Accepted Article

Synthesis of mannosylinositol phosphorylceramides is involved in maintenance of

1

cell integrity of yeast Saccharomyces cerevisiae1

Yuji Morimoto1, and Motohiro Tani1§

Department of Chemistry, Faculty of Sciences, Kyushu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan

§ To whom correspondence should be addressed: Department of Chemistry, Faculty of Sciences, Kyushu University, 6-10-1, Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan. Tel: +81-92-642-4182; Fax: +81-92-642-2607; E-mail: [email protected]

Running title: Role of MIPC synthase in cell integrity

Keywords: sphingolipids, complex sphingolipids, Saccharomyces cerevisiae, cell wall, cell integrity

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/mmi.12896

1 This article is protected by copyright. All rights reserved.

Accepted Article 1 2

SUMMARY Complex sphingolipids play important roles in many physiologically important events

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in yeast Saccharomyces cerevisiae.

In this study, we screened yeast mutant strains

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showing a synthetic lethal interaction with loss of mannosylinositol phosphorylceramide

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(MIPC) synthesis, and found that a specific group of glycosyltransferases involved in the

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synthesis of mannan-type N-glycans is essential for the growth of cells lacking MIPC

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synthases (Sur1 and Csh1). The genetic interaction was also confirmed by repression of

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MNN2, which encodes alpha-1,2-mannosyltransferase that synthesizes mannan-type

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N-glycans, by a tetracycline-regulatable system. MNN2-repressed sur1∆ csh1∆ cells

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exhibited high sensitivity to zymolyase treatment, and caffeine and SDS strongly

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inhibited the growth of sur1∆ csh1∆ cells, suggesting impairment of cell integrity due to

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the loss of MIPC synthesis. The phosphorylated form of Slt2, a MAP kinase activated by

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impaired cell integrity, increased in sur1∆ csh1∆ cells, and this increase was dramatically

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enhanced by the repression of Mnn2. Moreover, the growth defect of MNN2-repressed

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sur1∆ csh1∆ cells was enhanced by the deletion of SLT2 or RLM1 encoding a

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downstream transcription factor of Slt2. These results indicated that loss of MIPC

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synthesis causes impairment of cell integrity and this effect is enhanced by impaired

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synthesis of mannan-type N-glycans.

19 20

(195 words)

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2 This article is protected by copyright. All rights reserved.

Accepted Article 1

INTRODUCTION

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Sphingolipids are an essential component of biomembranes of eukaryotic organisms.

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Complex sphingolipids are each composed of a polar head group and a hydrophobic

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segment, ceramide, which comprises a fatty acid and a sphingoid long-chain base. They

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play important roles in signal transduction, membrane trafficking, stress adaptation, and

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formation of lipid microdomains (Simons & Sampaio, 2011, Dickson et al., 2006).

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The structure of the polar head groups in complex sphingolipids greatly differs among

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eukaryotic organisms.

Mammalian cells have sphingomyelin, which contains a

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phosphocholine head group, and glycosphingolipids. Glycosphingolipids contain one or

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more sugars attached to ceramides, there being over 100 structural variations of the sugar

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chains (Merrill, 2011). Drosophila melanogaster has ceramide phosphoethanolamine as

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the principal membrane complex sphingolipid instead of sphingomyelin (Acharya &

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Acharya, 2005).

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phosphoinositol, but not phosphocholine (Bure et al., 2014). In the budding yeast

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Saccharomyces cerevisiae, complex sphingolipids can carry one of three different types

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of polar head groups, and therefore complex sphingolipids in S. cerevisiae can be divided

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into inositol phosphorylceramide (IPC), mannosylinositol phosphorylceramide (MIPC),

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and mannosyldiinositol phosphorylceramide (M(IP)2C), all of which include

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phosphoinositol (Fig. S1) (Dickson et al., 2006).

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Schizosaccharomyces pombe has IPC and MIPC, but not M(IP)2C (Nakase et al., 2010).

