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Sec1p directly stimulates SNARE-mediated membrane fusion in vitro.

Scott BL, Van Komen JS, Irshad H, Liu S, Wilson KA, McNew JA - J. Cell Biol. (2004)

Bottom Line: We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae.Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells.Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, Rice University, Houston, TX, USA.

ABSTRACT
Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

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Sec1p stimulates in vitro fusion. Recombinant Sec1p was added to an in vitro fusion assay containing reduced levels of SNARE proteins. 10 μl of t-SNARE liposomes (Sso1p/Sec9c, ∼19.5 μg, ∼215 pmol of t-SNARE complex proteins, ∼22.5 nmol lipid) was mixed with 85 μl of recombinant His6-Sec1p (∼12.8 μg, ∼150 pmol, closed circles) or buffer A200 (open circles) for ∼15 h at 4°C. 5 μl of Snc1p liposomes (∼8.3 μg, 630 pmol Snc1p and 1.95 nmol lipid) were added and the NBD fluorescence measured for 2 h in a fluorescent plate reader at 37°C. The background values (solid and dashed lines) represent an inhibited reaction containing the same components as stimulated fusion reaction in addition to the soluble domain of Snc2p to inhibit vesicle fusion. The amount of fusion at 120 min was 0.98 rounds of fusion for the Sec1p stimulated curve (closed circles), 0.74 rounds of fusion for basal fusion (open circles), compared with an inhibited background of 0.05 rounds of fusion. This experiment was repeated three additional times using independent recombinant Sec1p purifications. The average stimulation observed of the four experiments was 39.3% ± an SEM of 3.3%. In addition, each sample was fused with protein free fluorescently labeled liposomes that showed a background fusion level of 0.059 rounds of fusion (not depicted).
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fig5: Sec1p stimulates in vitro fusion. Recombinant Sec1p was added to an in vitro fusion assay containing reduced levels of SNARE proteins. 10 μl of t-SNARE liposomes (Sso1p/Sec9c, ∼19.5 μg, ∼215 pmol of t-SNARE complex proteins, ∼22.5 nmol lipid) was mixed with 85 μl of recombinant His6-Sec1p (∼12.8 μg, ∼150 pmol, closed circles) or buffer A200 (open circles) for ∼15 h at 4°C. 5 μl of Snc1p liposomes (∼8.3 μg, 630 pmol Snc1p and 1.95 nmol lipid) were added and the NBD fluorescence measured for 2 h in a fluorescent plate reader at 37°C. The background values (solid and dashed lines) represent an inhibited reaction containing the same components as stimulated fusion reaction in addition to the soluble domain of Snc2p to inhibit vesicle fusion. The amount of fusion at 120 min was 0.98 rounds of fusion for the Sec1p stimulated curve (closed circles), 0.74 rounds of fusion for basal fusion (open circles), compared with an inhibited background of 0.05 rounds of fusion. This experiment was repeated three additional times using independent recombinant Sec1p purifications. The average stimulation observed of the four experiments was 39.3% ± an SEM of 3.3%. In addition, each sample was fused with protein free fluorescently labeled liposomes that showed a background fusion level of 0.059 rounds of fusion (not depicted).

Mentions: Recombinant Sec1p was added to in vitro fusion assays to determine its effects on fusion. Fusion was modestly stimulated when recombinant Sec1p was mixed for 12–15 h at 4°C with proteoliposomes that contained the t-SNARE complex Sso1p/Sec9c before the addition of fluorescently labeled v-SNARE liposomes containing Snc1p (Fig. 5). Due to the low concentration of Sec1p (<0.2 mg/ml), we had to reduce the overall amount of t-SNARE containing liposomes in the fusion assay and double the reaction volume. Even with these adaptations, we were unable to add Sec1p in excess of the t-SNARE complex. Sec1p was added at a molar ratio of ∼0.7. Under these conditions, the fusion obtained with Sso1p/Sec9c;Snc1p with buffer added instead of Sec1p (Fig. 5, open circles) was roughly 0.75 rounds of fusion at 120 min. The presence of Sec1p stimulated fusion to ∼1.0 round of fusion (Fig. 5, closed circles). The Sec1p mediated stimulation is SNARE dependent because soluble Snc2p completely inhibits fusion (Fig. 5, solid and dashed lines). The level of Sec1p stimulation was 39.3 ± 3.3% (mean ± SEM) above the background subtracted buffer controls for four independent preparations of Sec1p.


Sec1p directly stimulates SNARE-mediated membrane fusion in vitro.

