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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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Immunolocalization of endogenous Sec1p. Sec1p localizes to patches on the plasma membrane. (A) Differential interference contrast (DIC) image of S. cerevisiae. (B) Endogenous Sec1p is imaged in the field of cells shown in A using a polyclonal anti-Sec1p antibody. Arrowheads denote newly emerged buds. (C–V) Individual cells in different stages of the cell cycle are imaged: Small, unbudded cells (C–J), small-budded cells (K–R), and a large-budded cell (S–V). DIC images (C, G, K, O, and S) and indirect immunofluorescence images are shown for each cell. Sso1p localization was determined by staining a HA-tagged Sso1p with anti-HA (D, H, L, P, and T). Endogenous Sec1p localization in individual cells is illustrated (E, I, M, Q, and U) and a merge of both Sso1p and Sec1p staining is imaged (F, J, N, R, and V). We determined that 71 ± 15% of endogenous Sec1p colocalizes with Sso1p-HA (n = 62 cells) when total cell area is examined. Bars, 5 μm.
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fig2: Immunolocalization of endogenous Sec1p. Sec1p localizes to patches on the plasma membrane. (A) Differential interference contrast (DIC) image of S. cerevisiae. (B) Endogenous Sec1p is imaged in the field of cells shown in A using a polyclonal anti-Sec1p antibody. Arrowheads denote newly emerged buds. (C–V) Individual cells in different stages of the cell cycle are imaged: Small, unbudded cells (C–J), small-budded cells (K–R), and a large-budded cell (S–V). DIC images (C, G, K, O, and S) and indirect immunofluorescence images are shown for each cell. Sso1p localization was determined by staining a HA-tagged Sso1p with anti-HA (D, H, L, P, and T). Endogenous Sec1p localization in individual cells is illustrated (E, I, M, Q, and U) and a merge of both Sso1p and Sec1p staining is imaged (F, J, N, R, and V). We determined that 71 ± 15% of endogenous Sec1p colocalizes with Sso1p-HA (n = 62 cells) when total cell area is examined. Bars, 5 μm.

Mentions: pAbs raised against recombinant Sec1p allowed us to determine the localization of endogenous Sec1p, which is predicted to be a soluble protein with no physical attachments to the membrane. Our analysis suggests that Sec1p is mostly localized to the plasma membrane and broadly distributed as patches throughout the plasma membrane (Fig. 2). This localization is very similar to the plasma membrane SNAREs Sso1p (Fig. 2, D, H, L, P, and T) and Sec9p (Brennwald et al., 1994). In fact, Sec1p significantly colocalizes with Sso1p (Fig. 2, F, J, N, R, and V). Although Sec1p is seen in all parts of the plasma membrane in unbudded cells (Fig. 2, E and I), it seems to be concentrated in the bud neck of newly budded cells (Fig. 2 B, arrowheads; Fig. 2, M, Q, and U).


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)

Immunolocalization of endogenous Sec1p. Sec1p localizes to patches on the plasma membrane. (A) Differential interference contrast (DIC) image of S. cerevisiae. (B) Endogenous Sec1p is imaged in the field of cells shown in A using a polyclonal anti-Sec1p antibody. Arrowheads denote newly emerged buds. (C–V) Individual cells in different stages of the cell cycle are imaged: Small, unbudded cells (C–J), small-budded cells (K–R), and a large-budded cell (S–V). DIC images (C, G, K, O, and S) and indirect immunofluorescence images are shown for each cell. Sso1p localization was determined by staining a HA-tagged Sso1p with anti-HA (D, H, L, P, and T). Endogenous Sec1p localization in individual cells is illustrated (E, I, M, Q, and U) and a merge of both Sso1p and Sec1p staining is imaged (F, J, N, R, and V). We determined that 71 ± 15% of endogenous Sec1p colocalizes with Sso1p-HA (n = 62 cells) when total cell area is examined. Bars, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172507&req=5

fig2: Immunolocalization of endogenous Sec1p. Sec1p localizes to patches on the plasma membrane. (A) Differential interference contrast (DIC) image of S. cerevisiae. (B) Endogenous Sec1p is imaged in the field of cells shown in A using a polyclonal anti-Sec1p antibody. Arrowheads denote newly emerged buds. (C–V) Individual cells in different stages of the cell cycle are imaged: Small, unbudded cells (C–J), small-budded cells (K–R), and a large-budded cell (S–V). DIC images (C, G, K, O, and S) and indirect immunofluorescence images are shown for each cell. Sso1p localization was determined by staining a HA-tagged Sso1p with anti-HA (D, H, L, P, and T). Endogenous Sec1p localization in individual cells is illustrated (E, I, M, Q, and U) and a merge of both Sso1p and Sec1p staining is imaged (F, J, N, R, and V). We determined that 71 ± 15% of endogenous Sec1p colocalizes with Sso1p-HA (n = 62 cells) when total cell area is examined. Bars, 5 μm.
Mentions: pAbs raised against recombinant Sec1p allowed us to determine the localization of endogenous Sec1p, which is predicted to be a soluble protein with no physical attachments to the membrane. Our analysis suggests that Sec1p is mostly localized to the plasma membrane and broadly distributed as patches throughout the plasma membrane (Fig. 2). This localization is very similar to the plasma membrane SNAREs Sso1p (Fig. 2, D, H, L, P, and T) and Sec9p (Brennwald et al., 1994). In fact, Sec1p significantly colocalizes with Sso1p (Fig. 2, F, J, N, R, and V). Although Sec1p is seen in all parts of the plasma membrane in unbudded cells (Fig. 2, E and I), it seems to be concentrated in the bud neck of newly budded cells (Fig. 2 B, arrowheads; Fig. 2, M, Q, and U).

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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