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Interestingly, forced expression of M(IP)2C through expression of budding yeast M(IP)

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2C

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is toxic to the fission yeast (Nakase et al., 2012). In each organism, the structural

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diversity and complexity of the polar head group are thought to be important for the

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multiple biological functions of complex sphingolipids. Due to the limited molecular

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classes of polar head groups, the structural diversity of complex sphingolipids in yeast is

In plants and fungi, major complex sphingolipids contain

In contrast, the fission yeast

synthase causes strong growth inhibition of the fission yeast, indicating that M(IP) 2C

3 This article is protected by copyright. All rights reserved.

Accepted Article 1

relatively simple as compared with that in mammalian cells, making yeast a useful model

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for investigating the physiological significance of the structural complexity of complex

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sphingolipids.

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IPC, the simplest complex sphingolipid in yeast, is formed by IPC synthase (Aur1), an

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enzyme catalyzing the transfer of the head group of phosphatidylinositol to ceramides

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(Nagiec et al., 1997). There are five IPCs (IPC-A, -B, -B’, -C, and -D), which differ in

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the position and/or number of hydroxyl groups within the ceramide moiety (Fig. S1)

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(Dickson et al., 2006). Inhibition of Aur1 causes strong growth inhibition due to

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reductions in all the complex sphingolipid levels and accumulation of ceramides (Nagiec

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et al., 1997). MIPC is formed through the addition of mannose to IPC. Synthesis of

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MIPC is catalyzed by two homologous IPC mannosyltransferases, Sur1 and Csh1, and

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Csg2 functions as a regulatory subunit for both Sur1 and Csh1 (Zhao et al., 1994, Beeler

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et al., 1997, Uemura et al., 2003). MIPC is converted to M(IP)2C through the addition of

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another phosphoinositol, which is catalyzed by Ipt1 (Dickson et al., 1997).

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The biosynthesis of MIPC and M(IP)2C is basically non-essential for growth of S.

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cerevisiae; however, the structural diversity of the polar head groups of complex

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sphingolipids is suggested to be important for stress responses and drug sensitivity in S.

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cerevisiae. A defect of MIPC synthesis causes supersensitivity to Ca2+ (Zhao et al., 1994,

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Uemura et al., 2003), and rapid cell death under nitrogen starvation (Yamagata et al.,

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2013). The defect of MIPC synthesis also affects endosomal vesicular trafficking

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systems (Obara et al., 2013, Tani & Kuge, 2012). Deletion of IPT1 results in a complete

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loss of M(IP)2Cs and affects the sensitivity to phytotoxins, such as Dahlia merckii

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antimicrobial peptide 1 (DmAMP1) (Thevissen et al., 2000) and syringomycin E (Stock

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et al., 2000). In addition, gene expression of IPT1 is controlled by Pdr1 and Pdr3,

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transcriptional factors for multidrug-resistance genes, suggesting the involvement of

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M(IP)2C in drug resistance (Hallstrom et al., 2001). 4 This article is protected by copyright. All rights reserved.

Accepted Article 1

In the present study, we screened a collection of ~4800 yeast mutant strains lacking a

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non-essential gene for mutants showing a synthetic lethal interaction with loss of MIPC

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synthesis, and found that a specific enzyme group of glycosyltransferases, which are

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involved in the synthesis of mannan-type N-glycans frequently found in cell wall and

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periplasm proteins, is essential for the growth of sur1∆ csh1∆ cells. Furthermore, it was

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suggested that the synthesis of MIPC plays an important role in the maintenance of cell

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integrity.

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sphingolipids in cell integrity.

This is the first description of the involvement of specific complex

9

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5 This article is protected by copyright. All rights reserved.

