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Keywords:

  • fusion;
  • protein sorting;
  • SNARE;
  • vacuole;
  • Ykt6

Abstract

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

The dually lipidated SNARE Ykt6 is found on intracellular membranes and in the cytosol. In this study, we show that Ykt6 localizes to the Golgi as well as endosomal and vacuolar membranes in vivo. The ability of Ykt6 to cycle between the cytosol and the membranes depends on the intramolecular interaction of the N-terminal longin and C-terminal SNARE domains and not on either domain alone. A mutant deficient in this interaction accumulates on membranes and – in contrast to the wild-type protein – does not get released from vacuoles. Our data also indicate that Ykt6 is a substrate of the DHHC (Asp-His-His-Cys) acyltransferase network. Overexpression of the vacuolar acyltransferase Pfa3 drives the F42S mutant not only to the vacuole but also into the vacuolar lumen. Thus, depalmitoylation and release of Ykt6 are needed for its recycling and to circumvent its entry into the endosomal multivesicular body pathway.

The dynamics of vesicle budding and fusion along the endomembrane system are determined by several conserved protein families: among them, coat proteins, guanosine triphosphatases (GTPases) of the Rab and ADP ribosylation factor (Arf) family, SNAREs and tethering factors. Most of these proteins are peripheral membrane proteins, which cycle between a cytosolic and a membrane-bound state. Coat proteins require Arf-like proteins to get recruited, and tethers need Rab GTPases for proper function. In contrast, most SNAREs contain a C-terminal membrane anchor and a membrane proximal SNARE domain (1) and therefore permanently reside in membranes. SNAREs are grouped into Q (a, b and c)- and R-SNAREs depending on the central hydrophilic amino acid (2,3). They are found on vesicles and organelles, act after the Rab–tether-mediated vesicle docking stage and drive lipid bilayer mixing as a consequence of their assembly into a four helical trans complex/SNAREpin (4). In general, one R-SNARE combines with three Q-SNAREs. For fusion, at least one SNARE on each membrane requires a transmembrane anchor, whereas the other two SNAREs may be peripheral membrane proteins (5). Moreover, one SNARE may be involved in several fusion reactions as long as the assembly of a productive fusion-competent SNARE complex is possible (6).

The most versatile SNARE in eukaryotes is the R-SNARE Ykt6 (7,8). It is involved in multiple fusion reactions at the Golgi (together with the Q-SNAREs Sed5, Gos1 and Sft1), at the endosome (with Pep12, Vti1 and Syn8) and at the vacuole (with Vam3, Vti1 and Vam7) (9–14). Ykt6 is an essential protein in yeast and consists of an N-terminal longin domain and the membrane proximal R-SNARE domain, which is followed by a palmitoylation and prenylation sequence (9). A similar sequence is found in yeast Ras2 and mammalian N- and H-Ras (15). Prenylation occurs posttranslationally and is essentially irreversible. In contrast, palmitoylation at cysteine residues generates a reversible membrane anchor because the thioester bond can be cleaved by hydrolysis (16). Indeed, Ykt6 is found both on membranes and in the cytosol, and mutations within the conserved C-terminal cysteines inactivate Ykt6 (9,17–19). Both the membrane-associated and the cytoplasmic pool of Ykt6 are prenylated (9). This suggests that functional Ykt6 requires a cycle of dynamic membrane association and dissociation, which is intimately linked to palmitoylation. Our group recently showed that Ykt6 is released in an ATP-dependent reaction from yeast vacuoles and suggested that the release occurs by depalmitoylation (20). In addition, intramolecular interactions between the longin and the SNARE motifs within the Ykt6 protein have been observed and gave rise to the hypothesis that the Ykt6 prenyl anchor can be buried in a hydrophobic groove to facilitate the release from membranes (21,22). Furthermore, a mutation in the conserved amino acid F42 to any charged residue not only abolishes this interaction but also inactivates Ykt6 (21) (our unpublished observations). In mammalian cells, the F42E mutant is found on the Golgi (17) or on unidentified punctate structures in neuronal cells (19).

We decided to address the localization and dynamics of Ykt6 in yeast cells. In this study, we show that Ykt6 localizes to the Golgi, endosome and vacuole, and we provide evidence that its dynamics depend on intramolecular interactions between longin and SNARE domains. This interaction prevents Ykt6 from entering into the multivesicular body (MVB) pathway and thus unwanted degradation inside the vacuole.

Results

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Localization of Ykt6 in yeast cells

Despite its localization to the cytoplasm and membranes, the function of Ykt6 seems to be restricted to membranes because a transmembrane-anchored Ykt6 can partially complement the wild-type function (9) (our unpublished observations). In contrast, mutants of Ykt6 that can no longer stably associate with membranes cannot complement loss of the wild-type protein (17,20). However, attempts to determine the localization of membrane-associated wild-type Ykt6 failed because of the high cytosolic background. Therefore, we decided to place the YKT6 open reading frame (ORF) under the control of the inducible GAL1 promoter. If placed into glucose-containing medium, the GAL1-YKT6 strain grew like wild type for 13 h and then slowed down significantly (Figure 1A). We then compared the subcellular distribution of Ykt6 at three time-points after depletion. In wild-type cells, Ykt6 was found in the vacuole-containing membrane fraction (P13) and in the soluble S100 fraction throughout the experiment (Figure 1B). In GAL1-YKT6 cells, the depletion was most obvious for the cytosolic Ykt6 S100 fraction, which was successively lost after 9 and 13 h. The membrane-bound fraction remained fairly constant until the 9-h time point. We reason that cytosolic Ykt6 is recruited to membranes rather than being selectively degraded because cells still expanded their total membranes because of growth and division.

