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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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SNARE-bound Sec1p strongly stimulates in vitro fusion. A twofold dilution series of Sec1p was bound to t-SNARE complexes (Sso1p/Sec9c) in detergent solution before vesicle reconstitution. (A) Coomassie blue–stained gel of liposomes containing Sec1p bound t-SNARE complexes. Acceptor t-SNARE liposomes containing various amounts of bound His6-Sec1p were resolved on a 10% Bis-Tris NuPAGE gel (Invitrogen) and stained with Coomassie blue. Lane 1 contains 15 μl of liposomes derived from a reaction containing His8-Sso1p (∼160 μg, ∼4.7 nmol) and 60 μg (700 pmol) of His6-Sec1p. Lanes 2–6 contains 10 μl of liposomes derived from reactions including His8-Sso1p (∼160 μg, ∼4.7 nmol), GST-Sec9c (∼430 μg, ∼7.7 nmol) and decreasing amounts of Sec1p: lane 2, ∼60, μg, ∼700 pmol; lane 3, ∼30 μg, ∼350 pmol; lane 4, ∼15 μg, ∼175 pmol; and lane 5, ∼7.5 μg, ∼88 pmol. Lane 6 contained no Sec1p. Lane 7 contains 0.6 μg (∼7 pmol) of recombinant His6-Sec1p. (B) Kinetic fusion graph of Sec1p stimulated fusion. Vesicles (45 μl) containing t-SNARE complexes without Sec1p (open circles, ∼13 μg, 145 pmol Sso1p/Sec9c and 42 nmol lipid) and t-SNARE vesicles containing the highest amount of bound Sec1p (closed circles) were mixed with fluorescently labeled vesicles containing the v-SNARE Snc1p (5 μl, ∼8.3 μg, 630 pmol Snc1p and 1.95 nmol lipid) and incubated for 120 min at 37°C in a standard fusion reaction. The extent of fusion is represented as rounds of fusion, measured as fold lipid dilution in the reaction. The background values (solid and dashed lines) represent an inhibited reaction containing the same components in addition to the soluble domain of Snc2p to inhibit vesicle fusion. The amount of fusion at 120 min was 1.32 rounds of fusion for the Sec1p stimulated curve (closed circles), 0.42 rounds of fusion for basal fusion (buffer, open circles), and the inhibited fusion background, 0.1 rounds of fusion. Sec1p stimulated fusion ∼3.8-fold in this experiment after background subtraction. (C) Average fold stimulation caused by Sec1p. The amount of stimulation by Sec1p was examined using four independent preparations of recombinant His6-Sec1p. This histogram shows that His6-Sec1p stimulates fusion by 2.7-fold on average. The mean ± SEM are represented after the subtraction of an average background of 0.105 rounds of fusion. (D) Sec1p titration showing stimulation is concentration dependent. The extent of fusion observed at 120 min and the amount of Sec1p binding detected to t-SNARE liposomes is represented for independent in vitro fusion experiments relative to the amount of Sec1p added to the reaction. Rounds of fusion at 120 min are shown on the left y axis (open circles) and the amount of Sec1p binding (relative to His8-Sso1p) is quantified on the right y axis (closed circles). Both values are plotted relative to the concentration of Sec1p added to the reaction (nM). The binding values for Sec1p were determined by quantifying the gel shown in Fig. 6 A.
