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A t-SNARE of the endocytic pathway must be activated for fusion.

Paumet F, Brügger B, Parlati F, McNew JA, Söllner TH, Rothman JE - J. Cell Biol. (2001)

Bottom Line: The t-SNARE in a late Golgi compartment (Tlg2p) syntaxin is required for endocytosis and localization of cycling proteins to the late Golgi compartment in yeast.Activation can be mediated by a peptide derived from the v-SNARE, which likely bypasses additional regulatory proteins in the cell.Locking t-SNAREs creates the potential for spatial and temporal regulation of fusion by signaling processes that unleash their fusion capacity.

View Article: PubMed Central - PubMed

Affiliation: Cellular Biochemistry and Biophysics Program, Memorial Sloan Kettering Cancer Center, New York, NY 10021, USA.

ABSTRACT
The t-SNARE in a late Golgi compartment (Tlg2p) syntaxin is required for endocytosis and localization of cycling proteins to the late Golgi compartment in yeast. We show here that Tlg2p assembles with two light chains, Tlg1p and Vti1p, to form a functional t-SNARE that mediates fusion, specifically with the v-SNAREs Snc1p and Snc2p. In vitro, this t-SNARE is inert, locked in a nonfunctional state, unless it is activated for fusion. Activation can be mediated by a peptide derived from the v-SNARE, which likely bypasses additional regulatory proteins in the cell. Locking t-SNAREs creates the potential for spatial and temporal regulation of fusion by signaling processes that unleash their fusion capacity.

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Specificity of fusion between the endocytic t-SNARE and all potential v-SNAREs. (A) Fusion between reconstituted t-SNARE liposomes and reconstituted potential v-SNARE liposomes in the presence of Snc2-C peptide. Snc2-C-pept (3.5 nmol per reaction) was added to all reactions. (B) Fusion between reconstituted t-SNARE liposomes and reconstituted potential v-SNARE liposomes in the presence of each corresponding peptide. Peptides corresponding to the potential v-SNARE (Table I; 3.5 nmol per reaction) were added to all reactions. The extent of fusion at 120 min was normalized to the amount of cognate v-SNARE fusion (Snc2p; black bars). Fusion with protein free liposomes (0.0434 rounds) was subtracted from all the fusion results. The maximal extent of fusion (100%) with Snc2p donor liposomes and snc2-C-pept was 0.977 rounds (A), and 0.934 rounds (B). Ykt6p was anchored to the membrane by the attachment of a synthetic geranylgeranyl lipid anchor as described (McNew et al., 2000b).
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fig6: Specificity of fusion between the endocytic t-SNARE and all potential v-SNAREs. (A) Fusion between reconstituted t-SNARE liposomes and reconstituted potential v-SNARE liposomes in the presence of Snc2-C peptide. Snc2-C-pept (3.5 nmol per reaction) was added to all reactions. (B) Fusion between reconstituted t-SNARE liposomes and reconstituted potential v-SNARE liposomes in the presence of each corresponding peptide. Peptides corresponding to the potential v-SNARE (Table I; 3.5 nmol per reaction) were added to all reactions. The extent of fusion at 120 min was normalized to the amount of cognate v-SNARE fusion (Snc2p; black bars). Fusion with protein free liposomes (0.0434 rounds) was subtracted from all the fusion results. The maximal extent of fusion (100%) with Snc2p donor liposomes and snc2-C-pept was 0.977 rounds (A), and 0.934 rounds (B). Ykt6p was anchored to the membrane by the attachment of a synthetic geranylgeranyl lipid anchor as described (McNew et al., 2000b).

Mentions: The fusion activity of all of the potential v-SNAREs encoded in the yeast genome was tested by independently reconstituting each SNARE into donor liposomes and incubating them with Tlg2p/Tlg1p,Vti1p t-SNARE acceptor liposomes. Fusion was tested both in the presence or absence of the snc2-C peptide. No fusion activity was observed without the snc2-C peptide with any potential v-SNARE (unpublished data). In the presence of snc2-C peptide, significant fusion was seen with both Snc1p and Snc2p as v-SNAREs, but not with any other potential v-SNARE (Fig. 6 A). Snc2p is very similar to Snc1p and they are largely interchangeable functionally, since only when both genes are deleted is viability compromised (Protopopov et al., 1993). Each potential v-SNARE was also tested for fusion in the presence of a C-peptide based upon its own sequence (Table I). Even so, only the Snc2p and Snc1p liposomes were able to fuse with Tlg2p/Tlg1p,Vti1p acceptor liposomes (Fig. 6 B).