image

Figure 1. Depletion of Ykt6 from yeast cells. A) Growth curves. Wild-type (wt) yeast cells (CUY1598, see Table S1) or cells with YKT6 under the control of a GAL1 promoter (CUY1870) were grown in YP + galactose before being transferred into glucose-containing YPD medium for 17 h at 30°C. After 10 h, cells were diluted and grown again at 30°C. B) Subcellular fractionation. Yeast strains BY4741 and CUY1870 were subjected to subcellular fractionation at the indicated time-points. Cells were osmotically lysed and cleared by low-speed centrifugation (300 × g, 4°C). Lysates were then fractionated by centrifugation in two subsequent steps to yield P10 (10 000 × g, 4°C, 15 min) and P100 (100 000 × g, 4°C, 60 min) pellets and the S100 supernatant. These were analysed by SDS–PAGE and western blotting using antibodies against Vti1 and Ykt6. C) Localization of Ykt6-eGFP. Cells expressing eGFP-tagged Ykt6 (CUY2367) were grown in YPG or shifted to YPD for 9 h and then analysed by fluorescence microscopy. DIC, differential interference contrast.

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To visualize Ykt6, we generated a strain that expressed green fluorescent protein (GFP)-tagged Ykt6 from a GAL1 promoter and depleted cells for 9 h. The GFP-tagged Ykt6 was functional and behaved like the wild-type protein (data not shown). The cytosolic portion of Ykt6–GFP masked any membrane staining if expressed from its natural (data not shown) or the GAL1 promoter (Figure 1C). Upon depletion, Ykt6 accumulated on membranes (Figure 1B) and became visible on circular structures and dots scattered throughout the cell (Figure 1C).

To identify the compartments, to which Ykt6 localized under these conditions, we added C-terminal red fluorescent protein (RFP) tags to selected markers of the endoplasmic reticulum (ER) (Sec63), the Golgi (Mnn9), the endosome (Snx41) and the vacuole (Vac8). We observed a clear colocalization of GFP–Ykt6 with RFP-tagged Vac8 and Mnn9 (Figure 2A,C). Likewise, some Ykt6–GFP dots colocalized with the endosomal protein Snx41 (Figure 2D), but we did not observe Ykt6 on peripheral or nuclear ER structures stained by Sec63 (Figure 2B). It is possible that Ykt6 was present on the ER or plasma membrane prior to the depletion and that the quantities remaining after a 9-h depletion are below the detection limit of our system. Our localization data are consistent with the function of Ykt6 in Golgi, endosomal and vacuolar SNARE complexes.

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Figure 2. Localization of GFP–Ykt6 in live cells. Yeast strains CUY325, CUY331, CUY851 and CUY327 expressing Ykt6-eGFP and RFP-tagged Vac8 (A), Sec63 (B), Mnn9 (C) or Snx41 (D) were shifted from an overnight culture grown in galactose-containing medium to glucose-containing medium for 9 h before being analysed by fluorescence microscopy. Scale bar: 10 μm.

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Intramolecular interactions and localization of Ykt6

We screened mutants of conserved residues within the longin domain of Ykt6 to identify mutants that are affected in their dynamic localization. We introduced plasmids carrying the selected mutants into ykt6 deletion strains that were kept viable by the simultaneous presence of a plasmid coding for the Ykt6 wild-type protein. Upon loss of the wild-type plasmid, a ykt6 deletion mutant died, as did the F42S mutant (Figure 3A). These data are consistent with previous findings that mutations of F42 to a hydrophilic residue inside the hydrophobic pocket of the longin domain render Ykt6 unable to complement loss of the wild-type protein by preventing the interaction between longin and SNARE domains (21). Among the other mutants tested, only the introduction of a polar serine residue at position 17 (L17S) produced a non-functional protein. To determine the localization of these mutants, we generated GFP-tagged proteins and expressed them in the wild-type background. As shown in Figure 3B, the L17S and H67A mutants displayed a strong cytosolic background comparable to the wild type. However, the F42S mutant accumulated on several organelle membranes and inside the vacuole (Figure 3B), consistent with previous observations in mammalian cells (19). This suggests that the F42S mutant lost the ability to cycle between the membranes and the cytosol. We therefore decided to use this mutant as a tool to explore the sorting and palmitoylation of Ykt6 in more detail.

image

Figure 3. Analysis of Ykt6 longin domain mutants. A) ykt6Δ cells containing a URA3 plasmid coding for wild-type (wt) Ykt6 and plasmids expressing the indicated Ykt6 mutants (CUY2336, CUY1613, CUY2345, CUY2337, CUY2341, CUY2560, CUY2561 and CUY2559) were spotted as serial dilutions on synthetic complete dextrose without histidine (SDC-HIS) to select for the mutant plasmid or 5-Fluoroorotic acid (5-FOA) to displace the plasmid coding for wild-type Ykt6. B) Localization of GFP-tagged Ykt6 wild-type and mutant proteins by fluorescence microscopy in yeast strains CUY2484, CUY2485, CUY2486 and CUY2487.

