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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Sphingolipids play critical roles in many physiologically important events in the yeast Saccharomyces cerevisiae. In this study, we found that csg2Δ mutant cells defective in the synthesis of mannosylinositol phosphorylceramide exhibited abnormal intracellular accumulation of an exocytic v-SNARE, Snc1, under phosphatidylserine synthase gene (PSS1)-repressive conditions, although in wild-type cells, Snc1 was known to cycle between plasma membranes and the late Golgi via post-Golgi endosomes. The mislocalized Snc1 was co-localized with an endocytic marker dye, FM4-64, upon labelling for a short time. The abnormal distribution of Snc1 was suppressed by deletion of GYP2 encoding a GTPase-activating protein that negatively regulates endosomal vesicular trafficking, or expression of GTP-restricted form of Ypt32 GTPase. Furthermore, an endocytosis-deficient mutant of Snc1 was localized to plasma membranes in PSS1-repressed csg2Δ mutant cells as well as wild-type cells. Thus, the PSS1-repressed csg2Δ mutant cells were indicated to be defective in the trafficking of Snc1 from post-Golgi endosomes to the late Golgi. In contrast, the vesicular trafficking pathways via pre-vacuolar endosomes in the PSS1-repressed csg2Δ mutant cells seemed to be normal. These results suggested that specific complex sphingolipids and phosphatidylserine are co-ordinately involved in specific vesicular trafficking pathway.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Complex sphingolipids are a major component of the eukaryotic plasma membrane, and function in various cellular processes, such as proliferation, differentiation and adhesion (Hakomori and Igarashi, 1993). They consist of a hydrophilic head attached to a ceramide, which contains a fatty acid and a sphingolipid long-chain base. Complex sphingolipids dynamically cluster with sterols to form lipid microdomains, which function as platforms for effective signal transduction and localization of membrane proteins (Simons and Ikonen, 1997). In mammals, sphingolipids can carry phosphocholine or hundreds of carbohydrate chains as polar head groups, and this structural diversity and complexity is thought to attribute to their multiple biological functions.

In the budding yeast Saccharomyces cerevisiae, the fundamental structure of complex sphingolipids is similar to that in mammalian cells; however, the characteristics of the polar head groups of yeast sphingolipids is different. Yeast complex sphingolipids can carry one of only three different types of polar head group, and therefore complex sphingolipids in yeast can be divided into three types, IPCs (inositol phosphorylceramides), MIPCs (mannosylinositol phosphorylceramides), and M(IP)2Cs (mannosyldiinositol phosphorylceramides), all of which include phosphoinositol (Dickson and Lester, 2002). IPCs, the simplest complex sphingolipids in yeast, are formed by IPC synthase (Aur1), an enzyme catalysing the transfer of the head group of PI to ceramides (Nagiec et al., 1997). There are five IPCs (IPC-A, -B, -B′, -C and -D), which differ in the state of hydroxylation of the ceramide moiety (Dickson and Lester, 2002). Subsequently, IPCs are converted to MIPCs through the addition of mannose, and Csg1, Csh1 and Csg2 are involved in MIPC synthesis (Zhao et al., 1994; Beeler et al., 1997; Uemura et al., 2003). Finally, MIPCs are converted to M(IP)2Cs through the addition of another phosphoinositol, which is catalysed by Ipt1 (Dickson et al., 1997). Due to the limited molecular classes of polar head groups, the structural diversity of sphingolipids in yeast is relatively simple as compared with that in mammalian cells, making yeast a useful model for investigating the physiological significance of the structural complexity of sphingolipids. AUR1 is an essential gene, because a defect of it results in reductions in all the complex sphingolipid levels and in the accumulation of ceramides (Nagiec et al., 1997), whereas CSG1, CSH1, CSG2 and IPT1 are basically non-essential for yeast cell growth, indicating that loss of the synthesis of MIPCs and M(IP)2Cs does not cause a lethal phenotype if IPCs are synthesized.

It has been reported that sphingolipids are involved in vesicular trafficking systems in eukaryotes from yeast to mammals. For instance, sphingolipid long-chain bases are involved in the formation of eisosome, and then regulation of endocytosis via the Pkh1 and Pkh2 protein kinase-mediated signalling pathways in budding yeast (Walther et al., 2007; Luo et al., 2008). In the fission yeast Schizosaccharomyces pombe, the synthesis of MIPCs is required for endocytosis of a plasma membrane-localized amino acid transporter (Nakase et al., 2010). Complex sphingolipids appear to play a role in vesicular trafficking from the Golgi, because post-Golgi secretory vesicles are enriched in ergosterol and complex sphingolipids in budding yeast (Klemm et al., 2009). In addition, yeast cells lacking SNC genes encoding v-SNAREs exhibit a reduction in protein secretion, whereas a defect of the synthesis of very long-chain fatty acids in sphingolipids can suppress the defect of the secretory pathway (David et al., 1998). Finally, p24, a major transmembrane protein in COPI-coated transport vesicles in the Golgi, specifically interact with C18:0-sphingomyelin in mammalian cells (Contreras et al., 2012).

Previously, we found that a double mutation of SAC1 encoding phosphoinositides phosphatase and a non-essential sphingolipid-metabolizing enzyme gene (CSG1, CSG2, IPT1, or SCS7) causes a synthetic growth defect phenotype in budding yeast (Tani and Kuge, 2010). SAC1-repressed cells exhibited a reduced cellular phosphatidylserine (PS) level, and overexpression of PSS1 encoding PS synthase partly suppressed the synthetic growth defect phenotype of csg1Δ, ipt1Δ and scs7Δ cells under SAC1-repressive conditions. Furthermore, repression of PSS1 expression resulted in a synthetic growth defect with the deletion of each of CSG1, IPT1 and SCS7, indicating that PS synthesis is required for normal cell growth in the absence of specific non-essential sphingolipid-metabolizing enzyme genes. In this study, we focused on the genetic interaction between complex sphingolipids and PS synthesis, finding that the genetic interaction causes a defect of endosomal trafficking of a v-SNARE protein, Snc1, suggesting that complex sphingolipids and PS are co-ordinately involved in specific vesicular trafficking systems.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Abnormal distribution of v-SNARE Snc1 caused by deletion of sphingolipid-metabolizing enzymes and repression of PS synthase

To investigate the functional relationship between complex sphingolipids and PS, we used a mutant strain that carries the PSS1 gene under the control of a tetracycline-regulatable (tetO2) promoter (tetO2-PSS1). Figure 1A shows the growth curves of tetO2-PSS1 and tetO2-PSS1 csg2Δ cells in the presence or absence of doxycycline (Dox), which represses expression of the gene under the tetO2 promoter. Csg2 is a regulatory subunit of IPC mannosyltransferases containing a catalytic subunit, Csg1 or Csh1 (Uemura et al., 2007). Dox-treated tetO2-PSS1 cells showed a similar growth rate to Dox-untreated tetO2-PSS1 ones; however, the growth rate of tetO2-PSS1 csg2Δ cells began to slow at 6–9 h after the addition of Dox (Fig. 1A). Thus, tetO2-PSS1 csg2Δ cells exhibited a similar growth phenotype to that of tetO2-PSS1 csg1Δ, tetO2-PSS1 ipt1Δ, and tetO2-PSS1 scs7Δ cells (Tani and Kuge, 2010). To avoid non-specific effects caused by growth inhibition, we investigated the phenotype of Dox-treated cells harbouring tetO2-PSS1 at earlier time points for further analyses (around 8–9 h after the addition of Dox).