Accepted Article 1

EXPERIMENTAL PROCEDURES

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Yeast Strains and Media – The S. cerevisiae strains used are listed in Table 1. To

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generate a yeast in which the expression of MNN2 is regulated by doxycycline (Dox), the

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MNN2 upstream region was replaced with a tetracycline operator cassette containing a

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repressor binding site (tetO2), the gene encoding TetR-VP16 tTA transactivator, and a

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kanMX4 marker, as described previously (Belli et al., 1998). Disruption of SUR1, CSH1,

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CSG2, IPT1, SLT2, RLM1, MNN2, OCH1, MNN9, MNN5, KTR6, and MNN1 was

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performed by replacing their open reading frames with the URA3 marker from the

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pRS406 vector, the LEU2 marker from the pRS405 vector (Sikorski & Hieter, 1989), the

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kanMX4 marker from a genome from a yeast knockout collection or the pFA6a-kanMX4

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vector (Wach et al., 1994), or the natMX4 marker from the p4339 vector

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(pCRII-TOPO::natMX4) (Tong & Boone, 2006). Occasionally, the kanMX4 and natMX4

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were replaced with the hygromycin B-resistant gene (from the pFA6a-hphNTI vector

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(Janke et al., 2004)) to create hphMX4. For tagging of the C-terminus of MNN2 and SLT2

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with six copies of the HA epitope (6xHA), a 6xHA fusion cassette with the hphNT1

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marker from the pYM16 vector was introduced immediately upstream of the stop codon

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of chromosomal MNN2 or SLT2 as described previously (Janke et al., 2004). The cells

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were grown in either YPD medium (1% yeast extract, 2% peptone, and 2% glucose) or

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synthetic complete (SC/MSG) medium (0.17% yeast nitrogen base w/o amino acids and

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ammonium sulfate (BD Difco, Heidelberg, Germany), 0.1% L-glutamic acid sodium salt

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hydrate (MSG; Sigma), and 2% glucose) containing nutritional supplements (Tong &

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Boone, 2006).

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Synthetic Genetic Array Analysis – Synthetic genetic array analysis with the YMY12

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strain (Y7092, sur1∆::URA3 csh1∆::LEU2) and the MATa yeast knockout collection

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(parent strain BY4741; OpenBiosystems, Huntsville, AL) was performed as described

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previously (Tong & Boone, 2006). Briefly, the YMY12 cells were mated to the yeast 6 This article is protected by copyright. All rights reserved.

Accepted Article 1

knockout collection, the resulting diploids were allowed to sporulate, and then the

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haploid cells were selected. The haploid cells were spotted onto SC/MSG medium

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lacking histidine, arginine, and lysine, but containing 50 µg/ml canavanine, 50 µg/ml

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thialysine, and 200 µg/ml G418 (for kanMX4 selection); or SC/MSG medium lacking

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histidine, arginine, lysine, uracil, and leucine, but containing 50 µg/ml canavanine, 50

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µg/ml thialysine, and 200 µg/ml G418 (for kanMX4, URA3, and LEU2 selection).

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Random Spore Analysis – To observe the synthetic lethal genetic interaction, random

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spore analysis was performed according to the method of Tong and Boone (2006), using

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BY4741 (MATa) strains (Winzeler et al., 1999) and the Y7092 (MAT) strains (Tong &

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Boone, 2006). The selection media used for this study were as follows: SC/MSG lacking

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histidine, arginine, and lysine, but containing 50 µg/ml canavanine and 50 µg/ml

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thialysine (for haploid selection); SC/MSG lacking histidine, arginine, and lysine, but

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containing 50 µg/ml canavanine, 50 µg/ml thialysine, and 200 µg/ml G418 (for kanMX4

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selection); SC/MSG lacking histidine, arginine, lysine, and uracil, but containing 50

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µg/ml canavanine and 50 µg/ml thialysine (for URA3 selection); SC/MSG lacking

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histidine, arginine, lysine, uracil, and leucine, but containing 50 µg/ml canavanine and 50

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µg/ml thialysine (for URA3 and LEU2 selection); SC/MSG lacking histidine, arginine,

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lysine, and uracil, but containing 50 µg/ml canavanine, 50 µg/ml thialysine, and 200

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µg/ml G418 (for kanMX4 and URA3 selection); and SC/MSG lacking histidine, arginine,

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lysine, uracil, and leucine, but containing 50 µg/ml canavanine, 50 µg/ml thialysine, and

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200 µg/ml G418 (for kanMX4, URA3, and LEU2 selection).