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First, we introduced Ykt6 F42S as an additional GFP-tagged copy into selected temperature-sensitive mutants of the secretory pathway. If Ykt6 F42S associates with membranes initially at the ER, we would expect that a block in vesicular transport between the ER and the Golgi (sec12), within the Golgi (sec7) or to the plasma membrane (sec6) would lead to an accumulation of the protein. However, our data do not give a clear picture (Figure 4A). Under non-restrictive conditions, wild-type and mutant cells show the same distribution of Ykt6 F42S. If shifted to 37°C, some Ykt6 F42S accumulated in the vacuole in wild-type cells (discussed below). The same redistribution is also seen in the sec12 and sec6 mutants. Only in the sec7 mutant, some additional dot-like structures appear that are distinct from the vacuolar staining and could reflect a Golgi accumulation. Attempts to address this question using wild-type Ykt6 and our depletion assay described above failed because of technical reasons (data not shown).

image

Figure 4. Sorting and release of Ykt6 F42S. A) Localization of Ykt6 F42S in selected temperature-sensitive (ts) mutant strains CUY2727, CUY2728, CUY2730 and CUY2729. GFP-tagged Ykt6 F42S was expressed in wild-type (wt), sec6, sec7 or sec12 ts strains and grown in liquid medium at 23°C or 37°C for 1 h before being analysed by fluorescence microscopy. Scale bar: 10 μm. B) Analysis of Ykt6 release from membranes in yeast strains BJ3505 and CUY3346. Vacuoles were obtained after osmotic lysis of cells and flotation on a discontinuous Ficoll density gradient from the indicated strains. Wild-type vacuoles or vacuoles expressing myc-tagged Ykt6 F42S were incubated for 10 min at 26°C in reaction buffer together with 10 μm CoA and with or without ATP-regenerating system and centrifuged for 10 min at 12 000 × g. Pellets and TCA-precipitated supernatants were analysed by SDS–PAGE and western blotting using an antibody to Ykt6. C) Release of Ykt6 in a mutant lacking the acyltransferase Pfa3 in strains CUY1465 and CUY1494. The experiment was carried out as described for (B) except that membranes were probed for Vti1 and Sec17 in addition to Ykt6. P, pellet; S, supernatant.

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In a second approach to address Ykt6 dynamics, we isolated vacuoles carrying myc-tagged F42S Ykt6 and followed ATP-dependent release of the protein (20). As seen in Figure 4B, wild-type Ykt6 is released from membranes into the supernatant in a reaction dependent on addition of ATP and Sec18. In contrast, the myc-tagged Ykt6 F42S mutant protein was not released from membranes regardless of the absence or presence of ATP. This observation is consistent with the F42S mutant accumulating on membranes in vivo.

To address the question whether the vacuolar acyltransferase Pfa3 plays a role in the dynamics of Ykt6, we analysed release of the protein from pfa3Δ vacuoles but observed no change compared with wild-type membranes (Figure 4C). The well-described release of Sec17 was also unaltered under these conditions (23).

It was previously shown for wild-type Ykt6 that membrane localization is determined by its prenyl and palmitoyl anchors (20). To test whether this also applies to the F42S mutant, point mutations in the cysteine residues of the C-terminal CCIIM sequence were generated. We then localized the GFP-tagged Ykt6 wild-type (CC) and mutant (CA, AC and AA) proteins by fluorescence microscopy and subcellular fractionation (Figure 5A). The mutant lacking the palmitoyl anchor (AC) was still found on membranes, whereas loss of the prenyl anchor (CA) shifted a significant portion of Ykt6 to the cytosolic S100 fraction. However, complete cytosolic localization was only observed if both anchors (AA) were missing, indicating that the dual lipid anchor promotes membrane localization.

image

Figure 5. Analysis of Ykt6 membrane anchoring. A) GFP-tagged Ykt6 F42R proteins with mutations in the C-terminal CCIIM sequence (CC, palmitoylated and farnesylated; CA, non-farnesylated and AC, non-palmitoylated) were analysed by fluorescence microscopy in strains CUY2684, CUY2555, CUY2685 and CUY2556; Scale bar: 10 μm (left) and or subcellular fractionation (right). The analysis was performed as before. B) Localization of fusion proteins anchored to membranes through the Ykt6 membrane targeting CCIIM motif by fluorescence microscopy in strains CUY2332, CUY2397, CUY2334, CUY2398, CUY2573 and CUY2574. CaaX (yEGFP fused to CCIIM), Ykt6ΔC (Ykt6 longin domain fused to CCIIM), Ykt6ΔN (Ykt6 coiled-coil domain fused to CCIIM), Ykt6N–Vam3CC (fusion of Ykt6 longin domain with the Vam3 coiled-coil motif and CCIIM), Ykt6N–Nyv1CC (fusion of Ykt6 longin domain with the Nyv1 coiled-coil motif and CCIIM) and Vam3ΔTMD (Vam3 with CCIIM replacing the transmembrane domain). Scale bar: 10 μm. C) Scheme of constructs used for localization in (B).

