The SNARE Ykt6 is released from yeast vacuoles during an early stage of fusion

Abstract

The farnesylated SNARE (N-ethylmaleimide-sensitive factor attachment protein receptor) Ykt6 mediates protein palmitoylation at the yeast vacuole by means of its amino-terminal longin domain. Ykt6 is localized equally to membranes and the cytosol, although it is unclear how this distribution is mediated. We now show that Ykt6 is released efficiently from vacuoles during an early stage of yeast vacuole fusion. This release is dependent on the disassembly of vacuolar SNAREs (priming). In recent literature, it had been demonstrated for mammalian Ykt6 that the membrane-bound form is both palmitoylated and farnesylated at its carboxy-terminal CAAX box, whereas soluble Ykt6 is only farnesylated. In agreement with this, we find that yeast Ykt6 becomes palmitoylated in vitro at its C-terminal CAAX motif. Mutagenesis of the potential palmitoylation site in yeast Ykt6 prevents stable membrane association and is lethal. On the basis of these and other findings, we speculate that Ykt6 is released from membranes by depalmitoylation. Such a mechanism could enable recycling of this lipid-anchored SNARE from the vacuole independent of retrograde transport.

Figure 1

Palmitoylation of Ykt6. (A) In vitro palmitoylation of Ykt6. Recombinant proteins (His–Ykt6 and Vac8–GST) were incubated in PSK buffer (PS buffer plus 125 mM KCl) and [³H]Pal-CoA for 30 min at 26°C, and then precipitated and analysed by SDS–PAGE and fluorography. Ykt6 in lane 3 had been preheated for 10 min at 95°C. The Coomassiestained gel is shown on the right. All lanes shown are taken from the same gel but have been regrouped for clarity. (B) Palmitoylation occurs at the C-terminal CAAX box. Incubations were as in (A) with His–Ykt6 (3 μg). Samples were processed as before. Only Ykt6 bands are shown. (C) Model of the Ykt6 domain structure. Arrows indicate the palmitoylation targets of Ykt6.

Figure 2

Gel mobility of purified Ykt6 and the AAIIM mutant. His–Ykt6-WT and His–Ykt6-AAIIM were purified from Gal-overproducing strains as described in Methods, and analysed by reducing or nonreducing 4–10% gradient gels (Invitrogen, Karlsruhe, Germany). Indicated bands were identified by MALDI mass spectrometry.

Figure 3

Analysis of Ykt6 CAAX box mutants. (A) In vivo analysis. CUY434 (BY4743 ykt6Δ; pRS416.YKT6Pr-YKT6; contains Ykt6-WT on a plasmid with a URA selection marker) was transformed with plasmids encoding Ykt6 with the respective mutations in the CAAX box and plated on 5-FOA to induce loss of the original plasmid. CAIIM, ACIIM and AAIIM indicate the C-terminal amino acids that were mutated (WT=CCIIM). (B,C) Expression and membrane localization of CAAX box mutants. CUY434 was transformed with vectors encoding Ykt6 wild type or mutants (all are internally GFP-tagged to distinguish them from the endogenous protein). (B) Cells were lysed in 0.25 N NaOH, 140 mM β-mercaptoethanol (β-ME), 3 mM PMSF, and then TCA-precipitated and analysed by SDS–PAGE and western blotting. (C) To obtain membranes, cells were lysed as for vacuole purifications, except that the lysis buffer was 200 mM sorbitol, 50 mM KOAc, 2 mM EDTA, 20 mM Hepes–KOH (pH 6.8) and 1 × PIC (Haas, 1995). The total lysate was cleared by centrifugation at 400g (5 min, 4°C) and the resulting supernatant was centrifuged for 15 min at 13,000g. The resulting ‘P13' pellets were collected and diluted to 0.3 mg/ml in PS buffer containing 0.1 × PIC. Proteins were analysed as above.

Figure 4

Release of Ykt6 from yeast vacuoles. (A) ATP-dependent release of Ykt6. Vacuoles (30 μg), isolated from BJ3505, the vacuolar protease-deficient strain, were incubated for 10 min at 26°C in fusion buffer (20 mM PIPES, pH 6.8, 200 mM sorbitol, 125 mM KCl, 5 mM MgCl₂), CoA (10 μM), with or without an ATP-regenerating system (0.5 mM ATP, 40 mM creatine phosphate, 0.1 mg/ml creatine kinase), and then pelleted (10 min, 12,000g, 4°C). Proteins of the supernatant (Sup.) were precipitated with 13% trichloroacetic acid, and the pellet fraction (Pel.) was washed in 500 μl of PSK buffer before analysis by SDS–PAGE and western blotting. (B) Time course of Ykt6 release. Incubations and analysis were as for the pellet fractions in (A). At the indicated time points, samples were removed and set on ice. (C) Inhibitor analysis of Ykt6 release. An experiment showing Sec17 release (Dietrich et al, 2004) was redecorated with Ykt6 and Vam3 antibodies. (D) Ykt6 migrates as a single band on treatment with reducing agent (right) or release from the membrane (right). Right: isolated vacuoles were heated in sample buffer in the absence or presence of β-mercaptoethanol (β-ME). Left: protease-deficient vacuoles were incubated in standard fusion reactions (45 μl), in the absence or presence of ATP for 30 min. Vacuole membranes were re-isolated (pellet) and supernatants were TCA-precipitated (Sup.). In both experiments, Ykt6 was analysed by nonreducing 12% SDS–PAGE (10-cm-long gel). Note that mobility of Ykt6 forms depends on the gel system used.

Figure 5

Release of CCIIM-anchored vacuolar SNAREs. (A) Fractionation of lipid-anchored SNARE. Cells expressing CCIIM-anchored Nyv1 or Vam3 were prepared for vacuole preparations, but before centrifugation, aliquots of total cell extract (total) were removed and placed into sample buffer or centrifuged (30 min, 100,000g, 4°C). The supernatant fraction was TCA-precipitated. Proteins were analysed as before. Total and vacuoles, 30 μg each; supernatant was obtained from 150 μg of total. (B) Ykt6 release assay, performed as in Fig 4A. (C) Solubility of released Ykt6. Following the ATP-dependent release, reaction samples were centrifuged (10 min, 14,000g, 4°C). Half of the supernatant was TCA-precipitated (S14), and the other half was centrifuged again (30 min, 100,000g, 4°C). The resulting supernatant (S100) was TCA-precipitated. Pellets from each centrifugation (P14 and P100) were resuspended in sample buffer and analysed as before. Vti1 served as a control for a membrane-bound protein. (D) Model of the Ykt6 cycle. The Ykt6 longin domain is shown in yellow, and the SNARE domain in blue. The prenyl (black) and the palmitate anchor (red) are indicated. Vac8 is shown in purple.