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Yeast Protein Extraction, SDS-PAGE, and Western Blotting - Yeast cells grown in

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YPD medium were chilled on ice, collected by centrifugation, washed with distilled

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water, and then resuspended in 100 µl of 0.2 N NaOH containing 0.5%

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2-mercaptoethanol. The suspension was incubated on ice for 15 min. 1 ml of ice-cold

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acetone was added to the suspension, followed by incubation for 30 min at -30°C, and 7 This article is protected by copyright. All rights reserved.

Accepted Article 1

then proteins were precipitated by centrifugation for 10 min at 10,000 x g. The pellet was

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resuspended in 100 µl of SDS-sample buffer (125 mM Tris-HCl, pH6.8, containing 4%

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SDS, 20% glycerol, 5% 2-mercaptoethanol, and 0.001% bromophenol blue).

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suspension was mixed well, heated for 3 min at 95°C, and then centrifuged for 2 min at

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10,000 x g. Then the supernatant was separated by SDS-PAGE according to the method

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of Laemmli (Laemmli, 1970). For Western blotting, following separation by SDS-PAGE,

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proteins were electrotransferred to a nitrocellulose membrane. The membrane was

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blocked with PBS/0.1% Tween 20 (PBS-T) containing 3% dried milk. Proteins were

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identified by incubation with anti-HA (Sigma), anti-phospho-p44/42 MAPK (Cell

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Signaling Technology, Danvers, MA), or anti-Pgk1 (Molecular Probes, Carlsbad, CA) in

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PBS-T. Secondary antibodies (horseradish peroxidase-conjugated anti-mouse or

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anti-rabbit IgG (Biosource, Camarillo, CA)) were incubated in PBS-T. Labeling was

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visualized with Western Lightning Plus-ECL (PerkinElmer, Waltham, MA).

The

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Lipid Extraction and TLC Analysis - Lipids were extracted from S. cerevisiae by the

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method of (Hanson & Lester, 1980) with minor modification. Briefly, the cells were

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suspended in 300 µl of ethanol/water/diethyl ether/pyridine/15 M ammonia

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(15:15:5:1:0.018, v/v), and then incubated at 65°C for 15 min. The residue was

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centrifuged at 10,000 g for 1 min and extracted once more in the same manner. The

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resulting supernatants were dried and subjected to mild alkaline treatment using

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monomethylamine (MMA). For this reason, the lipid extracts were dissolved in 130 μl

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MMA (40% methanol solution)/water (10:3, v/v), incubated for 1 h at 53°C, and then

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dried. The lipids were suspended in 30 μl of chloroform/methanol/water (5:4:1, v/v), and

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then separated on Silica Gel 60 TLC plates (Merck, Whitehouse Station, NJ) with

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chloroform/methanol/4.2 M ammonia (9:7:2, v/v) as the solvent system. The TLC plates

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were sprayed with 10% copper sulphate in 8% orthophosphoric acid and then heated at

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180°C to visualize complex sphingolipids. Identification of each complex sphingolipid 8 This article is protected by copyright. All rights reserved.

Accepted Article 1

band was performed in previous study (Uemura et al., 2014).

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Calcofluor white staining – Calcofluor white (CFW) staining of yeast cells was

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performed as described in (Phelan et al., 2006) with minor modification. Briefly, yeast

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cells were collected by centrifugation and fixed with 500 μl of 2% paraformaldehyde in

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PBS (-) for 30 min. After washing two times with PBS (-), the cells were incubated with

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10 μg/ml CFW in PBS (-), and then incubated for 30 min at room temperature. Then, the

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cells were washed 2 times with PBS (-), and viewed under a fluorescence microscope

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(Leica DMRB; Leica, Solms, Germany).

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9 This article is protected by copyright. All rights reserved.