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In previous studies, we showed that the CCIIM sequence can be transplanted to the vacuolar SNAREs Vam3 and Nyv1, which we then co-purified with vacuoles (20,24). Both proteins did not get released from membranes, in contrast to the Ykt6 protein. To analyse whether the C-terminal anchor determines the subcellular localization of Ykt6, we fused CCIIM sequence of Ykt6 to the longin domain (Ykt6ΔC) and the coiled-coil domain (Ykt6ΔN). The Ykt6ΔC mutant localized to the plasma membrane and mostly punctate internal structures, whereas the Ykt6ΔN construct localized mainly to the plasma membrane (Figure 5B). Next, we linked the Ykt6 longin domain to the Vam3 and Nyv1 coiled-coil domains and replaced their respective transmembrane domains with CCIIM anchor of Ykt6 (Ykt6N–Vam3CC and Ykt6N–Nyv1CC, respectively) and compared it with Vam3 with its transmembrane domain replaced by CCIIM (Vam3ΔTMD; Figure 5B). None of the two chimaeras showed any remarkable alteration in localization when compared with Vam3ΔTMD. Apparently, the longin domain of Ykt6 is not sufficient to impart localization to internal membranes onto either the Vam3 or the Nyv1 coiled-coil domains. These observations support the notion that its intramolecular interaction and not either domain alone determines the sorting and dynamics of Ykt6.

Dynamics and palmitoylation of Ykt6

Dynamics of Ykt6 must be governed by a consecutive palmitoylation and depalmitoylation event, which seems to be linked to its intramolecular interactions (17). Several studies indicate that a family of acyltransferases with a common cytosolic DHHC (Asp-His-His-Cys) motif mediates palmitoylation. In yeast, seven isoforms exist. Specific targets have been reported for some DHHC proteins, but they also seem to have a substantial overlap in substrate recognition (25) (our unpublished observations). For instance, loss of the vacuolar DHHC Pfa3 leads to a mislocalization of the fusion factor Vac8 to the cytosol (26,27).

To address the question how Ykt6 localizes in the absence of single or multiple DHHCs, we employed our previously described depletion assay (Figure 1) in strains lacking selected acyltransferases (25). These depletions – contrary to those presented in Figures 1 and 2– were carried out with a strain still containing the genomic copy of wild-type YKT6 because we were not able to delete it in our DHHCΔ background strains. When we depleted the Ykt6 protein to observe potential changes in its membrane localization, only a deletion in five DHHC proteins, which included Pfa3, led to a dot-like accumulation of Ykt6 proximal to the vacuole (Figure S2A). This was not observed in all cells, and some accumulation was already seen in wild-type cells. We speculate that the presence of the untagged version of Ykt6 obstructed the accumulation of GFP-tagged Ykt6 on membranes.

We then tried to address acyltransferase-mediated palmitoylation of Ykt6 by expressing the GFP-tagged F42S mutant under the control of the GAL1 promoter in various DHHC deletion backgrounds. However, we were unable to detect a strong effect on the localization of GFP–Ykt6 F42S because of the loss of any one or multiple DHHC proteins (Figure S2B), suggesting that multiple DHHCs can palmitoylate Ykt6. In a further attempt to test the effect of deletions in DHHCs, we expressed the Ykt6ΔN construct (Figure 5C) in the same background strains. However, removal of the longin domain and expression of this construct in the different DHHCΔ backgrounds did not show any changes in its localization compared with the wild-type strain (Figure S2C).

Therefore, we decided to take advantage of a DHHC overexpression approach in yeast lacking five of the seven DHHC proteins to address the question of acyltransferase-mediated palmitoylation of Ykt6 F42S. If the vacuolar DHHC protein Pfa3 is overexpressed, it localizes to the vacuole and a prevacuolar compartment and can redirect proteins to the vacuolar surface (H. Hou, C. Meiringer, K. Subramanian and C. Ungermann, submitted). We expressed the GFP-tagged F42S mutant under the control of the constitutive NOP1 promoter in wild-type cells and the 5×DHHC deletion background overexpressing Pfa3 from the GAL1 promoter (Figure 6A). The latter strain grown in glucose is depleted of Pfa3, whereas in galactose-containing media, Pfa3 is strongly overexpressed compared with wild-type levels of the protein. Ykt6 F42 was found on the plasma membrane and on internal membranes including the vacuolar rim in the 5×DHHCΔ background under Pfa3 depletion conditions (Figure 6A). In contrast, some Ykt6 F42S appears to be sorted to the vacuolar lumen in wild-type cells under these conditions (compare also with Figure 5). Strikingly, overexpression of Pfa3 in the DHHC deletion background led to an almost complete shift of Ykt6 F42S into the vacuolar lumen (Figure 6A). In the wild-type background, some increase in vacuolar accumulation was also observed; however, the plasma membrane and most of the endomembranes still retained considerably more Ykt6 F42S compared with the Pfa3 overexpressor (Figure 6A). When we tested wild-type Ykt6 in the same strain backgrounds, localization of the protein was always cytosolic (Figure S1A).

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Figure 6. Depalmitoylation of Ykt6 is necessary for its recycling. A) Localization of eGFP-Ykt6 F42S in the wild-type (wt) yeast strain CUY3180 and the 5× DHHCΔ background (CUY2929) with Pfa3 under the control of the GAL1 promoter. Cells were grown either in glucose- – depletion of Pfa3 – or in galactose-containing medium – overexpression of Pfa3. In (B), an eGFP-Ykt6 F42S version lacking the palmitoylatable cysteine (CC[RIGHTWARDS ARROW]AC) is expressed in the same strain backgrounds as above (CUY3181 and CUY2968, respectively). C) Localization of GFP-tagged Vam3–CaaX in the absence or presence of overproduced Pfa3. The analysis was performed as in (A) with strains CUY3242 andCUY3065. Scale bar: 10 μm. D) Ykt6 F42S and Vam3–CaaX localization in strains overexpressing either Pfa4 or Pfa5 (CUY3079 and CUY3081 as well as CUY3082 and CUY3084, respectively).

