Limits...
Transition from hemifusion to pore opening is rate limiting for vacuole membrane fusion.

Reese C, Mayer A - J. Cell Biol. (2005)

Bottom Line: The LPC block reversibly prevented formation of the hemifusion intermediate that allows lipid, but not content, mixing.Transition from hemifusion to pore opening was sensitive to guanosine-5'-(gamma-thio)triphosphate.Pore opening was rate limiting for the reaction.

View Article: PubMed Central - PubMed

Affiliation: Département de Biochimie, Université de Lausanne, 1066 Epalinges, Switzerland.

ABSTRACT
Fusion pore opening and expansion are considered the most energy-demanding steps in viral fusion. Whether this also applies to soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE)- and Rab-dependent fusion events has been unknown. We have addressed the problem by characterizing the effects of lysophosphatidylcholine (LPC) and other late-stage inhibitors on lipid mixing and pore opening during vacuole fusion. LPC inhibits fusion by inducing positive curvature in the bilayer and changing its biophysical properties. The LPC block reversibly prevented formation of the hemifusion intermediate that allows lipid, but not content, mixing. Transition from hemifusion to pore opening was sensitive to guanosine-5'-(gamma-thio)triphosphate. It required the vacuolar adenosine triphosphatase V0 sector and coincided with its transformation. Pore opening was rate limiting for the reaction. As with viral fusion, opening the fusion pore may be the most energy-demanding step for intracellular, SNARE-dependent fusion reactions, suggesting that fundamental aspects of lipid mixing and pore opening are related for both systems.

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Related in: MedlinePlus

Trans-SNARE complex formation. Effects of LPC (A) and MED (B). 20× standard fusion reactions with vacuoles isolated from strains BJ3505 Δvam3 and BJ3505 Δnyv1 were started. Where indicated, the ATP-regenerating system was omitted and the sample was kept on ice. After 30 min, the reactions were stopped by chilling on ice. 380 μl of precipitation buffer was added (150 mM KCl, 1.3% [wt/vol] Triton X-100, 1.3 mM PMSF, 1.2× PIC, and 13 mM EDTA in PS). Samples were shaken for 10 min at 4°C and centrifuged at 21,000 g (10 min, 4°C). 450 μl of the solubilisate were transferred to a new reaction tube. 18 μg of affinity-purified anti-Vam3p antibody and 20 μl of protein A–Sepharose CL-4B (GE Healthcare) in 100 μl of washing buffer (150 mM KCl, 1% [wt/vol] Triton X-100, 1 mM PMSF, 1× PIC, and 10 mM EDTA in PS) were added to each sample. After 1 h of shaking at 4°C, the beads were washed with precipitation buffer and resuspended in SDS sample buffer. Aliquots were analyzed by SDS-PAGE and Western blotting. For A, trans-SNARE pairing was quantified by densitometry and compared with fusion activity that was determined from identical samples run in parallel (60 min, 27°C). The following inhibitors were used: 1 μM Gdi1p, 500 μM LPC-12, 120 μM LPC-14, 4 mM GTPγS, and 10 μM MED.
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fig4: Trans-SNARE complex formation. Effects of LPC (A) and MED (B). 20× standard fusion reactions with vacuoles isolated from strains BJ3505 Δvam3 and BJ3505 Δnyv1 were started. Where indicated, the ATP-regenerating system was omitted and the sample was kept on ice. After 30 min, the reactions were stopped by chilling on ice. 380 μl of precipitation buffer was added (150 mM KCl, 1.3% [wt/vol] Triton X-100, 1.3 mM PMSF, 1.2× PIC, and 13 mM EDTA in PS). Samples were shaken for 10 min at 4°C and centrifuged at 21,000 g (10 min, 4°C). 450 μl of the solubilisate were transferred to a new reaction tube. 18 μg of affinity-purified anti-Vam3p antibody and 20 μl of protein A–Sepharose CL-4B (GE Healthcare) in 100 μl of washing buffer (150 mM KCl, 1% [wt/vol] Triton X-100, 1 mM PMSF, 1× PIC, and 10 mM EDTA in PS) were added to each sample. After 1 h of shaking at 4°C, the beads were washed with precipitation buffer and resuspended in SDS sample buffer. Aliquots were analyzed by SDS-PAGE and Western blotting. For A, trans-SNARE pairing was quantified by densitometry and compared with fusion activity that was determined from identical samples run in parallel (60 min, 27°C). The following inhibitors were used: 1 μM Gdi1p, 500 μM LPC-12, 120 μM LPC-14, 4 mM GTPγS, and 10 μM MED.

