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1.
Figure 2

Figure 2. From: The V-ATPase proteolipid cylinder promotes the lipid-mixing stage of SNARE-dependent fusion of yeast vacuoles.

Fusion-deficient proteolipid alleles rescue yeast growth at alkaline pH. Wild-type and mutant strains were grown to logarithmic phase in selective medium. Cultures were diluted to an OD600 of 0.2. Drops of 1.5 μl of 1:7 serial dilutions were spotted on YPD plates buffered to pH 5.5 (50 mM MES) or pH 7.5 (50 mM HEPES). Pictures were taken after 48 h of incubation at 30 °C.

Bernd Strasser, et al. EMBO J. 2011 October 19;30(20):4126-4141.
2.
Figure 5

Figure 5. From: The V-ATPase proteolipid cylinder promotes the lipid-mixing stage of SNARE-dependent fusion of yeast vacuoles.

Proteolipid substitutions impede lipid flow. Vacuoles were labelled with Rh–PE at self-quenching concentrations, mixed with a six-fold excess of unlabelled vacuoles and incubated under fusion conditions in the presence or absence of ATP. Fluorescence was monitored for mutants in VMA3 (A, B), VMA11 (C, D) and VMA16 (E). (F) At the end of the fusion reactions, content-mixing signals were determined for all samples using the ALP assay. Lipid-mixing activities were calculated from the initial slope of the dequenching curves form (AE). Then, content-mixing and lipid-mixing activities were normalized to those of the wild-type cells in order to facilitate comparison (n=3).

Bernd Strasser, et al. EMBO J. 2011 October 19;30(20):4126-4141.
3.
Figure 8

Figure 8. From: The V-ATPase proteolipid cylinder promotes the lipid-mixing stage of SNARE-dependent fusion of yeast vacuoles.

Fusion defect of a covalently linked Vma16–Vma3 dimer. (A) BY4741 Δvma16 cells were reconstituted with HA-tagged alleles of VMA16 (Rec16–HA) or of a gene fusion of VMA16 and VMA3 (16–3–HA). Cells were grown in YPD pH 5.5 to logarithmic phase, stained with FM4-64 and analysed by spinning disc confocal microscopy. (B) Vacuoles were isolated from DKY6281 and BY4741Δpep4 expressing VMA16 (Rec16), VMA16–HA or the VMA16–VMA3 gene fusion as unique source of Vma16p and from BJ3505 and DKY6281 expressing VMA3–HA as unique source of Vma3p (Rec3-HA). Fusion activities were determined as in Figure 2 and are shown relative to Vma16–HA. ALP activity of this reference at 27 °C was between 7.5–9.5 U. Ice values varied from 0.25 to 1.8 U for all experiments and were subtracted from respective 27 °C values. *P<0.01 for the difference of the marked value relative to the reconstituted wild type (n=3).

Bernd Strasser, et al. EMBO J. 2011 October 19;30(20):4126-4141.
4.
Figure 7

Figure 7. From: The V-ATPase proteolipid cylinder promotes the lipid-mixing stage of SNARE-dependent fusion of yeast vacuoles.

Vma1p levels on vacuoles are increased by proteolipid mutations and by deletion of NYV1. Vacuoles were prepared from the indicated mutant cells for (A) VMA3, (B) VMA16 and (C) or from cells lacking NYV1 (Δnyv1) or overexpressing NYV1 from the ADH promotor (NYV1 overex.), and from their respective wild-type equivalents. Spheroplast samples were taken during vacuole isolation to control protein levels in entire cells (total). Proteins were analysed by SDS–PAGE and western blot. The V0 subunit Vma6p and the SNAREs Vam3p or Vti1p were used as loading controls. *P<0.01 for the difference of the marked value relative to the reconstituted wild type. n=>3. (D) HA-tagged VMA16 with or without the substitution F190Y or genetically fused to VMA3 were expressed in BJ3505. As a control for specificity of the antibodies, an untagged VMA16 was expressed in BJ3505. Vacuoles were isolated from these strains and the Vma16p variants were precipitated with antibodies to the HA epitope. Immunoprecipitates were analysed by western blotting for HA-tagged Vma16p variants.

Bernd Strasser, et al. EMBO J. 2011 October 19;30(20):4126-4141.
5.
Figure 3

Figure 3. From: The V-ATPase proteolipid cylinder promotes the lipid-mixing stage of SNARE-dependent fusion of yeast vacuoles.

Fusion-deficient proteolipid alleles support proton translocation in isolated vacuoles. Vacuoles were prepared from cells expressing proteolipids with the indicated substitutions in Vma3p (A, B), Vma11p (C, D) or Vma16p (E), a fusion protein of Vma16p and Vma3p (16–3–HA), or proteolipids carrying an HA-tag (Rec3–HA, Rec16–HA) (F). In each case, the expressed allele was the sole source of the respective proteins in the cell. The vacuoles were used to measure H+ translocation activity via fluorescence quenching of ACMA. Vacuoles were incubated with ACMA for 2 min in fusion buffer without ATP. Then, ATP was added and fluorescence was measured for 3 min, followed by addition of FCCP and recording for further 3 min. Control reactions were run in the absence of ATP, in the presence of 0.4 μM concanamycin A (Conc) or 12 μM archazolide (Arc), or using vacuoles from the subunit d deletion mutant Δvma6 or from vma3E137G, a strain lacking the H+-carrying glutamate.

