed5-interacting SNAREs required for Golgi function (Bos1, Sec22, Bet1, Gos1, Ykt6, and Sft1) were coreconstituted into liposomes at the molar ratios approximating those in the cis/trans Golgi compartments (see Materials and methods for details; Table I and Fig. 7 of Volchuk et al., 2004). The concentration of Sed5 was kept constant in all of the Golgi-mimetic liposomes. Each Golgi-mimetic “acceptor” liposome preparation (representing successively the CGN, C1–C5, and TGN) was then tested for the efficiency of its fusion with fluorescent probe–containing “donor” liposomes containing either the vcis (Bet1) or vtrans (Sft1).

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