Mentions: Docking results in the trans-association of v- and t-SNAREs from opposing membranes. The in vitro fusion reaction of yeast vacuoles offers the opportunity to assay this association and to determine whether trans-SNARE pairing correlates to fusion activity (Ungermann et al., 1998a,b; Merz and Wickner, 2004b). To this end, vacuoles are prepared from strains expressing only the vacuolar t-SNARE Vam3p or the v-SNARE Nyv1p. After mixing these two populations of vacuoles and incubating them in the presence of postdocking inhibitors of fusion, the membranes are diluted and solubilized. The trans-association of v- and t-SNAREs can then be assayed by coimmunoprecipitation of the v-SNARE Nyv1p with antibodies to the t-SNARE Vam3p. In the absence of a fusion inhibitor, these complexes represent a mixture of three species: trans-SNARE complexes between vacuoles that did not complete fusion; trans-SNARE complexes converted into cis-SNARE complexes because they triggered successful fusion of the vacuoles; and cis-SNARE complexes formed independently of docking by reassociation of solitary v- and t-SNAREs subsequent to the completion of fusion, i.e., as a simple result of membrane mixing between the v- and t-SNARE–carrying membranes. In the presence of late-acting fusion inhibitors, only the first species should accumulate. We tested for the formation of trans-SNARE complexes in the presence of LPCs. Without an ATP-regenerating system, priming and docking cannot occur and no Nyv1p could be coimmunoprecipitated with Vam3p (Fig. 4). In the presence of ATP, SNARE complexes formed. Simultaneous addition of the docking inhibitor Gdi1p completely blocked the formation of SNARE complexes, indicating that they resulted from docking. Similar to GTPγS, an established late-acting inhibitor of the postdocking phase, both LPC-14 and -12 allowed trans-SNARE pairing (Fig. 4), albeit only up to 50% of the uninhibited control. GTPγS permits docking as well as lipid mixing. Thus, the levels of trans-SNARE pairing reached in the presence of GTPγS should suffice to trigger lipid mixing. To assay the correlation between trans-SNARE pairing and fusion activity by independent means, we used another, recently identified inhibitor of vacuole fusion, myristoylated alanine-rich C kinase substrate effector domain peptide (MED; Fratti et al., 2004). MED sequesters phosphatidylinositol-4,5-bisphosphate, binds calmodulin with a Kd of 4 nM, and penetrates the acyl chain zone of lipid bilayers with its five phenylalanines (Sundaram et al., 2004). All three effects could contribute to its inhibitory activity in vacuole fusion. We observed that MED strongly promoted mutual binding and cluster formation of vacuoles (unpublished data). In accordance with this, MED permitted SNARE pairing to at least the same levels as in the uninhibited control, and in some experiments even twofold more (Fig. 4 B). Nevertheless, MED blocked content as well as lipid mixing (see next paragraph). Together, these results suggest that trans-SNARE complexes can efficiently form in the absence of lipid mixing. Furthermore, they show that LPCs and MED permit trans-SNARE association but interfere with vacuole fusion downstream of docking.


Transition from hemifusion to pore opening is rate limiting for vacuole membrane fusion.

Reese C, Mayer A - J. Cell Biol. (2005)

Trans-SNARE complex formation. Effects of LPC (A) and MED (B). 20× standard fusion reactions with vacuoles isolated from strains BJ3505 Δvam3 and BJ3505 Δnyv1 were started. Where indicated, the ATP-regenerating system was omitted and the sample was kept on ice. After 30 min, the reactions were stopped by chilling on ice. 380 μl of precipitation buffer was added (150 mM KCl, 1.3% [wt/vol] Triton X-100, 1.3 mM PMSF, 1.2× PIC, and 13 mM EDTA in PS). Samples were shaken for 10 min at 4°C and centrifuged at 21,000 g (10 min, 4°C). 450 μl of the solubilisate were transferred to a new reaction tube. 18 μg of affinity-purified anti-Vam3p antibody and 20 μl of protein A–Sepharose CL-4B (GE Healthcare) in 100 μl of washing buffer (150 mM KCl, 1% [wt/vol] Triton X-100, 1 mM PMSF, 1× PIC, and 10 mM EDTA in PS) were added to each sample. After 1 h of shaking at 4°C, the beads were washed with precipitation buffer and resuspended in SDS sample buffer. Aliquots were analyzed by SDS-PAGE and Western blotting. For A, trans-SNARE pairing was quantified by densitometry and compared with fusion activity that was determined from identical samples run in parallel (60 min, 27°C). The following inhibitors were used: 1 μM Gdi1p, 500 μM LPC-12, 120 μM LPC-14, 4 mM GTPγS, and 10 μM MED.
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Related In: Results  -  Collection

