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The Tlg SNARE complex is required for TGN homotypic fusion.

Brickner JH, Blanchette JM, Sipos G, Fuller RS - J. Cell Biol. (2001)

Bottom Line: Coimmunoisolation confirmed that the reaction resulted in cointegration of the two enzymes into the same bilayer.Antibody inhibition experiments coupled with antigen competition indicated a requirement for soluble NSF attachment protein receptor (SNARE) proteins Tlg1p, Tlg2p, and Vti1p in this reaction.Vps21p was sufficient if present on either the Kex2p or Ste13p membranes alone, indicative of an inherent symmetry in the reaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.

ABSTRACT
Using a new assay for membrane fusion between late Golgi/endosomal compartments, we have reconstituted a rapid, robust homotypic fusion reaction between membranes containing Kex2p and Ste13p, two enzymes resident in the yeast trans-Golgi network (TGN). Fusion was temperature, ATP, and cytosol dependent. It was inhibited by dilution, Ca+2 chelation, N-ethylmaleimide, and detergent. Coimmunoisolation confirmed that the reaction resulted in cointegration of the two enzymes into the same bilayer. Antibody inhibition experiments coupled with antigen competition indicated a requirement for soluble NSF attachment protein receptor (SNARE) proteins Tlg1p, Tlg2p, and Vti1p in this reaction. Membrane fusion also required the rab protein Vps21p. Vps21p was sufficient if present on either the Kex2p or Ste13p membranes alone, indicative of an inherent symmetry in the reaction. These results identify roles for a Tlg SNARE complex composed of Tlg1p, Tlg2p, Vti1p, and the rab Vps21p in this previously uncharacterized homotypic TGN fusion reaction.

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Characterization of in vitro TGN membrane fusion. (A) Processing of Ste13αHA requires an ATP regeneration system. Control reactions (±Kex2p) included 1.5 mM exogenous Mg-ATP, 40 mM phosphocreatine, and 0.125 mg ml−1 creatine kinase. Omission of Mg-ATP (−ATP), or phosphocreatine/creatine kinase, (−Regen) is indicated. (B) Temperature dependence of Ste13αHA processing in vitro. Reactions were performed at each temperature using lysates from either KEX2 or kex2Δ strains. (C) Dilution sensitivity of Ste13αHA processing. Reactions were diluted to the indicated extents before (Pre-reaction) or after (Post-reaction) incubation under reaction conditions. (D) Detergent sensitivity. MSS membranes from either KEX2 or kex2Δ strains were combined with MSS membranes from a Ste13αHA-expressing strain, and reactions were performed in the presence or absence of 1% Triton X-100.
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fig3: Characterization of in vitro TGN membrane fusion. (A) Processing of Ste13αHA requires an ATP regeneration system. Control reactions (±Kex2p) included 1.5 mM exogenous Mg-ATP, 40 mM phosphocreatine, and 0.125 mg ml−1 creatine kinase. Omission of Mg-ATP (−ATP), or phosphocreatine/creatine kinase, (−Regen) is indicated. (B) Temperature dependence of Ste13αHA processing in vitro. Reactions were performed at each temperature using lysates from either KEX2 or kex2Δ strains. (C) Dilution sensitivity of Ste13αHA processing. Reactions were diluted to the indicated extents before (Pre-reaction) or after (Post-reaction) incubation under reaction conditions. (D) Detergent sensitivity. MSS membranes from either KEX2 or kex2Δ strains were combined with MSS membranes from a Ste13αHA-expressing strain, and reactions were performed in the presence or absence of 1% Triton X-100.

Mentions: The cell-free membrane fusion that we observed exhibited features characteristic of other cell-free biological membrane fusion reactions. First, although Ste13αHA processing in the crude system did not require addition of exogenous ATP (Fig. 3 A), omission of the ATP regeneration system completely blocked processing (Fig. 3 A), indicating that the reaction was ATP dependent. Second, processing of Ste13αHA occurred with maximal efficiency between 25 and 30°C and was inhibited at temperatures below 15°C or above 35°C (Fig. 3 B), similar to the temperature dependence of other cell-free biological membrane fusion reactions (Baker et al., 1988; Latterich and Schekman, 1994). Third, dilution of the MSS membranes progressively inhibited Ste13αHA processing, indicating that membrane concentration was important for efficient fusion (Fig. 3 C). Fourth, addition of Triton X-100 (1% wt vol−1) completely inhibited Kex2p processing of Ste13αHA even though both Kex2p and DPAP were fully active under these conditions (unpublished data), indicating that membrane integrity was essential for efficient processing (Fig. 3 D). Furthermore, this result shows that for processing to be observed in this dilute system Kex2p must be coconcentrated along with the substrate Ste13αHA in the lumenal space of fused late Golgi membrane vesicles. Taken together, the characteristics of the reaction suggested that the observed Kex2p-dependent processing of Ste13αHA represented a biologically relevant vesicular transport or membrane fusion event between membrane compartments containing Kex2p and Ste13αHA.