Scott BL, Van Komen JS, Irshad H, Liu S, Wilson KA, McNew JA - J. Cell Biol. (2004)

Sec1p stimulates in vitro fusion. Recombinant Sec1p was added to an in vitro fusion assay containing reduced levels of SNARE proteins. 10 μl of t-SNARE liposomes (Sso1p/Sec9c, ∼19.5 μg, ∼215 pmol of t-SNARE complex proteins, ∼22.5 nmol lipid) was mixed with 85 μl of recombinant His6-Sec1p (∼12.8 μg, ∼150 pmol, closed circles) or buffer A200 (open circles) for ∼15 h at 4°C. 5 μl of Snc1p liposomes (∼8.3 μg, 630 pmol Snc1p and 1.95 nmol lipid) were added and the NBD fluorescence measured for 2 h in a fluorescent plate reader at 37°C. The background values (solid and dashed lines) represent an inhibited reaction containing the same components as stimulated fusion reaction in addition to the soluble domain of Snc2p to inhibit vesicle fusion. The amount of fusion at 120 min was 0.98 rounds of fusion for the Sec1p stimulated curve (closed circles), 0.74 rounds of fusion for basal fusion (open circles), compared with an inhibited background of 0.05 rounds of fusion. This experiment was repeated three additional times using independent recombinant Sec1p purifications. The average stimulation observed of the four experiments was 39.3% ± an SEM of 3.3%. In addition, each sample was fused with protein free fluorescently labeled liposomes that showed a background fusion level of 0.059 rounds of fusion (not depicted).
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fig5: Sec1p stimulates in vitro fusion. Recombinant Sec1p was added to an in vitro fusion assay containing reduced levels of SNARE proteins. 10 μl of t-SNARE liposomes (Sso1p/Sec9c, ∼19.5 μg, ∼215 pmol of t-SNARE complex proteins, ∼22.5 nmol lipid) was mixed with 85 μl of recombinant His6-Sec1p (∼12.8 μg, ∼150 pmol, closed circles) or buffer A200 (open circles) for ∼15 h at 4°C. 5 μl of Snc1p liposomes (∼8.3 μg, 630 pmol Snc1p and 1.95 nmol lipid) were added and the NBD fluorescence measured for 2 h in a fluorescent plate reader at 37°C. The background values (solid and dashed lines) represent an inhibited reaction containing the same components as stimulated fusion reaction in addition to the soluble domain of Snc2p to inhibit vesicle fusion. The amount of fusion at 120 min was 0.98 rounds of fusion for the Sec1p stimulated curve (closed circles), 0.74 rounds of fusion for basal fusion (open circles), compared with an inhibited background of 0.05 rounds of fusion. This experiment was repeated three additional times using independent recombinant Sec1p purifications. The average stimulation observed of the four experiments was 39.3% ± an SEM of 3.3%. In addition, each sample was fused with protein free fluorescently labeled liposomes that showed a background fusion level of 0.059 rounds of fusion (not depicted).
Mentions: Recombinant Sec1p was added to in vitro fusion assays to determine its effects on fusion. Fusion was modestly stimulated when recombinant Sec1p was mixed for 12–15 h at 4°C with proteoliposomes that contained the t-SNARE complex Sso1p/Sec9c before the addition of fluorescently labeled v-SNARE liposomes containing Snc1p (Fig. 5). Due to the low concentration of Sec1p (<0.2 mg/ml), we had to reduce the overall amount of t-SNARE containing liposomes in the fusion assay and double the reaction volume. Even with these adaptations, we were unable to add Sec1p in excess of the t-SNARE complex. Sec1p was added at a molar ratio of ∼0.7. Under these conditions, the fusion obtained with Sso1p/Sec9c;Snc1p with buffer added instead of Sec1p (Fig. 5, open circles) was roughly 0.75 rounds of fusion at 120 min. The presence of Sec1p stimulated fusion to ∼1.0 round of fusion (Fig. 5, closed circles). The Sec1p mediated stimulation is SNARE dependent because soluble Snc2p completely inhibits fusion (Fig. 5, solid and dashed lines). The level of Sec1p stimulation was 39.3 ± 3.3% (mean ± SEM) above the background subtracted buffer controls for four independent preparations of Sec1p.

Bottom Line: We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae.Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells.Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Cell Biology, Rice University, Houston, TX, USA.

ABSTRACT
Sec1 proteins are critical players in membrane trafficking, yet their precise role remains unknown. We have examined the role of Sec1p in the regulation of post-Golgi secretion in Saccharomyces cerevisiae. Indirect immunofluorescence shows that endogenous Sec1p is found primarily at the bud neck in newly budded cells and in patches broadly distributed within the plasma membrane in unbudded cells. Recombinant Sec1p binds strongly to the t-SNARE complex (Sso1p/Sec9c) as well as to the fully assembled ternary SNARE complex (Sso1p/Sec9c;Snc2p), but also binds weakly to free Sso1p. We used recombinant Sec1p to test Sec1p function using a well-characterized SNARE-mediated membrane fusion assay. The addition of Sec1p to a traditional in vitro fusion assay moderately stimulates fusion; however, when Sec1p is allowed to bind to SNAREs before reconstitution, significantly more Sec1p binding is detected and fusion is stimulated in a concentration-dependent manner. These data strongly argue that Sec1p directly stimulates SNARE-mediated membrane fusion.

Show MeSH
Related in: MedlinePlus