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fig4: Trans-SNARE complex formation. Effects of LPC (A) and MED (B). 20× standard fusion reactions with vacuoles isolated from strains BJ3505 Δvam3 and BJ3505 Δnyv1 were started. Where indicated, the ATP-regenerating system was omitted and the sample was kept on ice. After 30 min, the reactions were stopped by chilling on ice. 380 μl of precipitation buffer was added (150 mM KCl, 1.3% [wt/vol] Triton X-100, 1.3 mM PMSF, 1.2× PIC, and 13 mM EDTA in PS). Samples were shaken for 10 min at 4°C and centrifuged at 21,000 g (10 min, 4°C). 450 μl of the solubilisate were transferred to a new reaction tube. 18 μg of affinity-purified anti-Vam3p antibody and 20 μl of protein A–Sepharose CL-4B (GE Healthcare) in 100 μl of washing buffer (150 mM KCl, 1% [wt/vol] Triton X-100, 1 mM PMSF, 1× PIC, and 10 mM EDTA in PS) were added to each sample. After 1 h of shaking at 4°C, the beads were washed with precipitation buffer and resuspended in SDS sample buffer. Aliquots were analyzed by SDS-PAGE and Western blotting. For A, trans-SNARE pairing was quantified by densitometry and compared with fusion activity that was determined from identical samples run in parallel (60 min, 27°C). The following inhibitors were used: 1 μM Gdi1p, 500 μM LPC-12, 120 μM LPC-14, 4 mM GTPγS, and 10 μM MED.
Mentions: Docking results in the trans-association of v- and t-SNAREs from opposing membranes. The in vitro fusion reaction of yeast vacuoles offers the opportunity to assay this association and to determine whether trans-SNARE pairing correlates to fusion activity (Ungermann et al., 1998a,b; Merz and Wickner, 2004b). To this end, vacuoles are prepared from strains expressing only the vacuolar t-SNARE Vam3p or the v-SNARE Nyv1p. After mixing these two populations of vacuoles and incubating them in the presence of postdocking inhibitors of fusion, the membranes are diluted and solubilized. The trans-association of v- and t-SNAREs can then be assayed by coimmunoprecipitation of the v-SNARE Nyv1p with antibodies to the t-SNARE Vam3p. In the absence of a fusion inhibitor, these complexes represent a mixture of three species: trans-SNARE complexes between vacuoles that did not complete fusion; trans-SNARE complexes converted into cis-SNARE complexes because they triggered successful fusion of the vacuoles; and cis-SNARE complexes formed independently of docking by reassociation of solitary v- and t-SNAREs subsequent to the completion of fusion, i.e., as a simple result of membrane mixing between the v- and t-SNARE–carrying membranes. In the presence of late-acting fusion inhibitors, only the first species should accumulate. We tested for the formation of trans-SNARE complexes in the presence of LPCs. Without an ATP-regenerating system, priming and docking cannot occur and no Nyv1p could be coimmunoprecipitated with Vam3p (Fig. 4). In the presence of ATP, SNARE complexes formed. Simultaneous addition of the docking inhibitor Gdi1p completely blocked the formation of SNARE complexes, indicating that they resulted from docking. Similar to GTPγS, an established late-acting inhibitor of the postdocking phase, both LPC-14 and -12 allowed trans-SNARE pairing (Fig. 4), albeit only up to 50% of the uninhibited control. GTPγS permits docking as well as lipid mixing. Thus, the levels of trans-SNARE pairing reached in the presence of GTPγS should suffice to trigger lipid mixing. To assay the correlation between trans-SNARE pairing and fusion activity by independent means, we used another, recently identified inhibitor of vacuole fusion, myristoylated alanine-rich C kinase substrate effector domain peptide (MED; Fratti et al., 2004). MED sequesters phosphatidylinositol-4,5-bisphosphate, binds calmodulin with a Kd of 4 nM, and penetrates the acyl chain zone of lipid bilayers with its five phenylalanines (Sundaram et al., 2004). All three effects could contribute to its inhibitory activity in vacuole fusion. We observed that MED strongly promoted mutual binding and cluster formation of vacuoles (unpublished data). In accordance with this, MED permitted SNARE pairing to at least the same levels as in the uninhibited control, and in some experiments even twofold more (Fig. 4 B). Nevertheless, MED blocked content as well as lipid mixing (see next paragraph). Together, these results suggest that trans-SNARE complexes can efficiently form in the absence of lipid mixing. Furthermore, they show that LPCs and MED permit trans-SNARE association but interfere with vacuole fusion downstream of docking.

Bottom Line: The LPC block reversibly prevented formation of the hemifusion intermediate that allows lipid, but not content, mixing.Transition from hemifusion to pore opening was sensitive to guanosine-5'-(gamma-thio)triphosphate.Pore opening was rate limiting for the reaction.

View Article: PubMed Central - PubMed

Affiliation: Département de Biochimie, Université de Lausanne, 1066 Epalinges, Switzerland.

ABSTRACT
Fusion pore opening and expansion are considered the most energy-demanding steps in viral fusion. Whether this also applies to soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE)- and Rab-dependent fusion events has been unknown. We have addressed the problem by characterizing the effects of lysophosphatidylcholine (LPC) and other late-stage inhibitors on lipid mixing and pore opening during vacuole fusion. LPC inhibits fusion by inducing positive curvature in the bilayer and changing its biophysical properties. The LPC block reversibly prevented formation of the hemifusion intermediate that allows lipid, but not content, mixing. Transition from hemifusion to pore opening was sensitive to guanosine-5'-(gamma-thio)triphosphate. It required the vacuolar adenosine triphosphatase V0 sector and coincided with its transformation. Pore opening was rate limiting for the reaction. As with viral fusion, opening the fusion pore may be the most energy-demanding step for intracellular, SNARE-dependent fusion reactions, suggesting that fundamental aspects of lipid mixing and pore opening are related for both systems.

Show MeSH
Related in: MedlinePlus