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To control that relocalization of the F42S mutant was not because of differences in the growth conditions, but attributable to palmitoylation, we tested how a non-palmitoylable mutant (AC) would localize. Indeed, overexpression of Pfa3 had no effect on the localization of this mutant (Figure 6B). This is in agreement with our finding of intravacuolar accumulation of Ykt6 F42S carrying the wild-type membrane anchor, whereas the AC mutant was only found on vacuolar surfaces in the wild-type strain background (compare with Figure 5A).

The vacuolar t-SNARE Vam3 travels to the vacuole by the adaptor protein (AP)3 pathway (28). It thereby bypasses the endosome and also has no reported function on this organelle. To address the question whether any vacuolar protein carrying lipid anchor of Ykt6 (CCIIM) is sorted into the vacuolar lumen upon Pfa3 overexpression, we investigated the localization of GFP–Vam3 with its transmembrane domain replaced by CCIIM (Vam3–CaaX) in the same strains as the Ykt6 F42S mutant. Akin to Ykt6 F42S, Vam3–CaaX is found on vacuoles and the plasma membrane and does not get released during fusion (20), (Figure 6C). However, in the 5×DHHCΔ background, Vam3–CaaX localized exclusively to the plasma membrane when cells were depleted of Pfa3. Upon overexpression of Pfa3, Vam3–CaaX accumulated on the vacuolar membrane. However, Vam3–CaaX was not sorted into the vacuolar lumen. To control that interference of the AP3 pathway does not play a role in the sorting of Vam3–CaaX, we deleted the AP3 coat subunit gene APL5 in the 5×DHHCΔ background overexpressing Pfa3, thus forcing Vam3–CaaX into the MVB pathway. Neither Vam3–CaaX nor Ykt6 F42S was affected in its localization because of the apl5 deletion (Figure S1B). Therefore, both proteins take the same route to the vacuole by the endocytic pathway but encounter a different fate when MVBs are formed.

To address the question whether any other acyltransferase affects sorting of Ykt6 F42S and Vam3–CaaX, we overproduced ER-localized Pfa4 and plasma membrane-localized Pfa5. Ykt6 F42S was sorted into the vacuolar lumen upon overproduction of Pfa4 but not Pfa5 (Figure 6D). In contrast, both Pfa4 and Pfa5 imparted vacuolar localization upon Vam3–CaaX, although the effect was stronger for Pfa4 overproduction. We can only speculate that Vam3–CaaX may be more readily palmitoylated by Pfa5 than Ykt6 F42S.

Next, we directly investigated the palmitoylation state of Ykt6 F42S by the biotin switch method (29). Upon overproduction of Pfa3, GFP–Ykt6 F42S becomes strongly palmitoylated (Figure 7A), whereas a mere shift to galactose of the 5×DHHC deletion strain not overexpressing Pfa3, which showed some sorting of Ykt6 F42S into the vacuole, did not yield any palmitoylated Ykt6 F42S. Similarly, relocalization of Vam3–CaaX to the vacuole upon Pfa3 overproduction also coincided with an increase in palmitoylation (Figure 7B). Sorting into the vacuole through MVBs therefore coincides with an acyltransferase-mediated palmitoylation of the Ykt6 F42S mutant. This increase in palmitoylation is distinct from the Ykt6 autoacylation, which can be observed in vitro for wild-type protein but only poorly for the Ykt6 F42S mutant (Figure S3) (30). Thus, Ykt6 F42S is directly palmitoylated by Pfa3 and as a consequence gets sorted into the vacuole where it is then degraded.

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Figure 7. Sorting of Ykt6 F42S depends on palmitoylation and ESCRT. A) Palmitoylation of Ykt6 F42S in the 5×DHHCΔ strain with Pfa3 either under the control of the GAL1 promoter (CUY2929) or knocked out (CUY2966) were analysed by the biotin switch method after growth in galactose-containing medium. Free cysteines in lysates were quenched by incubation with 25 mm NEM followed by treatment with hydroxylamine (H3NO), pH 7.4, in the presence of 300 μm biotin-BMCC to specifically label palmitoylated cysteines. Afterwards, biotinylated proteins were precipitated using neutravidin–agarose. The 5% load corresponds to 5% of the input before the precipitation by neutravidin–agarose. Proteins were separated by SDS–PAGE, blotted and blots decorated with an antibody against Ykt6. B) Palmitoylation of Vam3–CaaX. Strain CUY3065 was grown in parallel in glucose- and galactose-containing medium and analysed by the biotin switch method as described above. C) ESCRT dependence for Ykt6 F42S sorting into MVBs. Yeast strains CUY3008 and CUY3086 were analysed as described in Figure 6.

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Finally, we asked whether sorting of Ykt6 F42S into the lumen of the vacuole depends on the ESCRT machinery. Indeed, luminal sorting of Ykt6 F42S under conditions were Pfa3 is overexpressed depends on palmitoylation and ESCRT function: deletion of the class E gene VPS4 relocalized Ykt6 F42S to the endosome and the vacuolar surface (Figure 7C) and prevented its entry into the vacuolar lumen. Deletion of VPS4 also resulted in an accumulation of Vam3–CaaX in distinct dots adjacent to the vacuole when Pfa3 was overexpressed (Figure 7C), indicating that it indeed travels by the endosomal pathway towards the vacuole.

