Ykt6 is involved in early and later steps of fusion. A 30 × scale fusion reaction was started in the presence of 0.5 mM ATP and 10 μM CoA at 26°C. Aliquots (30 μl) were removed at the indicated times, added to recombinant Ykt6-R71G, an N-terminal peptide of Ykt6 (Ykt6N) or antibodies to Ykt6, Ykt6N, Sec17, Sec18 or Vam3, and incubated at 26°C or placed on ice. Reactions were incubated for a total of 90 min before assaying for alkaline phosphatase activity. Inhibition by anti-Ykt6 (A), Ykt6-R71G (B), anti-Ykt6N (C) and Ykt6N (D).

Role of Sec18 beyond Sec17-dependent priming. (A) Vacuoles were incubated with anti-Sec18 antibody in a standard fusion reaction with cytosol at 26°C. After 10 min, one sample (no reisolation) was set aside, and the others were diluted 10-fold in PSK buffer, centrifuged (5 min, 9000 g, 4°C) and resuspended in the original volume of reaction buffer with cytosol and 0.2 μg Sec18, containing the indicated inhibitors. After an additional 60 min at 26°C, alkaline phosphatase activity was determined. Background activity was subtracted. The activity of control samples (no adds) was set to 100%. (B) His-Ykt6 was added instead of anti-Sec18 to vacuoles, and incubations and reisolation were performed as before. (C) Working model of vacuole fusion. For details, see text.

Ykt6 is sufficient for Vac8 palmitoylation. (A) In vitro reconstitution of the acyltransferase reaction depends on Ykt6. Indicated combinations of recombinant Vac8-GST (0.5 μM) and Ykt6-wt (2 μM) were incubated with 0.5 μM [³H]-Pal-CoA for 60 min at 26°C in the buffer of the standard fusion reaction. For inactivation, Ykt6 was heated for 5 min at 95°C before being added to the assay. In lanes 5 and 6, Ykt6(1–120)-GST (Ykt6 N-term.) or GST-Vti1(1–110) (Vti1-N-term.) were used instead of Ykt6-wt. After the assay, proteins were precipitated by chloroform/methanol, dried, resuspended in SDS sample buffer, resolved on SDS–PAGE and analyzed by fluorography. (B) Kinetics of the in vitro acylation reaction. Reactions were performed as described in (A). Either purified Ykt6 or a vacuolar lysate (Figure 4A) was used as the enzyme source. Fluorograms were quantified by laser densitometry. Maximal palmitoylation observed in the reaction was set to 100%. (C) Determination of the stoichiometry of the reaction. Increasing amounts of Ykt6 were titrated against 1.5 μM Vac8. The reaction was performed and analyzed as described in (A). (D) Indicated amounts of Vac8 were titrated against 0.25 μM Ykt6. (E) Ykt6 stimulates acylation under reducing conditions. Acylation was performed as in (A). Where indicated, 1 mM DTT was added to the reaction. Ykt6-mediated palmitoylation in the absence of DTT was set as 100%.

Acyltransferase activity in vitro corresponds to Ykt6. Sizing of the acyltransferase activity. Vacuoles (60 μg) were preincubated in PSK buffer for 10 min at 26°C without ATP, reisolated and solubilized in 300 μl 0.1% Triton X-100, 20 mM Tris/HCl (pH 7.4) and 150 mM NaCl for 10 min on ice. Unsolubilized material was removed by centrifugation (10 min, 14 000 × g, 4°C). The cleared detergent extract was loaded on top of a 10.5 ml 10–34% continuous glycerol gradient in PSK buffer and centrifuged (SW41, 40 000 g, 4°C, 18 h). Equal fractions (750 μl) were collected from the top of the gradient. Aliquots of the fractionated extract (10 μl) were collected and analyzed. To determine PAT activity, 10 μl extract and recombinant myr-Vac8-GST were incubated for 30 min at 26°C in a 100 μl reaction in the presence of [³H]-Pal-CoA (Pal-CoA) and then processed by SDS–PAGE of precipitated proteins. Fluorographs were quantified by laser densitometry. Aliquots of the same fraction were resolved on SDS–PAGE gels and blotted onto nitrocellulose to analyze the size of Sec18 and Ykt6 in the gradient. The distribution of Sec18 and Ykt6 is shown. For size determination, a control gradient with marker proteins was run in parallel. Sizes are indicated.