Accepted Article 1

RESULTS

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Screening of mutations that exhibit a synthetic lethal interaction with deletion of

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MIPC synthase genes

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To identify mutations that exhibit a synthetic lethal interaction with deletion of MIPC

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synthase genes, we performed synthetic genetic array analysis using the MATa yeast

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knockout collection (~4800 strains). S. cerevisiae has two MIPC synthases, Sur1 and

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Csh1, and thus sur1∆ csh1∆ cells were used as the query mutant. In the initial screening,

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92 mutants were found as candidates that show a synthetic lethal interaction when

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combined with the deletion of SUR1 and CSH1. Forty genes were chosen for retesting of

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the genetic interaction, because the rest of the genes encoded proteins involved in gene

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expression, DNA repair, and synthesis of ribosomes, which seemed to affect a broad

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range of cellular activities. Finally, retesting by means of random spore analysis (Tong &

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Boone, 2006) revealed that 16 genes showed a synthetic lethal interaction with SUR1 and

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CSH1 (Table 2). The 16 genes included SAC1 and VPS74, which were previously

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described as synthetic lethal genes with MIPC synthase genes (Tani & Kuge, 2010, Wood

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et al., 2012). Among them, MNN11 and KTR6, the genes encoding glycosyltransferases

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involved in the synthesis of polysaccharides of glycoproteins, attracted our attention,

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because it was reported that deletion of VPS74, the gene encoding a protein determining

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the localization of Golgi glycosyltransferase, exhibits a synthetic lethal interaction with

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sur1∆, csg2∆, elo2∆, and elo3∆, suggesting a functional connection between

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sphingolipids and the glycosylation of proteins (Tu et al., 2008, Wood et al., 2012).

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Mnn11 and Ktr6 are involved in synthesis of mannan-type N-glycans; that is, Mnn11 is a

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subunit of the mannosyltransferase complex, M-PolII, which is involved in extension of

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the polymannose backbone of mannan-type N-glycans, and Ktr6 is involved in addition

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of mannosylphosphate at the alpha-1,2-linked mannobiose of mannan-type N-glycans

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(Fig. 1A) (Munro, 2001, Dean, 1999). Thus, we investigated which step in the synthesis 10 This article is protected by copyright. All rights reserved.

Accepted Article 1

of mannan-type N-glycans is responsible for the synthetic lethal interaction with MIPC

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synthase genes. Deletion of OCH1, the gene encoding alpha-1,6-mannosyltransferase

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initiating elongation of the polymannose outer chains of mannan-type N-glycans, MNN9,

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a subunit of M-PolI and M-PolII, or MNN2, the alpha-1,2-mannosyltransferase

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responsible for the formation of branches on polymannose (Fig. 1A) (Munro, 2001, Dean,

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1999), exhibits a synthetic lethal interaction with sur1∆ csh1∆ (Fig. 1 B). In contrast,

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deletion of MNN5 or MNN1 did not cause synthetic lethality with sur1∆ csh1∆ (Fig. 1B).

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Both Mnn5 and Mnn1 are responsible for the addition of mannose to the branches formed

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by Mnn2 (Fig. 1A) (Munro, 2001, Dean, 1999). Thus, overall, it was suggested that

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addition of the first alpha-1,2-linked mannose to form the branches on the polymannoses

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of mannan-type N-glycans is essential for cell growth with deletion of SUR1 and CSH1.

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As Ktr6 is involved in the addition of mannosylphosphate to both N- and O-glycans

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(Nakayama et al., 1998), the synthetic lethal interaction between ktr6∆ and sur1∆ csh1∆

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may be caused by impaired synthesis of N- and/or O-glycans. Thus, we decided to

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analyze the genetic interaction between MNN2, SUR1, and CSH1 in further studies.

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Genetic interaction between MNN2 and genes encoding a sphingolipid-metabolizing

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enzyme

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Fig. 2A shows the effect of myriocin on the growth of wild-type and mnn2∆ cells.

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Myriocin is an inhibitor of serine palmitoyltransferase catalyzing the initial step of

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sphingolipid biosynthesis, and myriocin treatment causes reductions in all sphingolipid

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levels including those of complex sphingolipids (Dickson et al., 2006).

