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. 2015 Jan 15;26(2):305-15.
doi: 10.1091/mbc.E14-08-1298. Epub 2014 Nov 19.

Yeast Vacuolar HOPS, Regulated by Its Kinase, Exploits Affinities for Acidic Lipids and Rab:GTP for Membrane Binding and to Catalyze Tethering and Fusion

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Yeast Vacuolar HOPS, Regulated by Its Kinase, Exploits Affinities for Acidic Lipids and Rab:GTP for Membrane Binding and to Catalyze Tethering and Fusion

Amy Orr et al. Mol Biol Cell. .
Free PMC article

Abstract

Fusion of yeast vacuoles requires the Rab GTPase Ypt7p, four SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), the SNARE disassembly chaperones Sec17p/Sec18p, vacuolar lipids, and the Rab-effector complex HOPS (homotypic fusion and vacuole protein sorting). Two HOPS subunits have direct affinity for Ypt7p. Although vacuolar fusion has been reconstituted with purified components, the functional relationships between individual lipids and Ypt7p:GTP have remained unclear. We now report that acidic lipids function with Ypt7p as coreceptors for HOPS, supporting membrane tethering and fusion. After phosphorylation by the vacuolar kinase Yck3p, phospho-HOPS needs both Ypt7p:GTP and acidic lipids to support fusion.