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fig6: SNARE-bound Sec1p strongly stimulates in vitro fusion. A twofold dilution series of Sec1p was bound to t-SNARE complexes (Sso1p/Sec9c) in detergent solution before vesicle reconstitution. (A) Coomassie blue–stained gel of liposomes containing Sec1p bound t-SNARE complexes. Acceptor t-SNARE liposomes containing various amounts of bound His6-Sec1p were resolved on a 10% Bis-Tris NuPAGE gel (Invitrogen) and stained with Coomassie blue. Lane 1 contains 15 μl of liposomes derived from a reaction containing His8-Sso1p (∼160 μg, ∼4.7 nmol) and 60 μg (700 pmol) of His6-Sec1p. Lanes 2–6 contains 10 μl of liposomes derived from reactions including His8-Sso1p (∼160 μg, ∼4.7 nmol), GST-Sec9c (∼430 μg, ∼7.7 nmol) and decreasing amounts of Sec1p: lane 2, ∼60, μg, ∼700 pmol; lane 3, ∼30 μg, ∼350 pmol; lane 4, ∼15 μg, ∼175 pmol; and lane 5, ∼7.5 μg, ∼88 pmol. Lane 6 contained no Sec1p. Lane 7 contains 0.6 μg (∼7 pmol) of recombinant His6-Sec1p. (B) Kinetic fusion graph of Sec1p stimulated fusion. Vesicles (45 μl) containing t-SNARE complexes without Sec1p (open circles, ∼13 μg, 145 pmol Sso1p/Sec9c and 42 nmol lipid) and t-SNARE vesicles containing the highest amount of bound Sec1p (closed circles) were mixed with fluorescently labeled vesicles containing the v-SNARE Snc1p (5 μl, ∼8.3 μg, 630 pmol Snc1p and 1.95 nmol lipid) and incubated for 120 min at 37°C in a standard fusion reaction. The extent of fusion is represented as rounds of fusion, measured as fold lipid dilution in the reaction. The background values (solid and dashed lines) represent an inhibited reaction containing the same components in addition to the soluble domain of Snc2p to inhibit vesicle fusion. The amount of fusion at 120 min was 1.32 rounds of fusion for the Sec1p stimulated curve (closed circles), 0.42 rounds of fusion for basal fusion (buffer, open circles), and the inhibited fusion background, 0.1 rounds of fusion. Sec1p stimulated fusion ∼3.8-fold in this experiment after background subtraction. (C) Average fold stimulation caused by Sec1p. The amount of stimulation by Sec1p was examined using four independent preparations of recombinant His6-Sec1p. This histogram shows that His6-Sec1p stimulates fusion by 2.7-fold on average. The mean ± SEM are represented after the subtraction of an average background of 0.105 rounds of fusion. (D) Sec1p titration showing stimulation is concentration dependent. The extent of fusion observed at 120 min and the amount of Sec1p binding detected to t-SNARE liposomes is represented for independent in vitro fusion experiments relative to the amount of Sec1p added to the reaction. Rounds of fusion at 120 min are shown on the left y axis (open circles) and the amount of Sec1p binding (relative to His8-Sso1p) is quantified on the right y axis (closed circles). Both values are plotted relative to the concentration of Sec1p added to the reaction (nM). The binding values for Sec1p were determined by quantifying the gel shown in Fig. 6 A.

Mentions: Sec1p modestly stimulates fusion when added directly to an in vitro fusion assay. Given that Sec1p binds to t-SNARE complexes in detergent (Figs. 3 and 4), we determined if similar Sec1p containing complexes could be reconstituted into liposomes. His6-Sec1p was mixed with His8-Sso1p or His8-Sso1p/GST-Sec9c t-SNARE complexes in the presence of 0.6% octyl-glucoside for ∼15 h at 4°C. The overall amount of t-SNARE complex protein added to the reconstitution was reduced to favor the ratio of Sec1p to t-SNARE complex. The detergent solutions were then used to resuspend a lipid film to form unlabeled t-SNARE proteoliposomes. Vesicles were isolated by flotation in a density gradient and analyzed for the presence of specifically bound Sec1p by SDS-PAGE and Coomassie blue staining (Fig. 6 A). We found that significant amounts of His6-Sec1p were isolated with liposomes containing t-SNARE complexes (Fig. 6 A, lanes 2–5); whereas little or no Sec1p was isolated with liposomes containing free His8-Sso1p (Fig. 6 A, lane 1) or protein free liposomes (not depicted).