Both Ykt6 F42S and Vam3–CaaX are anchored identically to membranes and follow the same transport pathways. However, whereas Vam3–CaaX ends up on the vacuolar surface, Ykt6 F42S is internalized into the MVB. Internalization is dependent on the ESCRT machinery and stably membrane-anchored Ykt6. Ykt6 can be palmitoylated by the DHHC acyltransferase network and appears to depend on depalmitoylation to avoid sorting into MVBs and ultimately the vacuolar lumen.

Discussion

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Our study addresses the localization and dynamics of the dually lipidated SNARE Ykt6. Using a depletion approach, we were able to localize Ykt6 to the Golgi, endosome and vacuole. Impaired cycling of a mutant of Ykt6 (F42S) resulted in permanent membrane localization. Moreover, loss of depalmitoylation directed this mutant protein into the vacuolar lumen and misrouting required the ESCRT machinery. Taken together, this implies that release of Ykt6 at the endosome depends on a functional longin domain and probably its intramolecular interaction with the C-terminal SNARE/CCIIM domain.

Previous attempts to localize Ykt6 within yeast failed because of the abundant cytosolic population of Ykt6 (9). Our depletion approach reveals an in vivo localization of Ykt6 at membranes that is consistent with the previously identified presence of Ykt6 in selected SNARE complexes at the Golgi, at the endosome and at the vacuole (see previously). Our procedure might not be sufficiently sensitive to detect Ykt6 at other membranes; ER-localized Ykt6 might translocate to the Golgi during the depletion phase prior to the visual analysis. At least at the ER, an interaction of Ykt6 with the ER SNAREs Use1 and Ufe1 has been described (31). In agreement with this, we could show that Ykt6, and also Ykt6 F42S, binds to the Dsl tethering complex at the ER (Meiringer et al., manuscript in preparation). We also cannot exclude that wild-type Ykt6 is also present at the plasma membrane. Wild-type Ykt6 might get endocytosed or released from membranes during the depletion approach.

Ykt6 function clearly depends on a functional longin domain. Tochio et al. showed that the longin domain is a regulatory domain that binds to the C-terminal coiled-coil part. This interaction might be a prerequisite to accommodate the prenyl anchor in the cytosolic Ykt6 population. We could show that yeast Ykt6 F42S is strongly membrane bound (in accordance with data for the F42E mutant used in the study by Fukasawa et al.) and does not get released from membranes. Interestingly, Ykt6 F42S is also found on the plasma membrane under these conditions, suggesting a possible role of Ykt6 at this location. We can, however, not exclude that this localization is a consequence of its impaired cycling, which may redirect Ykt6 to several endomembranes, because other SNAREs equipped with the same anchor show a similar intracellular distribution (Figure 6C).

The interaction of N-terminus and C-terminus is linked to the ability of Ykt6 to loose its palmitoyl anchor. Palmitoylation of Ykt6 has been implicated by mutational analyses, although palmitoylation was demonstrated for the F42E mutant only (17,19,20). A previous study on the localization of Ykt6 in mammalian cells has shown that Ykt6ΔN and full-length Ykt6 carrying the F42E mutation are found on the Golgi (17). The non-prenylated CS and SS mutants of Ykt6 F42E localized to the cytoplasm and the nucleus, whereas the non-palmitoylated SC mutant showed cytoplasmic and Golgi localization. This is mostly in accordance with our data on the localization of Ykt6 in yeast, although we find Ykt6 F42S to be localized inside the vacuolar lumen, the plasma membrane and punctate structures, which are presumably the Golgi (Figure 5A). Our AC mutant localized to the ER, the vacuolar rim and the Golgi, indicating a broader membrane distribution compared with the situation in mammalian cells.

A second study on the localization of Ykt6 in neuronal cells presented a different picture (19). Ykt6 as well as its longin domain alone appear to localize to unidentifiable punctate structures with no apparent connection to the endomembrane system. The F42E mutation in the longin domain did not change its localization, but in the context of the full-length protein, this mutant relocalized to the Golgi and the plasma membrane. More importantly, this relocalization to the Golgi was reverted by removing the distal farnesylated cysteine residue or removing the entire CaaX anchor. Similarly, grafting various other longin domains onto the Ykt6 C-terminal domain caused all investigated chimaeras to localize to the Golgi and in the case of the Sec22b longin domain also to the plasma membrane. These data are more difficult to reconcile with our findings because we lack the described unidentified punctate structures in yeast (Figure 3B). In addition, our CA CaaX mutant does not revert to the wild-type localization but rather shows an accumulation on the vacuolar rim and ER (Figure 5A).

Additional insights into Ykt6 and the role of its farnesylation were provided by a recent structural study (22). Interestingly, farnesylated Ykt6 behaves like a non-modified protein in gel filtration, suggesting a tight packing of the farnesyl anchor to the longin domain. The binding most likely includes the hydrophobic pocket proximal to F42, which also forms an artificial dimerization interface in the crystal structure of the longin domain. Thus, the hydrophobic face at F42 has two functions: it binds the SNARE domain (21) and accommodates the farnesyl anchor (22), which in turn explains its strong membrane association.