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exhibited a more severe growth defect with myriocin as compared with wild-type cells,

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supporting the notion that the deletion of MNN2 causes a growth defect with aberrant

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metabolism of sphingolipids. To investigate more precisely how the defect in complex

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sphingolipid metabolism genetically interacts with mnn2∆, random spore analysis using

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mnn2∆ cells and sur1∆, csh1∆, csg2∆, or ipt1∆ cells was performed (Fig. 2B). The 11 This article is protected by copyright. All rights reserved.

mnn2∆ cells

Accepted Article 1

deletion of CSG2 causes drastic reductions in the MIPC and M(IP)2C levels (Zhao et al.,

2

1994). The deletion of SUR1 also causes reductions in the MIPC and M(IP)2C levels;

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however, the reductions are less than those in csg2∆ cells, because Csh1 is still active

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(Uemura et al., 2003, Tani & Kuge, 2012). ipt1∆ cells exhibit complete loss of the

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synthesis of M(IP)2Cs (Dickson et al., 1997). As shown in Fig. 2B, the double deletion of

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SUR1 and MNN2 did not cause a synthetic lethal phenotype, indicating that partial

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reduction in MIPC synthesis does not have a significant effect on the growth of mnn2∆

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cells. In contrast, a strong growth defect was observed for csg2∆ mnn2∆ cells. The

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deletion of IPT1 did not cause synthetic lethality with mnn2∆ (Fig. 2B). Thus, overall, it

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was suggested that loss of synthesis of MIPC, but not M(IP)2C, causes synthetic lethality

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with the deletion of MNN2.

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Growth defect on repression of MNN2 gene expression, and deletion of SUR1 and

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CSH1

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Since the triple-deletion mutant of MNN2, SUR1, and CSH1 could not be used for

15

further analyses due to the lethal phenotype, we created a mutant strain that carries the

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MNN2 gene under the control of a tetracycline-regulatable promoter (tetO2-MNN2) (Belli

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et al., 1998). To examine the effect of the tetO2 promoter on MNN2 expression, the

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3′-end of chromosomal MNN2 was tagged with 6xHA in wild-type, tetO2-MNN2, and

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tetO2-MNN2 sur1∆ csh1∆ cells (MNN2-6xHA). As shown in Fig. 3A, the expression

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levels of Mnn2-6xHA in tetO2-MNN2 and tetO2-MNN2 sur1∆ csh1∆ cells were

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moderately reduced as compared with that in wild-type cells even in the absence of Dox,

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and dramatic reduction of the expression was observed after the addition of Dox. Faint

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expression of Mnn2-6xHA was still observed after the addition of Dox, indicating that

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Mnn2 did not completely disappear on Dox treatment. It should be noted that the rate of

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decrease of Mnn2-6xHA caused by Dox was indistinguishable between tetO2-MNN2 and

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tetO2-MNN2 sur1∆ csh1∆ cells (Fig. 3A). Although the growth of tetO2-MNN2 cells was 12 This article is protected by copyright. All rights reserved.

Accepted Article 1

normal on YPD plates even in the presence of Dox, tetO2-MNN2 sur1∆ csh1∆ cells

2

exhibited a strong growth defect in the presence of Dox (Fig. 3B). Figure 3C shows the

3

time-courses of growth of tetO2-MNN2 and tetO2-MNN2 sur1∆ csh1∆ cells in the

4

presence and absence of Dox. Dox-treated tetO2-MNN2 cells showed a similar growth

5

rate to Dox-untreated tetO2-MNN2 ones; however, the growth rate of tetO2-MNN2 sur1∆

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csh1∆ cells began to slow at 6–9 h after the addition of Dox (Fig. 3C).

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Effect of Mnn2 repression on the complex sphingolipid composition

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Complex sphingolipids are essential for normal cell growth in yeast. To explore the

9

possibility that the repression of MNN2 expression has a profound effect on complex

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sphingolipid metabolism in sur1∆ csh1∆ cells, lipids were extracted from each strain, and

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complex sphingolipids were stained with a copper sulphate and orthophosphoric acid

12

reagent (Fig. 4A). As reported previously (Uemura et al., 2003), the deletion of SUR1

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and CSH1 caused a complete loss of MIPC and M(IP)2C. Although IPC level in

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MNN2-repressed cells were slightly decreased as compared with that in wild type cells,

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significant difference in IPC level was not observed between sur1∆ csh1∆ and

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tetO2-MNN2 sur1∆ csh1∆ cells (Fig. 4A and B).