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)

SNARE-bound Sec1p strongly stimulates in vitro fusion. A twofold dilution series of Sec1p was bound to t-SNARE complexes (Sso1p/Sec9c) in detergent solution before vesicle reconstitution. (A) Coomassie blue–stained gel of liposomes containing Sec1p bound t-SNARE complexes. Acceptor t-SNARE liposomes containing various amounts of bound His6-Sec1p were resolved on a 10% Bis-Tris NuPAGE gel (Invitrogen) and stained with Coomassie blue. Lane 1 contains 15 μl of liposomes derived from a reaction containing His8-Sso1p (∼160 μg, ∼4.7 nmol) and 60 μg (700 pmol) of His6-Sec1p. Lanes 2–6 contains 10 μl of liposomes derived from reactions including His8-Sso1p (∼160 μg, ∼4.7 nmol), GST-Sec9c (∼430 μg, ∼7.7 nmol) and decreasing amounts of Sec1p: lane 2, ∼60, μg, ∼700 pmol; lane 3, ∼30 μg, ∼350 pmol; lane 4, ∼15 μg, ∼175 pmol; and lane 5, ∼7.5 μg, ∼88 pmol. Lane 6 contained no Sec1p. Lane 7 contains 0.6 μg (∼7 pmol) of recombinant His6-Sec1p. (B) Kinetic fusion graph of Sec1p stimulated fusion. Vesicles (45 μl) containing t-SNARE complexes without Sec1p (open circles, ∼13 μg, 145 pmol Sso1p/Sec9c and 42 nmol lipid) and t-SNARE vesicles containing the highest amount of bound Sec1p (closed circles) were mixed with fluorescently labeled vesicles containing the v-SNARE Snc1p (5 μl, ∼8.3 μg, 630 pmol Snc1p and 1.95 nmol lipid) and incubated for 120 min at 37°C in a standard fusion reaction. The extent of fusion is represented as rounds of fusion, measured as fold lipid dilution in the reaction. The background values (solid and dashed lines) represent an inhibited reaction containing the same components in addition to the soluble domain of Snc2p to inhibit vesicle fusion. The amount of fusion at 120 min was 1.32 rounds of fusion for the Sec1p stimulated curve (closed circles), 0.42 rounds of fusion for basal fusion (buffer, open circles), and the inhibited fusion background, 0.1 rounds of fusion. Sec1p stimulated fusion ∼3.8-fold in this experiment after background subtraction. (C) Average fold stimulation caused by Sec1p. The amount of stimulation by Sec1p was examined using four independent preparations of recombinant His6-Sec1p. This histogram shows that His6-Sec1p stimulates fusion by 2.7-fold on average. The mean ± SEM are represented after the subtraction of an average background of 0.105 rounds of fusion. (D) Sec1p titration showing stimulation is concentration dependent. The extent of fusion observed at 120 min and the amount of Sec1p binding detected to t-SNARE liposomes is represented for independent in vitro fusion experiments relative to the amount of Sec1p added to the reaction. Rounds of fusion at 120 min are shown on the left y axis (open circles) and the amount of Sec1p binding (relative to His8-Sso1p) is quantified on the right y axis (closed circles). Both values are plotted relative to the concentration of Sec1p added to the reaction (nM). The binding values for Sec1p were determined by quantifying the gel shown in Fig. 6 A.