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Figure 1. Deletion of sphingolipid-metabolizing enzymes and repression of PS synthase cause a synthetic growth defect and an abnormal distribution of v-SNARE Snc1.

A. Time-course of growth of tetO2-PSS1, tetO2-PSS1 csg2Δ cells. Cells were cultured overnight in YPD medium and then diluted (0.02 A600 units ml−1) in fresh YPD medium with or without 10 μg ml−1 Dox, and aliquots of cell suspensions were subjected to cell density measurement (A600) at the indicated times. The results presented are for one experiment (triplicate) representative of at least three independent ones.

B. Localization of yeGFP-tagged Snc1. Wild-type, csg2Δ, tetO2-PSS1, and tetO2-PSS1 csg2Δ cells expressing yeGFP-Snc1 were cultured overnight in YPD medium, diluted (0.03 A600 units ml−1) in fresh YPD medium with or without 10 μg ml−1 Dox, and then incubated for 9 h at 30°C. GFP fluorescence was observed by fluorescent microscopy. Arrowheads and arrows indicate plasma membrane localization and intracellular punctate accumulation of yeGFP-Snc1 respectively.

C. Frequency distribution of internal punctate fluorescence intensity of yeGFP-Snc1 in individual cells. The fluorescence intensity was quantified with ImageJ software (NIH). Percentage of fluorescence of total puncate structures in each individual cell was calculated as follows: punctate fluorescence in individual cells (%) = percentage of punctate fluorescence in total cellular fluorescence. Data represent the value of 100 cells pooled from three independent experiments for each strain.

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A number of lines of evidence indicated that PS and/or PE flippase, Drs2–Cdc50 complex, regulates the trafficking of Snc1, an exocytic v-SNARE that is normally recycled from plasma membranes to the late Golgi through post-Golgi endosomes and then back to the plasma membranes (Lewis et al., 2000; Hua et al., 2002; Valdez-Taubas and Pelham, 2003; Saito et al., 2004). Thus, we investigated the subcellular localization of Snc1 in double mutants carrying tetO2-PSS1 (tetO2-PSS1 csg1Δ, tetO2-PSS1 csg2Δ, tetO2-PSS1 ipt1Δ, and tetO2-PSS1 scs7Δ) by tagging of Snc1 with a yeast-enhanced green fluorescent protein (yeGFP). In wild-type cells, yeGFP-Snc1 was largely localized to the plasma membrane of newly budded daughter cells and the bud sites, and slightly to intracellular punctate structures (Fig. 1B), which corresponds well with the normal localization pattern of Snc1 reported previously (Lewis et al., 2000; Valdez-Taubas and Pelham, 2003). In contrast to the wild-type cells, the tetO2-PSS1 csg2Δ cells exhibited a substantial decrease in yeGFP-Snc1 localized to the plasma membrane with a concomitant increase in the protein localized to internal punctate structures in both the presence and absence of Dox (Fig. 1B). Figure 1C shows quantification results of fluorescence intensity of the punctate structures of yeGFP-Snc1 in each strain. In wild-type, tetO2-PSS1, and Dox-treated tetO2-PSS1 cells, similar fluorescence pattern was observed. The fluorescence intensity of punctate structures was slightly increased in csg2Δ cells. In contrast, tetO2-PSS1 csg2Δ cells exhibited striking increase in the fluorescence intensity of punctate structures even in the absence of Dox (Fig. 1C). Thus, the severe mislocalization of yeGFP-Snc1 was caused by two genetic alterations, deletion of CSG2 and substitution of PSS1 promoter. As shown in Fig. S1, both tetO2-PSS1 csg1Δ and tetO2-PSS1 ipt1Δ cells exhibited a normal localization pattern of yeGFP-Snc1 in the absence of Dox. The treatment of these cells with Dox caused a significant increase in the mislocalization of yeGFP-Snc1. tetO2-PSS1 scs7Δ, and Dox-treated tetO2-PSS1 scs7Δ cells did not exhibit any abnormality in the yeGFP-Snc1 distribution (Fig. S1).

Collectively, these results indicated that double mutation of tetO2-PSS1 and deletion of one of the sphingolipid-metabolizing enzyme genes, including CSG1, CSG2 and IPT1, caused mislocalizaiton of yeGFP-Snc1 in the presence of Dox. It should be noted that the most severe phenotype was observed in tetO2-PSS1 csg2Δ cells, because the cells exhibited a severe mislocalization pattern even in the absence of Dox, whereas tetO2-PSS1 csg1Δ and tetO2-PSS1 ipt1Δ cells did not under the same conditions.

Phospholipids and complex sphingolipid analysis of sphingolipid-metabolizing enzymes and PS synthase mutants

To investigate the alteration of phospholipid metabolism in tetO2-PSS1 and tetO2-PSS1 csg2Δ cells, the cellular levels of major phospholipids (CL, PA, PE, PS, PI, and PC) were examined by means of steady-state radiolabelling with [32P]orthophosphate. As shown in Fig. 2A, the steady-state PS levels in tetO2-PSS1 and tetO2-PSS1 csg2Δ cells were reduced to less than half as compared with those in the wild-type, and csg2Δ cells, indicating that the expression of PSS1 under the tetracycline-regulatable promoter causes a partial reduction in the PS level even in the absence of Dox. The PE levels in cells harbouring tetO2-PSS1 were also reduced to less than 70% of those in wild-type and csg2Δ cells, probably due to reduced synthesis of PE from PS caused by PS reduction. The effect of Dox treatment on the cellular phospholipid levels was examined by pulse-radiolabelling with [32P]orthophosphate for 60 min after pretreatment with Dox for 8 h. As shown in Fig. 2B, Dox treatment caused a striking reduction in the PS level in both tetO2-PSS1 and tetO2-PSS1 csg2Δ cells. Similar alteration of phospholipid metabolism was also observed in tetO2-PSS1 csg1Δ and tetO2-PSS1 ipt1Δ cells (Fig. S2). To determine whether or not PSS1 repression affects the biosynthesis of complex sphingolipids, cells were pulse-labelled with [3H]inositol in the presence or absence of Dox (Fig. 2C). As reported previously, the deletion of CSG2 results in drastic reductions in the MIPC and M(IP)2C levels. The deletion of CSG1 also caused reductions in the MIPC and M(IP)2C levels; however, the reductions were less than those in csg2Δ cells. In ipt1Δ cells, complete loss of the synthesis of M(IP)2C was observed. In the csg1Δ, csg2Δ and ipt1Δ cells, repression of PS synthesis by tetO2-PSS1 did not cause significant alteration of complex sphingolipid synthesis (Fig. 2C).

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Figure 2. Metabolic labelling of glycerophospholipids and complex sphingolipids.

A. Steady-state radiolabelling of wild-type, csg2Δ, tetO2-PSS1, and tetO2-PSS1 csg2Δ cells with [32P]orthophosphate. The radiolabelled phospholipids were extracted and resolved by TLC. Major glycerophospholipids (CL, PA, PE, PS, PI, and PC) were quantified using a Bio Imaging analyser. Panel b shows the radiolabelled PS level in each lot of cells (extracted from panel a). Data shown are the averages of two independent experiments performed in duplicate determinations.

B. tetO2-PSS1, and tetO2-PSS1 csg2Δ cells treated with or without 10 μg ml−1 Dox for 8 h were pulse-radiolabelled for 1 h with [32P]orthophosphate at 30°C. Data shown are for one experiment (triplicate) representative of at least three independent experiments.