A t-SNARE of the endocytic pathway must be activated for fusion.

Paumet F, Brügger B, Parlati F, McNew JA, Söllner TH, Rothman JE - J. Cell Biol. (2001)

Specificity of fusion between the endocytic t-SNARE and all potential v-SNAREs. (A) Fusion between reconstituted t-SNARE liposomes and reconstituted potential v-SNARE liposomes in the presence of Snc2-C peptide. Snc2-C-pept (3.5 nmol per reaction) was added to all reactions. (B) Fusion between reconstituted t-SNARE liposomes and reconstituted potential v-SNARE liposomes in the presence of each corresponding peptide. Peptides corresponding to the potential v-SNARE (Table I; 3.5 nmol per reaction) were added to all reactions. The extent of fusion at 120 min was normalized to the amount of cognate v-SNARE fusion (Snc2p; black bars). Fusion with protein free liposomes (0.0434 rounds) was subtracted from all the fusion results. The maximal extent of fusion (100%) with Snc2p donor liposomes and snc2-C-pept was 0.977 rounds (A), and 0.934 rounds (B). Ykt6p was anchored to the membrane by the attachment of a synthetic geranylgeranyl lipid anchor as described (McNew et al., 2000b).
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Related In: Results  -  Collection

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fig6: Specificity of fusion between the endocytic t-SNARE and all potential v-SNAREs. (A) Fusion between reconstituted t-SNARE liposomes and reconstituted potential v-SNARE liposomes in the presence of Snc2-C peptide. Snc2-C-pept (3.5 nmol per reaction) was added to all reactions. (B) Fusion between reconstituted t-SNARE liposomes and reconstituted potential v-SNARE liposomes in the presence of each corresponding peptide. Peptides corresponding to the potential v-SNARE (Table I; 3.5 nmol per reaction) were added to all reactions. The extent of fusion at 120 min was normalized to the amount of cognate v-SNARE fusion (Snc2p; black bars). Fusion with protein free liposomes (0.0434 rounds) was subtracted from all the fusion results. The maximal extent of fusion (100%) with Snc2p donor liposomes and snc2-C-pept was 0.977 rounds (A), and 0.934 rounds (B). Ykt6p was anchored to the membrane by the attachment of a synthetic geranylgeranyl lipid anchor as described (McNew et al., 2000b).
Mentions: The fusion activity of all of the potential v-SNAREs encoded in the yeast genome was tested by independently reconstituting each SNARE into donor liposomes and incubating them with Tlg2p/Tlg1p,Vti1p t-SNARE acceptor liposomes. Fusion was tested both in the presence or absence of the snc2-C peptide. No fusion activity was observed without the snc2-C peptide with any potential v-SNARE (unpublished data). In the presence of snc2-C peptide, significant fusion was seen with both Snc1p and Snc2p as v-SNAREs, but not with any other potential v-SNARE (Fig. 6 A). Snc2p is very similar to Snc1p and they are largely interchangeable functionally, since only when both genes are deleted is viability compromised (Protopopov et al., 1993). Each potential v-SNARE was also tested for fusion in the presence of a C-peptide based upon its own sequence (Table I). Even so, only the Snc2p and Snc1p liposomes were able to fuse with Tlg2p/Tlg1p,Vti1p acceptor liposomes (Fig. 6 B).

Bottom Line: The t-SNARE in a late Golgi compartment (Tlg2p) syntaxin is required for endocytosis and localization of cycling proteins to the late Golgi compartment in yeast.Activation can be mediated by a peptide derived from the v-SNARE, which likely bypasses additional regulatory proteins in the cell.Locking t-SNAREs creates the potential for spatial and temporal regulation of fusion by signaling processes that unleash their fusion capacity.

View Article: PubMed Central - PubMed

Affiliation: Cellular Biochemistry and Biophysics Program, Memorial Sloan Kettering Cancer Center, New York, NY 10021, USA.

ABSTRACT
The t-SNARE in a late Golgi compartment (Tlg2p) syntaxin is required for endocytosis and localization of cycling proteins to the late Golgi compartment in yeast. We show here that Tlg2p assembles with two light chains, Tlg1p and Vti1p, to form a functional t-SNARE that mediates fusion, specifically with the v-SNAREs Snc1p and Snc2p. In vitro, this t-SNARE is inert, locked in a nonfunctional state, unless it is activated for fusion. Activation can be mediated by a peptide derived from the v-SNARE, which likely bypasses additional regulatory proteins in the cell. Locking t-SNAREs creates the potential for spatial and temporal regulation of fusion by signaling processes that unleash their fusion capacity.

Show MeSH