Figures

FIGURE 1:
FIGURE 1:
Lipid composition and Ypt7p:GTP control fusion. Fusion of RPLs of various lipid compositions, each with the four vacuolar SNAREs and either without Ypt7p (A) or with Ypt7p:GTP (B), was assayed as protected lumenal compartment mixing. RPLs, ATP, streptavidin, and buffer were mixed and preincubated for 10 min at 27ºC with GTP and EDTA in 7 μl to specifically exchange the Ypt7p-bound guanine nucleotide, then mixed with 3 μl of HOPS, Sec17p, Sec18p, and MgCl2 to initiate the fusion reaction. (C) The amount of lumenal compartment mixing after 5 min is shown as average ± SD for three independent experiments. Final concentrations in 10 μl: 1.3 mM ATP, 2.6 mM EDTA, 25.6 μM GTP, 12.6 μM streptavidin, 1.1 mM lipid, 3 mM MgCl2, 177 nM Sec17p, 907 nM Sec18p, and 218 nM HOPS.
FIGURE 2:
FIGURE 2:
Fusion is restored to RPLs that lack acidic lipids by the addition of diC8-PA or excess Vam7p. Fusion of RPLs composed of PC, PE, Erg, and DAG with the four vacuolar SNAREs and either without Ypt7p (A) or with Ypt7p:GTP (B) was assayed as protected lumenal compartment mixing. RPLs were preincubated without addition or with 2 μM Vam7p and/or 110 μM diC8-PA, as indicated, along with ATP, streptavidin, EDTA, and GTP in 7 μl for 10 min at 27ºC before the fusion reaction was started by addition of 3 μl of HOPS, Sec17p, Sec18p, and MgCl2. (C) The amount of lumenal compartment mixing after 5 min is shown as average ± SD for three independent experiments. Final concentrations in 10 μl: 0.9 mM ATP, 1.8 mM EDTA, 18 μM GTP, 9 μM streptavidin, 0.8 mM lipid, 2.5 mM MgCl2, 156 nM Sec17p, 800 nM Sec18p, and 193 nM HOPS.
FIGURE 3:
FIGURE 3:
Stimulation of fusion of PC/PE/Erg/DAG RPLs with four vacuolar SNAREs, including a transmembrane anchored form of Vam7p (Xu and Wickner, 2012), by addition of diC8-PA. RPLs were prepared as described in Materials and Methods, except that Ypt7p was stripped and reloaded with GTPγS before reconstitution, as described (Zick and Wickner, 2012). (A) RPLs without Ypt7p (circles) or with Ypt7p:GTPγS (triangles) were preincubated without addition (open symbols) or with 110 μM diC8-PA (filled symbols), along with MgCl2, ATP, and streptavidin, in 14 μl for 10 min at 27ºC before the fusion reaction was started by addition of 6 μl of HOPS, Sec17p, and Sec18p. Final concentrations in 20 μl: 1 mM ATP, 9.6 μM streptavidin, 0.5 mM lipid, 1 mM MgCl2, 100 nM Sec17p, 1 μM Sec18p, and 165 nM HOPS. (B) Fusion under these conditions after 5 min.
FIGURE 4:
FIGURE 4:
Acidic lipids and/or Ypt7p facilitate HOPS binding to membranes. Liposomes of various lipid compositions with or without the four vacuolar SNAREs and either without Ypt7p or with Ypt7p:GTP were assayed for their ability to bind HOPS as described in Materials and Methods.
FIGURE 5:
FIGURE 5:
HOPS requires acidic lipids and/or Ypt7p to induce membrane clustering. (A) Liposomes of various lipid compositions bearing either no Ypt7p or Ypt7p:GTP were assayed for HOPS-dependent tethering as described in Materials and Methods. (B, C) SNAREs do not make a major contribution to tethering. Liposomes of various lipid compositions with either all four vacuolar SNAREs (B) or only the R-SNARE or the three Q-SNAREs (C), which bore either no Ypt7p or Ypt7p:GTP, were assayed for HOPS-dependent tethering as described in Materials and Methods.
FIGURE 6:
FIGURE 6:
Salt sensitivity of tethering. (A) VML liposomes with either no Ypt7p or Ypt7p:GTP or (B) PC/PE/Erg/DAG liposomes with Ypt7p:GTP were assayed for HOPS-dependent tethering as described in Materials and Methods in the presence of increasing concentrations of NaCl.
FIGURE 7:
FIGURE 7:
Salt sensitivity of fusion. (A, B) Fusion of VML RPLs bearing the four vacuolar SNAREs and either no Ypt7p or Ypt7p:GTP was assayed as protected lumenal compartment mixing in the presence of increasing concentrations of NaCl. RPLs were preincubated with streptavidin and various amounts of NaCl in 15 μl for 10 min at 27ºC before the fusion reaction was started by addition of 5 μl of Sec17p, Sec18p, and HOPS, which had been preincubated for 10 min at 27ºC without (A) or with (B) Yck3p. (C) Fusion of RPLs composed of PC/PE/Erg/DAG, bearing four vacuolar SNAREs, including a transmembrane-anchored form of Vam7p, and Ypt7p:GTP was assayed as protected lumenal compartment mixing in the presence of increasing concentrations of NaCl. RPLs were preincubated without addition or with 100 μM diC8-PA, along with streptavidin and various amounts of NaCl, in 15 μl for 10 min at 27ºC before the fusion reaction was started by addition of 5 μl of Sec17p, Sec18p, and HOPS, preincubated for 10 min at 27ºC with or without Yck3p. Final concentrations in 20 μl: 1 mM ATP, 10 μM streptavidin, 0.5 mM lipid, 1 mM MgCl2, 600 nM Sec17p, 200 nM Sec18p, 100 nM HOPS, and 3 μM Yck3p.
FIGURE 8:
FIGURE 8:
Acidic lipids and Ypt7p:GTP can cooperate to support fusion at an elevated ionic strength where neither alone suffices. Proteoliposomes were prepared with (A) VML lipids, Ypt7p at the indicated molar ratios to lipid, and the four vacuolar SNAREs or (B) PC/PE/Erg/DAG lipids, Ypt7p, and the four vacuolar SNAREs, including a transmembrane- anchored form of Vam7p. The RPLs were incubated for 10 min at 27ºC with streptavidin, NaCl, EDTA, and GTP and assayed for fusion upon the addition of HOPS, Sec17p, Sec18p, ATP, and MgCl2. (C) The amount of lumenal compartment mixing after 5 min is shown as average ± SD for three independent experiments. Final concentrations in 20 μl: 0.5 mM lipid, 9.6 μM streptavidin, 225 mM NaCl, 1 mM EDTA, 10 μM GTP, 136 nM HOPS, 600 nM Sec17p, 200 nM Sec18p, 1 mM ATP, and 1 mM MgCl2.
FIGURE 9:
FIGURE 9:
Ypt7p allows phospho-HOPS to fulfill its nontethering functions. Fusion of RPLs composed of PC, PE, Erg, and DAG with the four vacuolar SNAREs, including a transmembrane-anchored form of Vam7p and either without Ypt7p (A) or with Ypt7p:GTP (B), was assayed as protected lumenal compartment mixing. RPLs were preincubated with streptavidin in 10 μl for 10 min at 27ºC before the fusion reaction was started by addition of 10 μl of Sec17p, Sec18p, MgCl2, and ATP, as well as HOPS, preincubated for 10 min at 27ºC with or without Yck3p and PEG, as indicated. (C) The amount of lumenal compartment mixing after 5 min is shown as average ± SD for three independent experiments. Final concentrations in 20 μl: 1 mM ATP, 10 μM streptavidin, 0.5 mM lipid, 1 mM MgCl2, 600 nM Sec17p, 200 nM Sec18p, 100 nM HOPS, 3 μM Yck3p, and 2% (wt/vol) PEG8000.
FIGURE 10:
FIGURE 10:
Ypt7p supports fusion only when in its GTP-bound state. Proteoliposomes of PC/PE/DAG/Erg were prepared with or without Ypt7p and with three wild-type vacuolar SNAREs plus the transmembrane-anchored Vam7p. After incubation for 10 min at 27°C with EDTA and either GTP (A) or GTPγS (B) to facilitate nucleotide exchange, they were mixed with MgCl2 and incubated for 10 min at 27°C with the indicated concentration of Gyp1-46. ATP, HOPS, Sec17p, and Sec18p were then added to initiate fusion, as described in Materials and Methods. Final concentrations in 20 μl: 1 mM ATP, 5 μM streptavidin, 0.5 mM lipid, 1 mM EDTA, 10 μM GTP or GTPγS, 100 nM HOPS, 600 nM Sec17p, 200 nM Sec18p, 2.25 mM MgCl2, and 0–4 μM Gyp1-46. (C) Average fusion after 10 min for triplicates of the experiments shown in panels A and B.
FIGURE 11:
FIGURE 11:
Ypt7p:GTP and PI can facilitate fusion in both homotypic and heterotypic manners. (A) Fusion of various combinations of RPLs composed of PC, PE, Erg, and DAG with the four vacuolar SNAREs, including a transmembrane-anchored form of Vam7p and Ypt7p:GTP, the acidic lipid PI, or neither, was assayed as protected lumenal compartment mixing. RPLs were preincubated with streptavidin, ATP, EDTA, and GTP in 7 μl for 10 min at 27ºC before the fusion reaction was started by addition of 3 μl of Sec17p, Sec18p, and MgCl2. (B) The amount of lumenal compartment mixing after 5 min is shown as average ±SD for three independent experiments. Final concentrations in 10 μl: 0.9 mM ATP, 1.2 mM EDTA, 17 μM guanine-nucleotide, 8.5 μM streptavidin, 0.8 mM lipid, 2.5 mM MgCl2, 156 nM Sec17p, 800 nM Sec18p, and 193 nM HOPS.

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