C. [3H]myo-inositol labelling. Cells were cultured overnight in YPD medium, diluted (0.1 A600 units ml−1) in fresh YPD medium with or without 10 μg ml−1 Dox, and then incubated for 8 h at 30°C. Cells were washed, resuspended in fresh SC/MSG medium with minimum inositol and with or without 10 μg ml−1 Dox, and then labelled with [3H]myo-inositol for 1 h at 30°C. Radiolabelled phospholipids were extracted, treated with monomethylamine, and resolved by TLC. The details are given under Experimental procedures.

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Reduction in the PS level per se is critical for the abnormal distribution of yeGFP-Snc1 in CSG2-deleted cells

In csg2Δ cells, the expression of PSS1 under control of the tetO2 promoter caused severe mislocalization of yeGFP-Snc1 even in the absence of Dox, conditions under which the slow growth rate was not induced (Fig. 1A). To investigate whether or not the reduction in the PS level is responsible for the abnormal distribution of yeGFP-Snc1 in tetO2-PSS1 csg2Δ cells, the promoter region of PSS1 was substituted by a tetO7 promoter, which exhibits a higher expression level in the absence of Dox as compared with the tetO2 promoter (Belli et al., 1998). As shown in Fig. 3A, the steady-state PS level in tetO7-PSS1 csg2Δ cells was approximately 2.2-fold higher than that in tetO2-PSS1 csg2Δ cells. Pulse-radiolabelling with [32P]orthophosphate revealed that Dox treatment caused a striking reduction in the PS level in tetO7-PSS1 csg2Δ cells (Fig. S3). In the absence of Dox, tetO7-PSS1 csg2Δ cells did not exhibit severe mislocalization of yeGFP-Snc1; however, treatment of tetO7-PSS1 csg2Δ cells with Dox caused a severe phenotype as to yeGFP-Snc1 mislocalization (Fig. 3B and C). Thus, it was suggested that the mislocalization of yeGFP-Snc1 in csg2Δ cells depends on the level of PS synthesis activity, but not on the promoter substitution itself.

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Figure 3. Phospholipid composition and localization of yeGFP-Snc1 in tetO2-PSS1 csg2Δ and tetO7-PSS1 csg2Δ cells.

A. Steady-state radiolabelling of tetO2-PSS1 csg2Δ and tetO7-PSS1 csg2Δ cells with [32P]orthophosphate. Panel b shows the radiolabelled PS level in each lot of cells (extracted from panel a). Data shown are the averages of two independent experiments performed in duplicate determinations.

B. Localization of yeGFP-tagged Snc1. Cells expressing yeGFP-Snc1 were cultured overnight in YPD medium, diluted (0.03 A600 units ml−1) in fresh YPD medium with or without 10 μg ml−1 Dox, and then incubated for 9 h at 30°C.

C. Frequency distribution of internal punctate fluorescence intensity of yeGFP-Snc1 in individual cells. Data represent the value of 100 cells pooled from three independent experiments for each strain.

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The mutation of tetO2-PSS1 also caused reductions in the PE level (Fig. 2A). To examine whether or not the reduction in PE level is related to the abnormal distribution of yeGFP-Snc1, PSD1 or PSD2, both of which catalyse PE synthesis from PS (Birner et al., 2001), was deleted in csg2Δ cells. As shown in Fig. S4, the PE levels in psd1Δ and psd1Δ csg2Δ cells were reduced by approximately 60% as compared with those in wild-type and csg2Δ cells; however, the PS levels were not decreased. The deletion of PSD2 caused an approximately 20% reduction in the PE level. Unlike tetO2-PSS1 csg2Δ cells, psd1Δ csg2Δ and psd2Δ csg2Δ cells did not exhibit severe mislocalization of yeGFP-Snc1, indicating that the combination of a reduction in the PE level and a defect of MIPC synthesis does not affect the localization of yeGFP-Snc1 (Fig. 4A and B). Collectively, these results suggested that the reduction in the PS level per se is critical for the abnormal distribution of yeGFP-Snc1 in CSG2-deleted cells.

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Figure 4. Localization of yeGFP-Snc1 in the double deletion mutant of CSG2 and PS decarboxylase genes.

A. Cells expressing yeGFP-Snc1 were cultured overnight in SC medium, diluted (0.3 A600 units ml−1) in fresh SC medium, and then incubated for 6 h at 30°C.

B. Frequency distribution of internal punctate fluorescence intensity of yeGFP-Snc1 in individual cells. Data represent the value of 100 cells pooled from three independent experiments for each strain.

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Impairment in trafficking of yeGFP-Snc1 between post-Golgi endosomes and the late Golgi in tetO2-PSS1 csg2Δ cells

Next, we investigated how yeGFP-Snc1 is mislocalized in PS- and sphingolipid-synthesis defective cells. The Snc1-V40A M43A mutant can be delivered to plasma membranes from the late Golgi, but can not be internalized through endocytosis for its recycling (Lewis et al., 2000). As shown in Fig. 5A, yeGFP-Snc1-V40A M43A was mainly localized to the whole region of plasma membranes in tetO2-PSS1 cells, due to a defect in endocytosis. A similar distribution pattern was observed when the mutant of yeGFP-Snc1 was expressed in tetO2-PSS1 csg2Δ cells in the presence or absence of Dox (Fig. 5A), indicating that trafficking of yeGFP-Snc1 from the late Golgi to plasma membranes was not impaired. In tetO2-PSS1 csg2Δ cells, most intracellularly accumulated yeGFP-Snc1 did not colocalized with Sec7-eqFP611, a marker protein for the late Golgi (Fig. 5B). The fluorescent endocytic marker FM4-64 stains vacuoles after long-time (∼ 2 h) incubation, but endosomes after short-time (∼ 10 min) incubation (Vida and Emr, 1995). When tetO2-PSS1 csg2Δ cells expressing yeGFP-Snc1 were labelled with FM4-64 on ice for 30 min, and chased at 30°C for 10 min, the intracellularly accumulated yeGFP-Snc1 was well-colocalized with FM4-64 (Fig. 5C), suggesting the localization of yeGFP-Snc1 in endosomes. Since Snc1 is normally transported from plasma membranes to the late Golgi through post-Golgi endosomes (Lewis et al., 2000; Valdez-Taubas and Pelham, 2003), these results suggested that yeGFP-Snc1 is accumulated in post-Golgi endosomes due to impairment of the trafficking from post-Golgi endosomes to the late Golgi in tetO2-PSS1 csg2Δ cells.

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Figure 5. Endosomal localization of yeGFP-Snc1 in tetO2-PSS1 csg2Δ cells.

A. Localization of yeGFP-Snc1-V40A M43A. tetO2-PSS1 and tetO2-PSS1 csg2Δ cells expressing yeGFP-Snc1 or yeGFP-Snc1-V40A M43A were cultured overnight in YPD medium, diluted (0.03 A600 units ml−1) in fresh YPD medium with or without 10 μg ml−1 Dox, and then incubated for 9 h at 30°C.

B. tetO2-PSS1 csg2Δ cells expressing yeGFP-Snc1 and Sec7-eqFP611 were cultured overnight in YPD medium, diluted (0.03 A600 units ml−1) in fresh YPD medium, and then incubated for 9 h at 30°C. GFP and RFP fluorescence was observed by fluorescent microscopy.

C. tetO2-PSS1 csg2Δ cells expressing yeGFP-Snc1 were pulse-labelled with FM4-64 on ice for 30 min and then chased at 30°C for 10 min. Arrows indicate colocalization of yeGFP-Snc1 and FM4-64. The details are given under Experimental procedures.