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Zymolyase sensitivity of Mnn2-repressed sur1∆ csh1∆ cells

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Mannan-type N-glycans are frequently found in cell wall and periplasm proteins

19

(Munro, 2001, Dean, 1999).

Thus, we next investigated whether or not multiple

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mutations of the MNN2 and MIPC synthase genes cause defects of cell wall functions.

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Zymolyase, a cell wall-digesting enzyme, has been used for evaluation of cell wall

22

integrity (Kitagaki et al., 2002), and we performed a zymolyase sensitivity assay for

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wild-type, tetO2-MNN2, sur1∆ csh1∆, and tetO2-MNN2 sur1∆ csh1∆ cells (Fig. 5).

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tetO2-MNN2 cells did not exhibit high sensitivity to zymolyase even in the presence of

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Dox. In contrast, sur1∆ csh1∆ cells were slightly but significantly more sensitive to

26

zymolyase than wild-type cells, indicating that a defect of synthesis of MIPC causes 13 This article is protected by copyright. All rights reserved.

Accepted Article 1

enhancement of cell lysis by the zymolyase treatment. tetO2-MNN2 sur1∆ csh1∆ cells

2

were much more sensitive to zymolyase than wild-type and sur1∆ csh1∆ cells even in the

3

absence of Dox, and the Dox treatment resulted in a more severe effect (Fig. 5). The

4

difference of sensitivity between sur1∆ csh1∆ and Dox-untreated tetO2-MNN2 sur1∆

5

csh1∆ cells may be caused by a decrease in the expression level of Mnn2 due to

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substitution of the promoter region (Fig. 3A). The cell lysis of Dox-treated tetO2-MNN2

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sur1∆ csh1∆ cells was not observed under the experimental conditions without the

8

addition of zymolyase (data not shown). These results suggested that multiple mutations

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of the MNN2 and MIPC synthase genes cause high sensitivity to digestion of cell walls,

10

implying the reduction of the cell integrity of the mutants.

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Effects of calcofluor white, SDS, and caffeine treatment on cell growth

12

In order to evaluate the impact of the disruption of SUR1 and CSH1 and/or MNN2 on

13

the structural integrity of the cell, the mutants were tested for sensitivity to several agents

14

that target the cell wall or membranes. Calcofluor white (CFW), a cell wall-perturbing

15

agent, binds with chitin and interferes with its polymerization (Ram & Klis, 2006). As

16

shown in Fig. 6A, sur1∆ csh1∆ cells exhibited significant resistance to CFW as compared

17

with wild type cells (This will be discussed later).

18

tetO2-MNN2 and tetO2-MNN2 sur1∆ csh1∆ cells exhibited high sensitivity to CFW (Fig.

19

6A). Next, we examined cell growth in the presence of SDS, a detergent compromising

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the integrity of the cell membrane (Levin, 2005). Cell wall defects lead to sensitivity to

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low levels of SDS because a weakened cell wall allows molecules of the detergent to

22

penetrate more easily (Levin, 2005). sur1∆ csh1∆ cells exhibited high sensitivity to SDS.

23

A severer growth defect was observed in Dox-treated tetO2-MNN2 sur1∆ csh1∆ cells;

24

that is, growth of Dox-treated tetO2-MNN2 sur1∆ csh1∆ cells, but not sur1∆ csh1∆ cells,

25

was hardly observed in the presence of 0.005 % SDS (Fig. 6B).

26

blocks cell wall biosynthesis in fungi, and thus fungi having abnormalities in cell surface 14 This article is protected by copyright. All rights reserved.

In contrast, both Dox-treated

Caffeine indirectly

Accepted Article 1

integrity show increased sensitivity to caffeine treatment (Levin, 2005). sur1∆ csh1∆

2

cells exhibited high sensitivity to caffeine (Fig. 6C). Fig. S2 shows the effects of single

3

deletion of SUR1, CSH1, CSG2, or IPT1 on the SDS and caffeine sensitivities.

4

Significantly high sensitivity to SDS or caffeine was only seen in csg2∆ cells (Fig. S2).