The polytopic membrane proteins of the DHHC family have been implicated in palmitoylation (32), and we show in this study that overproduction of the vacuolar (and possibly endosomal) Pfa3 protein directly affects the localization of Ykt6 F42S (Figure 6A). This is a consequence of palmitoylation; a non-palmitoylated Ykt6 F42S (AC) shows similar localization regardless of Pfa3 overexpression and does not get sorted into the vacuolar lumen. Likewise, we could not observe wild-type Ykt6 inside the vacuole if Pfa3 was overproduced (Figure S1A). Apparently, depalmitoylation of Ykt6 is linked to its recycling. Whether this release occurs exclusively at the endosome or also at the vacuole cannot be deduced from our data. It is possible that Ykt6 undergoes several palmitoylation–depalmitoylation cycles, which we cannot detect with our assays. Consistent with this idea, we observe wild-type Ykt6 during our depletion approach not only at endosomes but also on the entire vacuolar surface (Figure 2). In vivo, Ykt6 may reach the vacuolar surface by an alternative route, for example the Cvt pathway or may be directly recruited to the vacuole from the cytosol.

It is remarkable that the autoacylation reaction using non-prenylated Ykt6 shows exactly the reverse preference; the wild-type protein but not the F42S mutant is efficiently autoacylated (Figure S3) (30). Within our experimental set-up, we could not observe any palmitate turnover on wild-type Ykt6. We speculate that the in vitro assay could mirror the reverse, depalmitoylation reaction, which requires the interaction of longin and SNARE domains. Future experiments need to address the mechanism of Ykt6 deacylation in vivo.

We believe that this ESCRT-dependent luminal sorting of Ykt6 at the endosome is a consequence of the poor release of Ykt6, which suggests an important function of Ykt6 at the endosome. Indeed, Ykt6 forms a SNARE complex with the vacuolar SNAREs Vam3, Vam7 and Vti1 and has been implicated in protein trafficking between the endosome and the vacuole (10,11,13). We postulate that this complex is required for endosome–vacuole fusion, which may include changes in the palmitoylation status of Ykt6. At the vacuole, Ykt6 has been linked to early stages of fusion (18). How Ykt6 function is related to its endosomal sorting and whether it is part of an as yet unidentified protein complex involved in MVB formation need to be determined in future studies. Likewise, the molecular details of Ykt6 release from membranes will need to be clarified.

Methods

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Molecular biology and yeast strains

Yeast strains used in this study are listed in Table S1. These were generated either by homologous recombination of polymerase chain reaction-amplified fragments or by transformation with plasmids (see subsequently). Pfu polymerase as well as DNA modification and restriction enzymes were obtained from Fermentas. The boundary for the Ykt6 longin domain (Ykt6LD) was used as described (21) and extends from amino acid 1 to 140, and the coiled-coil domain (Ykt6CC) is from I141 to M200. Vam3 coiled coil (Vam3CC) is from T190 to V265, and Nyv1 coiled coil (Nyv1CC) is from I167 to N221. Point mutations in Ykt6 were made using the QuickChange Site-Directed Mutagenesis Kit from Stratagene.

For depletion of Ykt6 from cells, cells were initially grown in YPG to log phase, then diluted and grown for 13 h to an optical density (OD)600 of 0.5. Serial dilutions of yeast cells were carried out as described (H. Hou et al., submitted).

For Ykt6 tagged with GFP, emerald GFP (eGFP) or yeast enhanced GFP (yEGFP) was cloned in frame either at the N-terminus of Ykt6 (GFP–Ykt6) or into an unstructured loop found in the longin domain of Ykt6 (Ykt6-eGFP).

Genes for PFA3, PFA4 and PFA5 were subcloned from the Yeast Overexpression ORF Library (Open Biosystems) into plasmid pRS406 as a SacI–KpnI fragment containing the GAL1 promoter and the C-terminally 6×HIS-HA-PrtA tagged DHHC. The plasmids were then digested with AgeI inside the GAL1 promoter and integrated genomically.

Microscopy

Yeast cells were grown to early log phase, harvested, washed once with phosphate buffered saline (PBS) and mounted on object slides. For the depletion assay of Ykt6–GFP, cells were grown overnight in yeast peptone (YP) + 2% galactose, followed by an 8-h chase in YP + 2% glucose. Visualization was performed using a fluorescence microscope (Leica DM5500 B; Leica Microsystems GmbH) equipped with an internal filter wheel (D460sp, BP460-515 and D580lp; Leica Microsystems GmbH) and fluorescence filters (fluorescein isothiocyanate: Exc. D480/30, Em. D535/40; Wide Green: Exc. D535/50, Em. E590lp; Chroma Technology Corp.), captured with a digital camera (Spot Pursuit-XS Monochrome; Diagnostic Instruments, Inc.) and processed using metamorph v6.3 (Molecular Devices Corp.) and autoquant x v1.3.3 (Media Cybernetics, Inc.).

Subcellular fractionation

Fractionation of yeast membranes was carried out as described (33). Yeast cells were osmotically lysed, and membranes fractionated by differential centrifugation. These were resuspended in sodium dodecyl sulphate (SDS) sample buffer and proteins separated by SDS–PAGE, followed by immunoblotting and detection using primary antibodies derived from rabbits and secondary antibodies coupled with either Alexa 680 or IRDye 800.