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Trafficking via pre-vacuolar endosomes in tetO2-PSS1 csg2Δ cells

To investigate whether or not other endosomal protein trafficking is impaired in tetO2-PSS1 csg2Δ cells, we examined the transport of carboxypeptidase Y (CPY) by pulse-chase metabolic labelling of cells with [35S]methionine/cysteine. CPY is initially synthesized in the ER as a precursor form (p1 form), glycosylated in the Golgi (p2 form), and finally transported to vacuoles to give the mature form through pre-vacuolar endosomes (Robinson et al., 1988). In the wild-type, csg2Δ, tetO2-PSS1, and Dox-treated tetO2-PSS1 cells, CPY had largely been converted to the mature form after a 20 min chase (Fig. 6A). In Dox-treated tetO2-PSS1 csg2Δ cells, CPY processing was slightly delayed, that is, the rate of conversion of p1 and p2 to the mature form after 20 min chase was 79.2% ± 0.6 (n = 3), whereas in wild-type cells it was 90.9% ± 0.6 (n = 3). After a 45 min chase the CPY processing in Dox-treated tetO2-PSS1 csg2Δ cells reached 88.8% ± 1.0 (n = 3). In contrast, a deletion mutant of VPS10 encoding the sorting receptor for CPY exhibited significant accumulation of the precursor form even after a 45 min chase (Fig. 6A). These results indicated that the CPY processing is not severely impaired in Dox-treated tetO2-PSS1 csg2Δ cells.

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Figure 6. Pre-vacuolar endosomal trafficking in tetO2-PSS1 csg2Δ cells.

A. Wild-type, csg2Δ, tetO2-PSS1, tetO2-PSS1 csg2Δ, and vps10Δ cells were metabolically labelled with [35S]methionine/cysteine for 10 min, and then chased with excess non-radiolabelled methionine and cysteine at 30°C for 10, 20, or 45 min. Then, cell extracts were immunoprecipitated with CPY antiserum and analysed by SDS-PAGE. p1, ER precursor of CPY; p2, Golgi precursor of CPY; m, vacuolar mature form of CPY.

B. Wild-type, tetO2-PSS1 csg2Δ, and vps17Δ cells expressing Vps10-yeGFP were cultured overnight in YPD medium, diluted (0.03 A600 units ml−1) in fresh YPD medium with or without 10 μg ml−1 Dox, and then incubated for 9 h at 30°C. GFP fluorescence was observed by fluorescent microscopy.

C. snx4Δ, tetO2-PSS1 csg2Δ snx4Δ, and tetO2-PSS1 csg2Δ cells expressing yeGFP-Snc1 were cultured overnight in YPD medium, diluted (0.1 A600 units ml−1) in fresh YPD medium with or without 10 μg ml−1 Dox, and then incubated for 6 h at 30°C. Cells were pulse-labelled with FM4-64 at 30°C for 15 min and then chased at 30°C for 2 h. Arrows indicate colocalization of yeGFP-Snc1 and FM4-64. The details are given under Experimental procedures.

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Vps10 cycles between the late Golgi and pre-vacuolar endosomes (Cooper and Stevens, 1996). In wild-type cells Vps10-yeGFP exhibited a punctate fluorescence pattern, a characteristic of Golgi-localized yeast proteins, whereas deletion of VPS17, which is required for the endosome-to-Golgi retrieval of Vps10 (Horazdovsky et al., 1997), resulted in missorting of Vps10-yeGFP to vacuoles (Fig. 6B). In Dox-untreated or treated tetO2-PSS csg2Δ cells, Vps10-yeGFP exhibited a punctate fluorescence pattern, as in wild-type cells, suggesting that Vps10-yeGFP was normally retrieved from pre-vacuolar endosomes in the mutant.

SNX4 encoding sorting nexin is involved in the sorting of Snc1 from post-Golgi endosomes to the late Golgi, and the deletion causes missorting of Snc1 from post-Golgi endosomes to vacuoles via pre-vacuolar endosomes (Hettema et al., 2003). To examine the transport pathway from post-Golgi endosomes to vacuoles via pre-vacuolar endosomes in tetO2-PSS1 csg2Δ cells, the distribution of yeGFP-Snc1 was observed under the deletion of SNX4. To visualize vacuoles, cells were labelled with FM4-64 for 15 min, and then chased for 2 h at 30°C, conditions under which the dye is transported from plasma membranes to vacuoles (Vida and Emr, 1995). As shown in Fig. 6C, yeGFP-Snc1 was colocalized with FM4-64 in snx4Δ cells, indicating missorting of the protein to vacuoles. Although yeGFP-Snc1 was not colocalized with FM4-64 in tetO2-PSS1 csg2Δ cells, tetO2-PSS1 csg2Δ snx4Δ cells exhibited colocalization of yeGFP-Snc1 with FM4-64 in vacuoles in both the presence and absence of Dox, indicating that the vesicular trafficking from post-Golgi endosomes to vacuoles is not impaired (Fig. 6C). Taken together, these results suggested that anterograde and retrograde vesicular trafficking via pre-vacuolar endosomes is not impaired in Dox-treated or untreated tetO2-PSS1 csg2Δ cells.

Defect of GTPase-activating protein Gyp2 suppresses the abnormal distribution of yeGFP-Snc1 in tetO2-PSS1 csg2Δ cells

The F-box protein Rcy1 is involved in trafficking of Snc1 from post-Golgi endosomes to the late Golgi for recycling (Wiederkehr et al., 2000). The localization pattern of yeGFP-Snc1 in tetO2-PSS1 csg2Δ cells was similar to that in rcy1Δ cells, which exhibited high accumulation of yeGFP-Snc1 in post-Golgi endosomes (Wiederkehr et al., 2000). The defect in trafficking of Snc1 in rcy1Δ cells is suppressed by deletion of GYP2, which encodes a Ypt/Rab GTPase-activating protein (GAP) and negatively regulates endosomal vesicular trafficking (Lafourcade et al., 2003). Thus, we next investigated whether or not the abnormal distribution of yeGFP-Snc1 in tetO2-PSS1 csg2Δ cells is suppressed by the deletion of GYP2. As shown in Fig. 7A and B, the abnormal distribution of yeGFP-Snc1 in tetO2-PSS1 csg2Δ cells was almost completely suppressed by GYP2 deletion in both the presence and absence of Dox. In contrast, the abnormal distribution was not suppressed by deletion of GYP3 encoding a GAP toward Sec4, a Rab GTPase essential for exocytic secretion (Gao et al., 2003). These results support the notion that the trafficking from post-Golgi endosomes to the late Golgi is impaired in tetO2-PSS1 csg2Δ cells as well as in rcy1Δ cells. However, the deletion of GYP2 had no effect on the growth defect of Dox-treated tetO2-PSS1 csg2Δ cells (Fig. 7C), suggesting that factor(s) other than the defect of endosomal trafficking may be involved in induction of the growth inhibition.

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Figure 7. Suppression of the abnormal distribution of yeGFP-Snc1 in tetO2-PSS1 csg2Δ cells by deletion of GYP2.

A. tetO2-PSS1 csg2Δ, tetO2-PSS1 csg2Δ gyp2Δ, and tetO2-PSS1 csg2Δ gyp3Δ cells expressing yeGFP-Snc1 were cultured overnight in YPD medium, diluted (0.03 A600 units ml−1) in fresh YPD medium with or without 10 μg ml−1 Dox, and then incubated for 9 h at 30°C. GFP fluorescence was observed by fluorescent microscopy.