Ykt6 is implicated in a Sec17-independent early reaction. (A) Palmitoylation of Vac8 is specifically inhibited by antibodies to Ykt6 and Sec18. Fusion reactions (300 μl) containing 60 μg of vacuoles from BJ3505 were incubated for 10 min at 26°C in the presence of ATP (0.5 mM), cytosol (0.5 μg/μl), coenzyme A (CoA, 10 μM), [³H]-palmitate (150 μCi), and either purified IgGs to the indicated proteins or recombinant Ykt6-R71G (15 μM) or an equal volume of PS buffer (20 mM PIPES/KOH, 200 mM sorbitol). Vacuoles were then isolated by centrifugation (10 min, 12 000 g, 4°C), washed with 500 μl of PSK buffer (20 mM PIPES/KOH, pH 6.8, 200 mM sorbitol, 150 mM KCl) and resuspended in SDS sample buffer without 2-mercaptoethanol. Palmitoylated Vac8 was identified by SDS–PAGE and fluorography. Bands were quantified by laser densitometry (IPLab GelH, Scientific Image Processing 1.5, Signal Analytics). The positive control was set to 100%. (B) Interference with Ykt6 blocks fusion. Standard fusion reactions were incubated for 60 min at 26°C in the presence of Ykt6-R71G (15 μM) or anti-Ykt6 antibodies. The inhibiting amounts of protein were determined by titration (see Figure 1C). Background activity was subtracted from all measurements. Fusion activity in the absence of inhibitor was set to 100%. (C) Inhibition of fusion by Ykt6-R71G, but not Ykt6-wt. Indicated amounts of purified Ykt6 proteins were added to standard fusion reactions. Alkaline phosphatase activity was determined after 90 min at 26°C. (D) Sec17 release is unaffected by Ykt6-R71G. BJ3505 vacuoles were incubated for 10 min at 26°C in a 150 μl reaction containing 200 ng/ml Sec18 in the presence or absence of Ykt6-R71G (15 μM), anti-Ykt6 (inactive), anti-Ykt6, anti-Vam3, anti-Nyv1, anti-Sec17 or anti-Sec18. Then vacuoles were placed on ice, centrifuged (5 min, 16 000 g, 4°C) and washed twice with 500 μl PSK buffer. Vacuoles were analyzed by immunoblotting with anti-Sec17 and anti-Vam3 antibodies. (E) Excess recombinant Sec17 blocks palmitoylation. Palmitoylation reactions were performed as in (A). Recombinant Sec17 (7 μmol) or Sec18 (12.5 μmol) was added where indicated. At the same concentration, Sec17 inhibited the fusion reaction completely (data not shown), in agreement with previous studies (Wang et al, 2000).

Essential function of the N-terminal domain of Ykt6. (A) Inhibition of fusion by the N-terminal domain. Fusion reactions were performed as described in Materials and methods in the presence of antibodies to Ykt6N or recombinant Ykt6N (22 μM) according to the titration (see the inset). (B) Inhibition of palmitoylation by N-terminal antibodies or recombinant Ykt6N. Palmitoylation in the absence or presence of Ykt6N (22 μM) or anti-Ykt6N was performed as in Figure 1A. (C) Sequence alignment of Ykt6 (accession number P36015) and human fatty acid synthase (P49327) as revealed by Psi-BLAST. The alignment of homologous regions is shown. Identical residues are in black and conserved ones in gray (32% identical, 49% conserved). (D) Ykt6 is proximal to its substrate Vac8 on vacuoles. Isolated BJ3505 vacuoles (60 μg) were incubated with 200 μM of the cleavable crosslinker DSP for 30 min on ice. Vacuoles were then reisolated, lysed in 10 μl 10% SDS, boiled for 5 min at 95°C and then detergent solubilized in 1 ml IP buffer (1% Triton X-100, 300 mM NaCl, 10 mM Tris/HCl, pH 7.4). Immunoprecipitation and analysis was carried out with protein A-coupled antibodies to Vac8 as described (Veit et al, 2001). Immunoblots were decorated with antibodies to Vti1 and Ykt6. (E,F) The N-terminal domain of Ykt6 binds to CoA. [³H]-Pal-CoA (1.7 fmol (0.1 μCi); American Radiolabeled Chemicals, Inc. (E)) or [³H]-acetyl-CoA (same concentration (F)) was incubated alone or together with 20 μg of the indicated protein in 500 μl PS buffer at room temperature for 10 min. Then, a piece of nitrocellulose (1 cm²; Protran, Schleicher and Schuell) was added to the reaction. After 30 min, the nitrocellulose was washed three times for 10 min in PSK buffer. The amount of [³H]-Pal-CoA/[³H]-acetyl-CoA attached to the nitrocellulose was determined by scintillation counting. Unspecific binding of Pal-CoA/acetyl-CoA to nitrocellulose (no protein) was subtracted and set to 0% (CoA: 0%=30 cpm, 100%=170 cpm; Pal-CoA: 0%=2800 cpm, 100%=5000 cpm). The signal obtained for BSA, which binds at least six acyl chains per protein, was divided by six in (E) and set to 100%. BSA was not able to bind to acetyl-CoA (F). (G) Growth kinetics. BJ Gal-Ykt6 (full-length) strains containing chromosomally integrated pRS406 plasmids encoding full-length Ykt6, a deletion mutant lacking amino acids 60–90 (Ykt6Δ60–90) or one lacking the N-terminus (Ykt6ΔN) were grown overnight in galactose-containing YP medium, and then diluted to an OD₆₀₀ of 0.002 in fresh YP medium containing glucose in order to switch off the expression of Gal-Ykt6. The cultures were incubated at 30°C and OD₆₀₀ was determined at the indicated time points.