The Tlg SNARE complex is required for TGN homotypic fusion.

Brickner JH, Blanchette JM, Sipos G, Fuller RS - J. Cell Biol. (2001)

Characterization of in vitro TGN membrane fusion. (A) Processing of Ste13αHA requires an ATP regeneration system. Control reactions (±Kex2p) included 1.5 mM exogenous Mg-ATP, 40 mM phosphocreatine, and 0.125 mg ml−1 creatine kinase. Omission of Mg-ATP (−ATP), or phosphocreatine/creatine kinase, (−Regen) is indicated. (B) Temperature dependence of Ste13αHA processing in vitro. Reactions were performed at each temperature using lysates from either KEX2 or kex2Δ strains. (C) Dilution sensitivity of Ste13αHA processing. Reactions were diluted to the indicated extents before (Pre-reaction) or after (Post-reaction) incubation under reaction conditions. (D) Detergent sensitivity. MSS membranes from either KEX2 or kex2Δ strains were combined with MSS membranes from a Ste13αHA-expressing strain, and reactions were performed in the presence or absence of 1% Triton X-100.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2150899&req=5

fig3: Characterization of in vitro TGN membrane fusion. (A) Processing of Ste13αHA requires an ATP regeneration system. Control reactions (±Kex2p) included 1.5 mM exogenous Mg-ATP, 40 mM phosphocreatine, and 0.125 mg ml−1 creatine kinase. Omission of Mg-ATP (−ATP), or phosphocreatine/creatine kinase, (−Regen) is indicated. (B) Temperature dependence of Ste13αHA processing in vitro. Reactions were performed at each temperature using lysates from either KEX2 or kex2Δ strains. (C) Dilution sensitivity of Ste13αHA processing. Reactions were diluted to the indicated extents before (Pre-reaction) or after (Post-reaction) incubation under reaction conditions. (D) Detergent sensitivity. MSS membranes from either KEX2 or kex2Δ strains were combined with MSS membranes from a Ste13αHA-expressing strain, and reactions were performed in the presence or absence of 1% Triton X-100.
Mentions: The cell-free membrane fusion that we observed exhibited features characteristic of other cell-free biological membrane fusion reactions. First, although Ste13αHA processing in the crude system did not require addition of exogenous ATP (Fig. 3 A), omission of the ATP regeneration system completely blocked processing (Fig. 3 A), indicating that the reaction was ATP dependent. Second, processing of Ste13αHA occurred with maximal efficiency between 25 and 30°C and was inhibited at temperatures below 15°C or above 35°C (Fig. 3 B), similar to the temperature dependence of other cell-free biological membrane fusion reactions (Baker et al., 1988; Latterich and Schekman, 1994). Third, dilution of the MSS membranes progressively inhibited Ste13αHA processing, indicating that membrane concentration was important for efficient fusion (Fig. 3 C). Fourth, addition of Triton X-100 (1% wt vol−1) completely inhibited Kex2p processing of Ste13αHA even though both Kex2p and DPAP were fully active under these conditions (unpublished data), indicating that membrane integrity was essential for efficient processing (Fig. 3 D). Furthermore, this result shows that for processing to be observed in this dilute system Kex2p must be coconcentrated along with the substrate Ste13αHA in the lumenal space of fused late Golgi membrane vesicles. Taken together, the characteristics of the reaction suggested that the observed Kex2p-dependent processing of Ste13αHA represented a biologically relevant vesicular transport or membrane fusion event between membrane compartments containing Kex2p and Ste13αHA.

Bottom Line: Coimmunoisolation confirmed that the reaction resulted in cointegration of the two enzymes into the same bilayer.Antibody inhibition experiments coupled with antigen competition indicated a requirement for soluble NSF attachment protein receptor (SNARE) proteins Tlg1p, Tlg2p, and Vti1p in this reaction.Vps21p was sufficient if present on either the Kex2p or Ste13p membranes alone, indicative of an inherent symmetry in the reaction.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.

ABSTRACT
Using a new assay for membrane fusion between late Golgi/endosomal compartments, we have reconstituted a rapid, robust homotypic fusion reaction between membranes containing Kex2p and Ste13p, two enzymes resident in the yeast trans-Golgi network (TGN). Fusion was temperature, ATP, and cytosol dependent. It was inhibited by dilution, Ca+2 chelation, N-ethylmaleimide, and detergent. Coimmunoisolation confirmed that the reaction resulted in cointegration of the two enzymes into the same bilayer. Antibody inhibition experiments coupled with antigen competition indicated a requirement for soluble NSF attachment protein receptor (SNARE) proteins Tlg1p, Tlg2p, and Vti1p in this reaction. Membrane fusion also required the rab protein Vps21p. Vps21p was sufficient if present on either the Kex2p or Ste13p membranes alone, indicative of an inherent symmetry in the reaction. These results identify roles for a Tlg SNARE complex composed of Tlg1p, Tlg2p, Vti1p, and the rab Vps21p in this previously uncharacterized homotypic TGN fusion reaction.

Show MeSH
Related in: MedlinePlus