B. Frequency distribution of internal punctate fluorescence intensity of yeGFP-Snc1 in individual cells. Data represent the value of 100 cells pooled from three independent experiments for each strain.

C. tetO2-PSS1, tetO2-PSS1 gyp2Δ, tetO2-PSS1 csg2Δ, and tetO2-PSS1 csg2Δ gyp2Δ cells were cultured overnight in YPD medium, and then spotted onto YPD plates with or without the indicated concentrations of Dox, in 10-fold serial dilutions starting with a density of 0.7 A600 units ml−1. All plates were incubated at 30°C and photographed after 2 days.

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Expression of GTP-restricted form of Ypt32-GTPase suppresses the abnormal distribution of yeGFP-Snc1 in tetO2-PSS1 csg2Δ cells

It is suggested that Gyp2 exhibits GAP activity toward Ypt31/32 and Ypt6 GTPases (Lafourcade et al., 2003; Sciorra et al., 2005), which are involved in the regulation of several vesicular trafficking pathways including the recycling pathway of Snc1 (Siniossoglou and Pelham, 2001; Chen et al., 2005). Thus, we next investigated whether or not expression of GTP-restricted form of Ypt32 (Ypt32-Q72L) or Ypt6 (Ypt6-Q69L) suppresses the abnormal distribution of yeGFP-Snc1 in tetO2-PSS1 csg2Δ cells. Ypt32-Q72L or Ypt6-Q69L was expressed under the control of a TEF promoter in tetO2-PSS1 csg2Δ cells. As shown in Fig. 8A and B, the abnormal distribution of yeGFP-Snc1 in tetO2-PSS1 csg2Δ cells was partly suppressed by the expression of Ypt32-Q72L in the absence of Dox. Treatment of the cells with Dox resulted in a dramatic increase in the ratio of cells with a severe phenotype as to yeGFP-Snc1 mislocalization. In contrast, the expression of Ypt6-Q69L did not affect the abnormal distribution of yeGFP-Snc1 in Dox-untreated tetO2-PSS1 csg2Δ cells (Fig. 8A and B). These results indicated that constitutive activation of Ypt32 GTPase can restore the defect of the trafficking of Snc1 from post-Golgi endosomes to the late Golgi in tetO2-PSS1 csg2Δ cells.

figure

Figure 8. Effect of expression of GTP-restricted form of Ypt32 and Yp6-GTPases on the abnormal distribution of yeGFP-Snc1 in tetO2-PSS1 csg2Δ cells.

A. tetO2-PSS1 csg2Δ cells expressing yeGFP-Snc1 and tetO2-PSS1 csg2Δ cells expressing yeGFP-Snc1 and Ypt32-Q72L or Ypt6-Q69L were cultured overnight in YPD medium, diluted (0.03 A600 units ml−1) in fresh YPD medium with or without 10 μg ml−1 Dox, and then incubated for 9 h at 30°C. GFP fluorescence was observed by fluorescent microscopy.

B. Frequency distribution of internal punctate fluorescence intensity of yeGFP-Snc1 in individual cells. Data represent the value of 100 cells pooled from three independent experiments for each strain.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

In the present study, we showed that double mutation of complex sphingolipid-metabolizing enzyme genes, i.e. CSG1, CSG2 and IPT1, and repression of PSS1 cause an abnormal distribution of yeGFP-Snc1. csg2Δ cells did not exhibit severe mislocalization of yeGFP-Snc1; however, tetO2-PSS1 csg2Δ cells exhibited severe phenotype even in the absence of Dox, conditions under which the cells did not show slow growth rate (Fig. 1A and C). Even in the absence of Dox, cells harbouring tetO2-PSS1 exhibited a reduced PS level of less than half compared with that in wild-type cells (Fig. 2A). Thus, the abnormal distribution of yeGFP-Snc1 in tetO2-PSS1 csg2Δ cells seemed to be caused by the partial reduction in PS synthesis. In contrast to tetO2-PSS1 csg2Δ cells, the majority of tetO2-PSS1 csg1Δ and tetO2-PSS1 ipt1Δ cells exhibited normal yeGFP-Snc1 localization in the absence of Dox (Fig. S1). Therefore, the most severe phenotype was observed for tetO2-PSS1 csg2Δ cells. Csg2 is a regulatory subunit of IPC mannosyltransferases containing a catalytic subunit, Csg1 or Csh1, and the deletion of CSG2 causes drastic reductions in the MIPC and M(IP)2C levels (Uemura et al., 2003; 2007). The deletion of CSG1 causes only a partial reduction in the MIPC and M(IP)2C levels, because Csh1 is still active (Uemura et al., 2003). The deletion of IPT1 results in complete loss of M(IP)2Cs but not MIPCs (Dickson et al., 1997). Thus, collectively, it was suggested that the synthesis of MIPCs (and maybe M(IP)2Cs) is an important factor for correct localization of yeGFP-Snc1 under PS synthesis-repressive conditions.

In tetO2-PSS1 csg2Δ cells, the mislocalized yeGFP-Snc1 was co-localized with the endocytic marker FM4-64 upon short time labelling (Fig. 5C) but not with Sec7-eqFP611, a marker protein for the late Golgi (Fig. 5B), suggesting endosomal localization. Furthermore, an endocytosis-deficient mutant of Snc1 (Snc1-V40A M43A) was localized to plasma membranes in tetO2-PSS1 csg2Δ cells (Fig. 5A). Since Snc1 is normally transported from plasma membranes to the late Golgi through post-Golgi endosomes for its recycling (Lewis et al., 2000; Valdez-Taubas and Pelham, 2003), these results suggested that the yeGFP-Snc1 is accumulated in post-Golgi endosomes in tetO2-PSS1 csg2Δ cells due to a defect in the Snc1 recycling pathway (Fig. 9). On the other hand, the transport of CPY from the Golgi to vacuoles via pre-vacuolar endosomes and pre-vacuolar endosome-to-late Golgi retrieval of Vps10 were not severely impaired in tetO2-PSS1 csg2Δ cells even in the presence of Dox (Fig. 6A and B). Furthermore, in tetO2-PSS1 csg2Δ cells, yeGFP-Snc1 was transported to vacuoles when SNX4 encoding sorting nexin in post-Golgi endosomes was deleted (Fig. 6C). These results suggested that the vesicular trafficking pathways via pre-vacuolar endosomes are nearly normal in PS and complex sphingolipid synthesis-defective cells (Fig. 9). This also suggested that the defect in the recycling of yeGFP-Snc1 of tetO2-PSS1 csg2Δ cells is not caused by a global defect of endosomal trafficking pathways.

figure

Figure 9. Defect of specific endosomal trafficking in tetO2-PSS1 csg2Δ cells. The exocytic v-SNARE Snc1 is normally transported from the plasma membrane to the late Golgi through post-Golgi endosomes for its recycling. In tetO2-PSS1 csg2Δ cells, yeGFP-Snc1 is accumulated in post-Golgi endosomes, due to a defect in the Snc1 recycling pathway. However, the transport of CPY from the Golgi to vacuoles via pre-vacuolar endosomes and pre-vacuolar endosome-to-Golgi retrieval of Vps10 were not severely impaired in tetO2-PSS1 csg2Δ cells. In addition, yeGFP-Snc1 was transported to vacuoles from post-Golgi endosomes when SNX4 was deleted. Thus, it was suggested that vesicular trafficking pathways via pre-vacuolar endosomes are nearly normal in tetO2-PSS1 csg2Δ cells and that the defect in the recycling of yeGFP-Snc1 is not caused by a global defect of endosomal trafficking pathways.