Release of Ykt6 from vacuole membranes

Yeast vacuoles from indicated strains were isolated by diethylaminoethyl–dextran-mediated lysis and floted on a discontinuous Ficoll gradient as described (34). ATP-dependent release of Ykt6 and Ykt6 (F42S) from vacuoles was performed as described (20). In brief, 30 μg of vacuoles was incubated in fusion buffer (20 mm PIPES/KOH, pH 6.8, 200 mm sorbitol, 125 mm KCl (PSK) and 5 mm MgCl2) at 26°C with 10 μm CoA and with or without ATP-regenerating system (0.5 mm ATP, 40 mm creatine phosphate and 0.1 mg/mL creatine kinase) for 10 min. Vacuoles were pelleted at 12 000 × g for 10 min, washed once with 500 μL of PSK buffer and resuspended in SDS sample buffer. The supernatant after the first spin was trichloroacetic acid (TCA) precipitated and equally resuspended in sample buffer.

Biotin switch

The biotin switch experiment was performed as described (35). In brief, 60 OD600 units of yeast cells grown overnight in either YP + 2% glucose or 2% galactose was harvested, resuspended in PBS containing 5 mm ethylenediaminetetraacetic acid and 0.5× protease inhibitor cocktail and lysed using glass beads on a disruptor genie device (33). Unbroken cells were pelleted and the protein concentration determined. Total protein (500 μg) was extracted using 1% Triton-X-100, and free cysteines were quenched by addition of 25 mm n-ethylmaleimide (NEM). Following chloroform/MetOH precipitation, pellets were resuspended in resuspension buffer (50 mm Tris, pH 7.4, 100 mm NaCl, 8 m urea and 2% SDS) and treated with either 1 m Tris or 1 m hydroxylamine, pH 7.4, together with 300 μm biotin-BMCC (1-Biotinamido-4-[4′-(maleimidomethyl)cyclohexanecarboxamido]butane, Pierce Biotechnology). Proteins were again precipitated with chloroform/MetOH, resuspended as before and diluted with 1 mL of lysis buffer containing 0.1% Triton-X-100. Samples were then incubated on neutravidin–agarose (Pierce Biotechnology), washed and eluted by boiling in SDS sample buffer.

Protein purification

The full-length ORFs of YKT6 and YKT6 carrying the F42S mutation were cloned into the bacterial expression vector pETHIS (derived from pET32a(+) by removal of the enterokinase site and the thioredoxin tag) using BamHI–HindIII. Proteins were expressed in BL21 Rosetta by addition of 0.3 mm isopropyl-thiogalactoside (IPTG) overnight at 20°C. Purification was performed using Ni-NTA agarose (Qiagen) and eluted using 250 mm imidazole, pH 7.5. Buffer exchange on the purified proteins into a buffer containing 20 mm Tris, pH 7.4, 5% glycerol and 150 mm NaCl was performed with a PD10 column (GE Healthcare).

In vitro palmitoylation

Purified Ykt6 and Ykt6 containing the F42S mutation were incubated in a 100 μL reaction buffer containing 20 mm Tris, pH 8.0, 100 mm NaCl, 0.015% Triton-X-100 and 1 mm DTT with or without 45 pmol [3H] Pal-CoA (2.2 μCi) for 15 min at 30°C. The reaction was quenched by addition of 900 μL reaction buffer without DTT but with 2.5 mm NEM and 10 μm Pal-CoA and incubated for 0, 10, 30 and 60 min, followed by TCA precipitation. Samples were resuspended in SDS sample buffer without reducing agent and separated on 13% SDS acrylamide gels. Gels were then stained with Coomassie, destained and dried onto a layer of Whatman paper and subsequently exposed to film.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

We thank Matthias Geyer and Clemens Ostrowicz for comments on the manuscript and members of the laboratory for fruitful discussions. This study was supported by the Deutsche Forschungsgemeinschaft (SFB 431), the Fonds der Chemischen Industrie and the Hans-Mühlenhoff foundation (to C. U.).

References

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Figure S1: Localization of Ykt6 F42S in DHHC deletion strains. Localization of Ykt6-eGFP (A, after shift to glucose-containing medium for 9 h) or eGFP-Ykt6 (F42S) under control of the GAL1 promoter (B) was done in the indicated strains (CUY2523, CUY2524, CUY2527, CUY2525 and CUY2526 as well as CUY2324, CUY2323, CUY2321 and CUY2326) using fluorescence microscopy as described previously. C) Localization of yEGFP-tagged Ykt6▵N was analyzed in the indicated DHHC▵ strains CUY2540, CUY2541, CUY2542 and CUY2718.

Figure S2: Depalmitoylation of Ykt6 is necessary for its recycling. A) Localization of GFP–Ykt6 was analyzed in the same strains as described in Figure 6A (CUY2967, CUY3017 and CUY3015). B) Localization of GFP–Ykt6 F42S and Vam3–CCIIM was analyzed in strains overexpressing Pfa3 and deleted for APL5 (CUY3191 and CUY3192, respectively). C) Ykt6 F42S localization in strains deleted for VPS4 in the wild-type background (CUY3239) and in the 5× DHHC▵ background (CUY2990).

Figure S3: Dynamics of Ykt6 palmitoylation in vitro. A) Recombinant Ykt6 and Ykt6 F42S were incubated with 2.2 μCi of [3H] Pal-CoA in the presence of 1 mm DTT (pulse) for 15 min at 30°C. DTT and [3H] Pal-CoA were then quenched by an excess of NEM and cold Pal-CoA for the indicated times. Proteins were precipitated, separated by SDS–PAGE and analysed by radiography. ML represents maximum labeling by [3H] Pal-CoA, N the negative control without [3H] Pal-CoA and MQ maximum quenching where NEM and cold and [3H] Pal-CoA were premixed before the addition of recombinant proteins.

Table S1: Yeast strains used in this study

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