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Supplementary Data
1. Labelling of vacuoles with different fluorescent lipids
We tested several fluorescent phospholipids for incorporation into isolated vacuolar membranes upon dilution out of organic solvents. Pairs of Rhodamine-/NBD-labelled or of BODIPY-labelled phospholipids are common for measuring fusion of liposomes via fluorescence energy transfer (FRET) 1,2. These lipid pairs were unsuitable for intact vacuoles since they incorporated into our biological membranes with unequal efficiencies. Furthermore, NBD-lipids produced a strong contact- and temperature-dependent fluorescence increase which was unrelated to the authentic fusion pathway (data not shown). Rhodamine labelled phosphatidylethanolamine (Rh-PE) incorporated efficiently into isolated vacuolar membranes when added out of a saturated solution in DMSO (Fig. S1a). After addition of the dye, vacuoles were floated through a Ficoll gradient to remove DMSO and unincorporated Rh-PE. Xanthene dyes such as rhodamine undergo concentration-dependent fluorescence self-quench 3. Formation of non-fluorescent dimers 4 and energy transfer to non-fluorescent dimers and collisional quenching interactions between dye monomers have been discussed as mechanisms 3. Concentration-dependent Rhodamine self-quenching is characterized by a blue shift in absorbance. Accordingly, absorbance of Rh-PE incorporated into vacuolar membranes was increased at 530 nm (Fig. S1b). This blue shift disappeared if the membranes were solubilized by Triton X-100 to disperse Rh-PE and reduce its local concentration. Thus, Rh-PE dimers form after incorporation into vacuolar membranes in a similar fashion as characterized earlier 4.
Incorporation of increasing concentrations of Rh-PE led to a decrease in relative fluorescence of vacuolar Rh-PE (Fig. S1c). At 400 pmol Rh-PE per µg vacuoles (equivalent to 3 mol% of total lipid) fluorescence was quenched to 10% of the control signal obtained after solubilizing the membranes in Triton X-100. For the lipid-mixing assay Rh-PE labelled vacuoles from the maturase carrying strains were mixed with a sixfold excess of unlabelled vacuoles from the pro-phosphatase expressing strain. This maximizes the probability that a labelled vacuole fuses with an unlabelled vacuole. The unlabelled pro-phosphatase carrying vacuoles were also significantly bigger than the labelled species (64 +/-9 µm2 vs. 24 +/-3.6 µm2 surface). A single round of fusion thus dilutes Rh-PE approximately 3-fold. The resulting dequenching was quantified in a fluorescence microplate reader. Since the vacuoles carried also the reporters for contents mixing via alkaline phosphatase activity we could simultaneously assay lipid and contents-mixing on identical samples.
We note that the assay we use provides an indirect readout of lipid transition caused by diluting Rh-PE into the acceptor vacuole. We interpret the resulting increase in fluorescence as an indicator of membrane continuity between the fusion partners because the reporter Rh-PE is firmly anchored in the membrane by its two long fatty acyl chains. Therefore, we do not expect that spontaneous deinsertion and reinsertion into the acceptor vacuole or other forms of lipid transfer independent of membrane continuity are a major problem in the system. We cannot, however, rule this out completely.
2. GTPgS blocks maturation of ALP by preventing fusion of vacuoles
GTPgS could suppress the contents-mixing signal by inhibiting fusion but also by preventing maturation of the reporter pro-ALP or by inhibiting its activity. We confirmed that GTPgS blocked pro-ALP maturation in a fusion-dependent manner by monitoring the conversion of pro-ALP to mature m-ALP by Western blotting. m-ALP has a lower molecular weight and can be distinguished from pro-ALP by its electrophoretic mobility. After a 60 min incubation under fusion conditions, 30 % of pro-ALP had been converted to m-ALP (Fig. S2). This maturation depended on fusion because it occurred neither in the absence of ATP nor in the presence of the docking inhibitor Gdi1p. Also GTPgS suppressed pro-ALP conversion. In order to check that this block of maturation was due to a block of fusion and not to a block of the maturation enzyme Pep4p we performed the same incubation and test in the presence of Triton X-100. The detergent solubilizes the membranes and permits access of Pep4p to pro-ALP independently of fusion. Under these conditions maturation of pro-ALP was influenced neither by ATP or Gdi1p, nor by GTPgS. Therefore, GTPgS blocks pro-ALP conversion only by blocking fusion of intact vacuoles, but not by blocking the maturation activity of Pep4p.
Since contents mixing is ususally not measured by Western blotting but by m-ALP activity we also considered potential effects of GTPgS on m-ALP activity. That this is no problem under the conditions of our fusion assay is apparent from previous results 5,6: If GTPgS blocked m-ALP activity it should suppress the fusion signals when added in the midst or at the end of a fusion reaction, i.e. it should abolish ALP activity already created by fusion. This does not occur. At all timepoints, GTPgS addition only blocks further increase of ALP activity, similarly as chilling the fusion reactions on ice, which blocks completion of fusion at any point of the reaction. It does not, however, reduce the signals to less than those of the ice-curve. Therefore, GTPgS inhibits contents mixing rather than ALP maturation or activity.
This conclusion is further supported by the results from morphological assays of vacuole fusion. In a qualitative assessment of vacuole structure during in vitro vacuole fusion fluorecein-labelled vacuoles did not enlarge in the presence of GTPgS 7. Also our assays show that GTPgS blocked both the fusion-dependent increase in vacuole size (Figs. 5a and S3) and the transfer of the fluorescent reporter calcein between vacuoles. The latter is apparent as a block in the fusion-dependent increase of the frequency of calcein-labelled vacuoles in the total population (Fig. 5b).
GTPgS blocked contents-mixing with an IC50 of 1 mM. This is significantly higher than the concentrations usually necessary to influence GTPases via GTPgS. This might reflect very slow GTP exchange if a GTPase were the target. One can also consider this as an indication that the effect of GTPgS in vacuole fusion is not explained by action on a GTPase. In line with this, kinetic analysis of the requirements for all four GTPases involved in vacuole fusion, Ypt7p, Rho1p, Cdc42p, and Vps1p, have so far revealed only functions in early steps of the fusion reaction 6,8-10. We assume that GTPgS has another non-GTPase target of unknown nature. The fact that GTPgS may not act via a GTPase will complicate the identification of its target but does not influence its validity as a tool to arrest fusion at a late stage.
Supplementary References
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