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The Rab/Ypt-GTPases are key regulators of various vesicular trafficking systems. They cycle between GDP-bound inactive and GTP-bound active forms by guanine nucleotide exchange factors (GEFs) and GAPs, which are critical for their functions. The abnormal distribution of yeGFP-Snc1 in tetO2-PSS1 csg2Δ cells was suppressed by the deletion of GYP2 encoding a GAP, which negatively regulates endosomal vesicular trafficking through regulation of activity of Ypt31/32 and Ypt6 GTPases (Lafourcade et al., 2003; Sciorra et al., 2005) (Fig. 7). Furthermore, the expression of GTP-restricted form of Ypt32, but not Ypt6, suppressed the abnormal distribution of yeGFP-Snc1 in tetO2-PSS1 csg2Δ cells (Fig. 8), suggesting that the double defects of PS and complex sphingolipid synthesis may cause impairment of Ypt31/32 GTPases-mediated vesicular trafficking system. Ypt6 GTPase is localized to the late Golgi and assist fusion between endosome-derived vesicles and the late Golgi (Siniossoglou and Pelham, 2001). In contrast, Ypt31/32 GTPases are involved in formation of vesicles destined for the late Golgi in post-Golgi endosomes (Chen et al., 2005; 2011; Furuta et al., 2007). In tetO2-PSS1 csg2Δ cells yeGFP-Snc1 was suggested to be accumulated in post-Golgi endosomes but not in the late Golgi (Fig. 5B and C), these results implied that PS and complex sphingolipids are co-ordinately involved in the formation of transport vesicles in post-Golgi endosomes in a Ypt31/32 GTPases-dependent manner.

Rcy1 is suggested to be involved in the vesicular trafficking from post-Golgi endosomes to the late Golgi (Wiederkehr et al., 2000). Rcy1 regulates ubiquitination and phosphorylation of Snc1, which are required for the Snc1 recycling through the late Golgi (Chen et al., 2005; 2011), and active form of Ypt31/32 GTPases modulates protein level and localization of Rcy1 (Chen et al., 2005). Thus, Rcy1 is suggested to function as a downstream effector of Ypt31/32 GTPases in the trafficking of Snc1 from post-Golgi endosomes to the late Golgi. Furthermore, Rcy1 binds to putative PS/PE flippase Drs2–Cdc50 complex to regulate Ypt31/32 GTPases-mediated Snc1 recycling pathway, suggesting functional connection between these aminophospholipids and Rcy1 (Furuta et al., 2007). Thus, taken together, a close functional relationship between complex sphingolipids, PS, and Rcy1 is suggested. Further investigation of the functional relationship would allow elucidation of the role of complex sphingolipids and PS in vesicular trafficking.

Very recently, a PS-binding protein, evectin-2, has been identified as a key regulator of retrograde vesicular trafficking from recycling endosomes to the Golgi in African green monkey kidney COS1 cells (Uchida et al., 2011). In COS1 cells, PS is most concentrated in recycling endosomes, and evectin-2 is recruited there through binding of its pleckstrin homology domain to PS. Furthermore, sphingomyelin has been shown to be enriched in recycling endosomes with cholesterol and PS in Madin-Darby canine kidney MDCK cells (Gagescu et al., 2000), suggesting the possible role of complex sphingolipids in recycling endosomes. Although a homologue of evectin-2 and recycling endosomes were not identified in yeast cells, our present results raised the possibility that a specific protein, like evectin-2, that directly interacts with PS and/or complex sphingolipids in post-Golgi endosomes and regulates the endosomal trafficking exists in yeast.

In summary, the present study indicated that complex sphingolipids and PS are co-ordinately involved in specific endosomal trafficking system from post-Golgi endosomes to the late Golgi. Elucidation of detailed molecular mechanisms underlying regulation of the vesicular trafficking by complex sphingolipids and PS will provide a new insight into the physiological significance of these phospholipids.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Yeast strains and media

The S. cerevisiae strains used are listed in Table 1. To generate a yeast in which the expression of PSS1 is regulated by doxycycline (Dox), the PSS1 upstream region from −1 to −10 bp was replaced with a tetracycline operator cassette containing a repressor binding site (tetO2 or tetO7), the gene encoding TetR-VP16 tTA transactivator, and a kanMX4 marker, as described previously (Belli et al., 1998). Occasionally, the G418-resistant gene in kanMX4 was replaced with the hygromycin B-resistant gene [from the pFA6a-hphNTI vector (Janke et al., 2004)] to create hphMX4. Disruption of CSG1, CSG2, IPT1, SCS7, PSD1, PSD2, SNX4, VPS17, GYP2, and GYP3 was performed by replacing their open reading frames with the URA3 marker from the pRS406 vector (Sikorski and Hieter, 1989), the kanMX4 marker from a genome from a yeast knockout library or the pFA6a-kanMX4 vector (Wach et al., 1994), or the natMX4 marker from the p4339 vector (pCRII-TOPO::natMX4) (Tong and Boone, 2006). For tagging of the N-terminus of Snc1 with a yeast-enhanced green fluorescent protein (yeGFP) tag, a yeGFP fusion cassette with the TEF promoter and natNT2 marker from the pYM-N21 vector was introduced immediately downstream of the initiator ATG of chromosomal SNC1 as described previously (Janke et al., 2004). For tagging of the C-terminus of Vps10 with the yeGFP tag, a yeGFP fusion cassette with the kanMX4 marker from the pKT127 vector was introduced immediately upstream of the stop codon of chromosomal VPS10 as described previously (Sheff and Thorn, 2004). For tagging of the C-terminus of Sec7 with a red fluorescent protein [eqFP611 (Wiedenmann et al., 2002)] tag, a eqFP611 fusion cassette with the kanMX4 marker from the pYM51 vector was introduced immediately upstream of the stop codon of chromosomal SEC7 as described previously (Janke et al., 2004). Introduction of the V40A and M43A mutations into SNC1 is described below. The yeGFP-SNC1::natNT2 fragment was amplified with a 5′ primer (5′-TCAACCTTCAGCTTCCAATCACTC-3′), a 3′ primer (5′-AAGCGCAACGTATTCATTGTCCATG-3′), and genome DNA from yeGFP-SNC1::natNT2 cells as a template, and the resultant DNA fragment was inserted into the pGEM-T Easy vector (Promega). The mutations were performed by fusing the upstream and downstream DNA fragments of yeGFP-SNC1::natNT2. The upstream fragment was amplified with a 5′ primer (5′-TCAACCTTCAGCTTCCAATCACTC-3′), a 3′ primer (5′-ATGTTATCTCTCGCTATTCCCGCGGTATCATCAA-3′), and pGEM-T Easy yeGFP-SNC1::natNT2 as a template. The downstream fragment was amplified with a 5′ primer (5′-AAGCGCAACGTATTCATTGTCCATG-3′), a 3′ primer (5′-ATTATGCTTTCTGCCCAATTTGACT-3′), and pGEM-T Easy yeGFP-SNC1::natNT2. These fragments were extended by PCR and transformed into yeast cells. For expression of GTP-restricted form of Ypt32 (Ypt32-Q72L) with TEF promoter, chromosomal YPT32 were replaced as described below. A DNA fragment of the YPT32-Q72L ORF was obtained by fusing the upstream and downstream DNA fragments of YPT32. The upstream fragment was amplified with a 5′ primer (5′-ATGAGCAACGAAGATTACGGATACG-3′), a 3′ primer (5′-CTGTAACGTTCTAGACCTGCCGTGT-3′), and yeast genomic DNA as a template. The downstream fragment was amplified with a 5′ primer (5′-ACACGGCAGGTCTAGAACGTTACAG-3′), a 3′ primer (5′-CGACCTTAGTAGGGCACAAAATTT-3′), and yeast genomic DNA as a template. These fragments were extended by PCR. A TEF promoter cassette with the kanMX4 marker from pYM-N18 (Janke et al., 2004) was amplified with a 5′ primer (5′-GAAAAGCGTGGTGATAAAGAGCGAACCAAGCATATTGTTTTCCAAGAATGCGTACGCTGCAGGTCGAC-3′), a 3′ primer (5′-AGAACTATCTTAAATAAATAATCGTAGTCGTATCCGTAATCTTCGTTGCTCATCGATGAATTCTCTGTCG-3′). These two DNA fragments (the TEF promoter cassette with kanMX4 and the YPT32-Q72L ORF) were extended by PCR, and the resultant DNA fragment (YPT32-Q72L::kanMX4) was used to transform into yeast cells. For expression of GTP-restricted form of Ypt6 (Ypt6-Q69L) with TEF promoter was performed by the same protocol as described in Ypt32-Q72L. Primers used in the PCR were listed below. A 5′ primer (5′-ATGAGCAGATCCGGGAAATC-3′), a 3′ primer (5′-ATCTAAATCTTTCCAGACCTGCTGTATCC-3′), A 5′ primer (5′-GGATACAGCAGGTCTGGAAAGATTTAGAT-3′), and a 3′ primer (5′-CTAACACTGACAAGCGCTTTGTTC-3′) for amplification of YPT6-Q69L ORF. 5′ primer (5′-AGCTGTTGATTCTGAACAGTAAAAGATAAACAAAGAAGAGATTAACAATGCGTACGCTGCAGGTCGAC-3′), a 3′ primer (5′-TCTCCCAAAAAAACAATTTTGTACTTTGTCAATGATTTCCCGGATCTGCTCATCGATGAATTCTCTGTCG-3′) for amplification of A TEF promoter cassette with the kanMX4 marker. The sequences were verified with an ABI3100 DNA sequencer. The cells were grown in either YPD medium (1% yeast extract, 2% peptone, and 2% glucose), synthetic complete (SC) medium (0.67% yeast nitrogen base w/o amino acids (BD Difco, Heidelberg, Germany), and 2% glucose) containing nutritional supplements, or SC/MSG medium (0.17% yeast nitrogen base w/o amino acids and ammonium sulphate (BD Difco, Heidelberg, Germany), 0.1% L-glutamic acid sodium salt hydrate (MSG; Sigma), and 2% glucose) containing nutritional supplements (Tong and Boone, 2006).