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fig6: SNARE-bound Sec1p strongly stimulates in vitro fusion. A twofold dilution series of Sec1p was bound to t-SNARE complexes (Sso1p/Sec9c) in detergent solution before vesicle reconstitution. (A) Coomassie blue–stained gel of liposomes containing Sec1p bound t-SNARE complexes. Acceptor t-SNARE liposomes containing various amounts of bound His6-Sec1p were resolved on a 10% Bis-Tris NuPAGE gel (Invitrogen) and stained with Coomassie blue. Lane 1 contains 15 μl of liposomes derived from a reaction containing His8-Sso1p (∼160 μg, ∼4.7 nmol) and 60 μg (700 pmol) of His6-Sec1p. Lanes 2–6 contains 10 μl of liposomes derived from reactions including His8-Sso1p (∼160 μg, ∼4.7 nmol), GST-Sec9c (∼430 μg, ∼7.7 nmol) and decreasing amounts of Sec1p: lane 2, ∼60, μg, ∼700 pmol; lane 3, ∼30 μg, ∼350 pmol; lane 4, ∼15 μg, ∼175 pmol; and lane 5, ∼7.5 μg, ∼88 pmol. Lane 6 contained no Sec1p. Lane 7 contains 0.6 μg (∼7 pmol) of recombinant His6-Sec1p. (B) Kinetic fusion graph of Sec1p stimulated fusion. Vesicles (45 μl) containing t-SNARE complexes without Sec1p (open circles, ∼13 μg, 145 pmol Sso1p/Sec9c and 42 nmol lipid) and t-SNARE vesicles containing the highest amount of bound Sec1p (closed circles) were mixed with fluorescently labeled vesicles containing the v-SNARE Snc1p (5 μl, ∼8.3 μg, 630 pmol Snc1p and 1.95 nmol lipid) and incubated for 120 min at 37°C in a standard fusion reaction. The extent of fusion is represented as rounds of fusion, measured as fold lipid dilution in the reaction. The background values (solid and dashed lines) represent an inhibited reaction containing the same components in addition to the soluble domain of Snc2p to inhibit vesicle fusion. The amount of fusion at 120 min was 1.32 rounds of fusion for the Sec1p stimulated curve (closed circles), 0.42 rounds of fusion for basal fusion (buffer, open circles), and the inhibited fusion background, 0.1 rounds of fusion. Sec1p stimulated fusion ∼3.8-fold in this experiment after background subtraction. (C) Average fold stimulation caused by Sec1p. The amount of stimulation by Sec1p was examined using four independent preparations of recombinant His6-Sec1p. This histogram shows that His6-Sec1p stimulates fusion by 2.7-fold on average. The mean ± SEM are represented after the subtraction of an average background of 0.105 rounds of fusion. (D) Sec1p titration showing stimulation is concentration dependent. The extent of fusion observed at 120 min and the amount of Sec1p binding detected to t-SNARE liposomes is represented for independent in vitro fusion experiments relative to the amount of Sec1p added to the reaction. Rounds of fusion at 120 min are shown on the left y axis (open circles) and the amount of Sec1p binding (relative to His8-Sso1p) is quantified on the right y axis (closed circles). Both values are plotted relative to the concentration of Sec1p added to the reaction (nM). The binding values for Sec1p were determined by quantifying the gel shown in Fig. 6 A.
Mentions: Sec1p modestly stimulates fusion when added directly to an in vitro fusion assay. Given that Sec1p binds to t-SNARE complexes in detergent (Figs. 3 and 4), we determined if similar Sec1p containing complexes could be reconstituted into liposomes. His6-Sec1p was mixed with His8-Sso1p or His8-Sso1p/GST-Sec9c t-SNARE complexes in the presence of 0.6% octyl-glucoside for ∼15 h at 4°C. The overall amount of t-SNARE complex protein added to the reconstitution was reduced to favor the ratio of Sec1p to t-SNARE complex. The detergent solutions were then used to resuspend a lipid film to form unlabeled t-SNARE proteoliposomes. Vesicles were isolated by flotation in a density gradient and analyzed for the presence of specifically bound Sec1p by SDS-PAGE and Coomassie blue staining (Fig. 6 A). We found that significant amounts of His6-Sec1p were isolated with liposomes containing t-SNARE complexes (Fig. 6 A, lanes 2–5); whereas little or no Sec1p was isolated with liposomes containing free His8-Sso1p (Fig. 6 A, lane 1) or protein free liposomes (not depicted).

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