Bernd Strasser, et al. EMBO J. 2011 October 19;30(20):4126-4141.
6.
Figure 6

Figure 6. From: The V-ATPase proteolipid cylinder promotes the lipid-mixing stage of SNARE-dependent fusion of yeast vacuoles.

Activation and trans-complex formation of SNAREs. (A) SNARE activation. Fusion reactions with vacuoles from proteolipid point mutants were incubated in the presence or absence of ATP (15 min, 27 °C) and centrifuged. Supernatants and pellets were separated, TCA-precipitated and analysed for the presence of Sec17p/α-SNAP by western blot. Vacuoles from all analysed candidate mutants released Sec17p/α-SNAP in presence of ATP. (B) Trans-SNARE pairing. Vacuoles were prepared from proteolipid mutants carrying either Nyv1–HA or Vam3–VSV. The two vacuole populations were mixed into one fusion reaction that was incubated in the presence or absence of ATP (30 min, 27 °C). After solubilization in Triton X-100, Nyv1–HA was adsorbed to anti-HA beads. Adsorbed proteins were analysed by SDS–PAGE and western blotting. Trans-SNARE pairing is assessed via the amount of Vam3–VSV from one fusion partner that co-adsorbs with Nyv1–HA from the other fusion partner. (C) The signals from (B) were detected by fluorescent secondary antibodies and quantified with a Licor Odyssey scanner (n=3). *P<0.01 for the difference of the marked value relative to the reconstituted wild type.

Bernd Strasser, et al. EMBO J. 2011 October 19;30(20):4126-4141.
7.
Figure 4

Figure 4. From: The V-ATPase proteolipid cylinder promotes the lipid-mixing stage of SNARE-dependent fusion of yeast vacuoles.

Positioning of substitutions affecting vacuole fusion. Side view (A) and top view (B) of a homology model of the proteolipid cylinder, based on the proteolipid structure 2bl2 from E. hirae (Murata et al, 2005). Only half of the cylinder is shown for clarity. The positions in the cylinder that are not shown are occupied by three further copies of the c-subunit Vma3p. Mutations are marked as yellow spheres in the ribbon representations of Vma3p (red), Vma11p (green) and Vma16p (blue). The essential glutamic acid residues are shown as yellow patches within the ribbons. (C) Helical wheel projections of the proteolipid transmembrane domains. The orientation of the helical wheels corresponds to that of the transmembrane domains in the top view of the homology model. Residues affecting fusion are coloured in red. (D) Sequence coverage of the mutagenesis approach. Plasmids were retrieved from 10 random clones of each mutagenized proteolipid library that had not undergone the screening procedure. The ensemble of affected residues found in all 10 clones is labelled as yellow spheres in each of the proteolipid sequences (upper panel). Their random distribution contrasts the clustering of fusion-deficient point mutations in the cytosolic half of the transmembrane domains, shown for comparison (lower panel).

Bernd Strasser, et al. EMBO J. 2011 October 19;30(20):4126-4141.
8.
Figure 9

Figure 9. From: The V-ATPase proteolipid cylinder promotes the lipid-mixing stage of SNARE-dependent fusion of yeast vacuoles.

Working hypothesis. We assume that hydrophobic surfaces in the proteolipid cylinder initiate lipid reorientation and hemifusion. They are exposed by a transient conformational change, illustrated in the form of an orange crevice between the yellow proteolipids (collectively designated as ‘c'). V0 subunit e is not shown because there are no data implicating it in fusion. The hydrophobic crevice could be invaded by lipid acyl chains, changing lipid orientation and bilayer curvature. A lipidic fusion pore could then form at the periphery of the cylinder and expand laterally. The conformational change of proteolipids may be promoted by SNAREs. Association with the SM protein and Rab-GTPase associated tether factors might restrict the rotational freedom of SNAREs and arrest their complex in a tense, twisted conformation (Pieren et al, 2010), which should exert mechanical strain on the bilayer. In addition, the size of the SNARE/SM complex should drive it away from a site of close membrane apposition or hemifusion, thus creating lateral tension on the fusion site (Mayer, 2001; Rizo et al, 2006).

Bernd Strasser, et al. EMBO J. 2011 October 19;30(20):4126-4141.
9.
Figure 1

Figure 1. From: The V-ATPase proteolipid cylinder promotes the lipid-mixing stage of SNARE-dependent fusion of yeast vacuoles.

Vacuole structure and vacuole fusion activity in proteolipid mutants. BJ Δvma3, BJ Δvma11, BY Δvma16 reconstituted with plasmid-borne wild-type (designated as ‘Rec x') (A) or mutant alleles (B, C) were grown in YPD pH 5.5 to logarithmic phase, stained with FM4-64 and analysed by confocal microscopy. Vacuoles were isolated from mutants found in the screen (D) and from strains expressing alleles with single substitutions derived from these mutants (E) and tested for their in vitro fusion activity relative to wild type. Vacuoles were incubated in standard fusion reactions in the presence of 1 mg/ml cytosol for 60 min on ice or at 27 °C. Then, ALP activities were determined as a tracer for fusion. ALP activity of the reconstituted wild types at 27 °C was 3.4–6.4 U for Vma3p, 2.4–5.7 U for Vma11p and 2.8–4.8 U for Vma16p. Ice values varied from 0.28–1.0 U for all experiments and were subtracted from respective 27 °C values (n⩾3). *P<0.01 for the difference of the marked value relative to the reconstituted wild type.

Bernd Strasser, et al. EMBO J. 2011 October 19;30(20):4126-4141.

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