Table 1. Strains used in this study
StrainGenotypeSource
BY4741MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0Brachmann et al. (1998)
MTY582BY4741, csg1Δ::URA3This study
MTY719BY4741, csg2Δ::URA3This study
MTY584BY4741, ipt1Δ::URA3This study
MTY304BY4741, tetO2-PSS1::kanMX4Tani and Kuge (2010)
MTY323BY4741, tetO2-PSS1::kanMX4 csg1Δ::URA3Tani and Kuge (2010)
MTY723BY4741, tetO2-PSS1::kanMX4 csg2Δ::URA3This study
MTY325BY4741, tetO2-PSS1::kanMX4 ipt1Δ::URA3Tani and Kuge (2010)
MTY324BY4741, tetO2-PSS1::kanMX4 scs7Δ::URA3Tani and Kuge (2010)
MTY620BY4741, yeGFP-SNC1::natNT2This study
MTY710BY4741, tetO2-PSS1::kanMX4 yeGFP-SNC1::natNT2This study
MTY713BY4741, tetO2-PSS1::kanMX4 csg1Δ::URA3 yeGFP-SNC1::natNT2This study
MTY725BY4741, tetO2-PSS1::kanMX4 csg2Δ::URA3 yeGFP-SNC1::natNT2This study
MTY715BY4741, tetO2-PSS1::kanMX4 ipt1Δ::URA3 yeGFP-SNC1::natNT2This study
MTY714BY4741, tetO2-PSS1::kanMX4 scs7Δ::URA3 yeGFP-SNC1::natNT2This study
MTY916BY4741, tetO7-PSS1::kanMX4 csg2Δ::URA3This study
MTY912BY4741, tetO7-PSS1::kanMX4 csg2Δ::URA3 yeGFP-SNC1::natNT2This study
MTY744BY4741, tetO2-PSS1::kanMX4 yeGFP-snc1-V40A-M43A::natNT2This study
MTY745BY4741, tetO2-PSS1::kanMX4 csg2Δ::URA3 yeGFP-snc1-V40A-M43A::natNT2This study
MTY961BY4741, psd1Δ::kanMX4This study
MTY962BY4741, psd2Δ::kanMX4This study
MTY963BY4741, psd1Δ::kanMX4 csg2Δ::URA3This study
MTY964BY4741, psd2Δ::kanMX4 csg2Δ::URA3This study
MTY749BY4741, psd1Δ::kanMX4 yeGFP-SNC1::natNT2This study
MTY741BY4741, psd2Δ::kanMX4 yeGFP-SNC1::natNT2This study
MTY750BY4741, psd1Δ::kanMX4 csg2Δ::URA3 yeGFP-SNC1::natNT2This study
MTY742BY4741, psd2Δ::kanMX4 csg2Δ::URA3 yeGFP-SNC1::natNT2This study
MTY868BY4741, tetO2-PSS1::hphMX4 csg2Δ::URA3 yeGFP-SNC1::natNT2 SEC7- eqFP611::kanMX4This study
MTY879BY4741, VPS10-yeGFP::kanMX4This study
MTY881BY4741, tetO2-PSS1::hphMX4 csg2Δ::URA3 VPS10-yeGFP::kanMX4This study
MTY882BY4741, vps17Δ::hphMX4 VPS10-yeGFP::kanMX4This study
MTY859BY4741, tetO2-PSS1::hphMX4 csg2Δ::URA3 snx4Δ::kanMX4 yeGFP-SNC1::natNT2This study
MTY870BY4741, snx4Δ::kanMX4 yeGFP-SNC1::natNT2This study
MTY905BY4741, tetO2-PSS1::hphMX4 gyp2Δ::kanMX4This study
MTY906BY4741, tetO2-PSS1::hphMX4 csg2Δ::URA3 gyp2Δ::kanMX4This study
MTY884BY4741, tetO2-PSS1::hphMX4 csg2Δ::URA3 gyp2Δ::kanMX4 yeGFP-SNC1::natNT2This study
MTY931BY4741, tetO2-PSS1::hphMX4 csg2Δ::URA3 gyp3Δ::kanMX4 yeGFP-SNC1::natNT2This study
MTY1052BY4741, tetO2-PSS1::hphMX4 csg2Δ::URA3 yeGFP-SNC1::natNT2 ypt32-Q72L::kanMX4This study
MTY1053BY4741, tetO2-PSS1::hphMX4 csg2Δ::URA3 yeGFP-SNC1::natNT2 ypt6-Q69L::kanMX4This study
vps10ΔBY4741, vps10Δ::kanMX4Winzeler et al. (1999)

Fluorescent microscopy

Yeast cells grown in YPD or SC medium were collected by centrifugation and then viewed under a fluorescence microscope (Leica DMRB; Leica, Solms, Germany) with a 100× Plan-Neofluar objective lens. Fluorescence and differential interference contrast images were captured with a CCD camera (Retiga EXi; Nippon Roper; Tokyo, Japan) using QCapture (Nippon Roper; Tokyo, Japan).

FM4-64 staining

Yeast cells grown in YPD medium were collected by centrifugation and then resuspended in fresh YPD medium to 10 A600 units ml−1. For staining of the endocytic pathway, FM4-64 (Molecular Probes, Eugene, OR) was added to the cells to a final concentration of 20 μM, followed by reaction on ice for 30 min, washing with cold YPD medium three times, and then chasing in fresh YPD medium (5 A600 units ml−1) without FM4-64 at 30°C for 10 min. For staining of vacuoles, FM4-64 was added to the YPD medium to a final concentration of 20 μM, followed by reaction at 30°C for 15 min, washing with YPD medium three times, and then chasing in fresh YPD medium (5 A600 units ml−1) without FM4-64 at 30°C for 2 h. Endocytosis was stopped by the addition of NaF and NaN3 to final concentrations of 10 mM. Cells were collected by centrifugation and viewed under a fluorescence microscope.

[32P]orthophosphate labelling

For pulse labelling, cells were cultured overnight in YPD medium, diluted (0.1 A600 units ml−1) in YPD medium with or without 10 μg ml−1 Dox, and then incubated for 8 h. The cells were collected by centrifugation, resuspended in 500 μl of fresh YPD medium with or without Dox to 2 A600 units ml−1, and then labelled with [32P]orthophosphate (1 μCi 1−1 A600 unit of cells; MP Biomedicals, Morgan Irvine, CA) for 1 h at 30°C. For steady-state labelling, cells were cultured overnight in YPD medium, diluted (0.1 A600 units ml−1) in 2 ml of fresh YPD medium, and then labelled with 4 μCi [32P]orthophosphate for 12 h at 30°C. The cells were resuspended in 1 ml of fresh YPD medium to 0.3 A600 units ml−1 and then labelled with 2 μCi [32P]orthophosphate for 5 h. The radiolabelled cells were chilled on ice, collected by centrifugation, washed with distilled water, and then suspended in 150 μl of 80% ethanol. After heating at 95°C for 30 min, 800 μl of chloroform/methanol (1:1, v/v) and subsequently 330 μl of PBS were added to the sample. The sample was mixed well and then centrifuged at 10 000 g for 1 min. The lower phase was collected, dried and applied to a LK5 silica gel 150 A TLC plate (Whatman, Clifton, NJ), which had been prewashed in chloroform/methanol (1:1, v/v) and treated with 2% boric acid in ethanol. The TLC plate was developed two times with chloroform/ethanol/water/triethylamine (30:35:7:35, v/v) and then analysed with a Bio Imaging analyser FLA-2000 (Fuji Photo Film, Kanagawa, Japan).

[3H]myo-inositol labelling

Yeast cells grown in YPD medium were collected by centrifugation and then washed with SC/MSG medium with minimum inositol (2 μg ml−1). Then the cells were resuspended in 500 μl of SC/MSG medium with minimum inositol to 2 A600 units ml−1 and labelled with [3H]myo-inositol (1 μCi 1−1 A600 unit of cells; PerkinElmer Life Sciences, Norwalk, CT) for 1 h at 30°C. The cells were chilled on ice, collected by centrifugation, washed with distilled water, and then suspended in 150 μl of ethanol/water/diethylether/pyridine/15 N ammonia (15:15:5:1:0.018, v/v). Radioactivity was measured using a liquid scintillation system, and samples containing lipids exhibiting equal levels of radioactivity were used for further experiments. After 15 min incubation at 60°C, the residue was centrifuged at 10 000 g for 1 min and extracted once more in the same manner. The resulting supernatants were dried. For mild alkaline treatment, the lipid extracts were dissolved in 150 μl monomethylamine (40% methanol solution)/water (10:3, v/v) incubated for 1 h at 53°C, and then dried. The lipids were suspended in 20 μl of chloroform/methanol/water (5:4:1, v/v) and then separated on Silica Gel 60 TLC plates (Merck, Whitehouse Station, NJ) with chloroform/methanol/4.2 N ammonia (9:7:2, v/v) as the solvent system.

Pulse-chase labelling of CPY with [35S]methionine/[35S]cysteine

Yeast cells were cultured overnight in YPD medium, diluted (0.1 A600 units ml−1) in fresh YPD medium with or without 10 μg ml−1 Dox, and then incubated for 6.5 h at 30°C. They were again collected by centrifugation, washed with distilled water, resuspended in SC/MSG medium lacking methionine and cysteine with or without 10 μg ml−1 Dox, and then incubated at 30°C for 1 h. Cells were collected by centrifugation, and resuspended in 700 μl of fresh SC/MSG medium lacking methionine and cysteine with or without 10 μg ml−1 Dox to 1.4 A600 units ml−1. Then the cells were pulse-labelled with [35S]methionine/[35S]cysteine (25 μCi 1−1 A600 unit of cells; EXPRESSTM protein labelling mix; 1000 Ci mmol−1; PerkinElmer Life Sciences) for 10 min, and then chased in the presence of cold methionine (final concentration, 0.5 mg ml−1) and cysteine (0.1 mg ml−1) for the indicated times. Intracellular protein transport was stopped by the addition of NaF and NaN3 to final concentrations of 10 mM. Cells were collected by centrifugation, washed with distilled water, and then resuspended in 100 μl of 0.2 N NaOH containing 0.5% 2-mercaptoethanol. The suspension was incubated on ice for 15 min, and 1 ml of ice-cold acetone was added to the suspension, followed by incubation for 30 min at −20°C. The proteins were precipitated by centrifugation for 10 min at 10 000 g. The pellets were dissolved in 100 μl of 20 mM Tris-HCl (pH 7.5) containing 1% SDS and 1% 2-mercaptoethanol, and then heated for 3 min at 95°C. Each cell lysate was centrifuged at 10 000 g for 5 min, and the supernatant was mixed with 1 ml of 50 mM Tris-HCl (pH 7.5) containing 150 mM NaCl, 2 mM EDTA, and 1% Triton X-100 (buffer A). The mixture was incubated with 5 μl of CPY antiserum (Rockland, Gilbertsville, PA) at 4°C for 12 h. Then 5 μl of protein A/G agarose (Santa Cruz Biotechnology, Santa Cruz, CA) was added, and the mixture was incubated at 4°C for 3 h. The precipitate was spun-down by centrifugation, washed five times with buffer A, and then suspended in 20 μl of SDS-sample buffer (62.5 mM Tris-HCl, pH 6.8, containing 2% SDS, 10% glycerol, 1% 2-mercaptoethanol, and 0.001% bromophenol blue). After heating for 3 min at 95°C, the sample was subjected to SDS-PAGE according to the method of Laemmli (Laemmli, 1970). The gel was fixed, treated with Amplify Fluorographic Reagent (GE Healthcare), dried, and then analysed with the FLA-2000.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We wish to thank Dr T. Ogishima (Kyushu University) for the valuable suggestions throughout this study. This study was funded by a KAKENHI (19870017) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, The Naito Foundation in Japan, The Agricultural Chemical Research Foundation in Japan and The Cosmetology Research Foundation in Japan.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
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