5

The results as to SDS and caffeine sensitivity strongly suggested that a defect of MIPC

6

synthesis causes impairment of cell integrity, and that this phenotype is enhanced by the

7

repression of Mnn2. However, the growth defect of Dox-treated tetO2-MNN2 sur1∆

8

csh1∆ cells was not suppressed on the addition of 1 M sorbitol, which is known as an

9

osmostabilizer protecting the growth defects caused by impaired cell integrity (Fig. 6D).

10

The growth defect of Dox-treated tetO2-MNN2 sur1∆ csh1∆ cells in the presence of 1 M

11

sorbitol was also confirmed by liquid culture (Fig. S3). Treatment with 1 M sorbitol also

12

induces hyper-osmotic stress (Hohmann, 2002). Thus, the effects of 1 M KCl, which also

13

induces hyper-osmotic stress (Hohmann, 2002), on growth of the mutants were examined.

14

As shown Fig. S4, growth of Dox-treated tetO2-MNN2 sur1∆ csh1∆ cells was hardly

15

observed in the presence of 1 M KCl. Thus, it is likely that the increase in sensitivity to

16

hyper-osmotic stress in Dox-treated tetO2-MNN2 sur1∆ csh1∆ cells prevents the

17

suppression of cell growth on the addition of 1 M sorbitol.

18

Increases in cell wall chitin levels in MNN2-repressed and MNN2-repressed sur1∆

19

csh1∆ cells

20

The chitin level is dynamically regulated in response to cell wall stress, and the deletion

21

of MNN2 causes an increase in the cell wall chitin level due to compensation for impaired

22

synthesis of mannan-type N-glycans (Lesage et al., 2005). To investigate whether or not

23

the deletion of SUR1 and CSH1 affects the increased level of chitin in MNN2-repressed

24

cells, cell wall chitin was stained with CFW, followed by observation under a

25

fluorescence microscope (Fig. 7). The fluorescence intensity at the cell surface of

26

Dox-treated tetO2-MNN2 cells was much higher than that in the case of wild-type cells, 15 This article is protected by copyright. All rights reserved.

Accepted Article 1

indicating an increase in the chitin level at the cell wall. Notable differences in the

2

fluorescence intensity were not observed between wild-type and sur1∆ csh1∆ cells. An

3

increase in the cell wall chitin level was also observed in Dox-treated tetO2-MNN2 sur1∆

4

csh1∆ cells, indicating that the loss of MIPC synthesis did not affect the increase in the

5

chitin level caused by MNN2 repression (Fig. 7).

6

Enhancement of phosphorylation of Slt2 on deletion of SUR1 and CSH1 and/or

7

repression of MNN2

8

Cellular integrity in S. cerevisiae is mainly controlled by signal transduction through a

9

MAPK (Mitogen-Activated Protein Kinases) cascade, named the cell wall integrity

10

(CWI) pathway (Levin, 2005). This signaling pathway ends with phosphorylation of

11

Slt2/Mpk1 MAPK, which subsequently phosphorylates and activates the transcription

12

factors involved in the cell wall biogenesis, such as Rlm1 (Levin, 2005). To investigate

13

whether or not the CWI pathway is activated in sur1∆ csh1∆, tetO2-MNN2, and

14

tetO2-MNN2 sur1∆ csh1∆ cells, phosphorylated Slt2 was detected by Western blotting

15

using anti phospho-p44/42 MAPK antibody (Corcoles-Saez et al., 2012). To detect

16

expression of Slt2, the 3′-end of chromosomal SLT2 was tagged with 6xHA in all cells.

17

As shown in Fig. 8A and B, an approximately two-fold increase of phosphorylated

18

Slt2-6xHA was observed in sur1∆ csh1∆ cells as compared with in wild-type cells when

19

the level of phosphorylated Slt2-6xHA was normalized to that of Pgk1. tetO2-MNN2 and

20

Dox-treated tetO2-MNN2 also exhibited an increase in the phosphorylated Slt2-6xHA

21

level. Notably, in Dox-untreated tetO2-MNN2 sur1∆ csh1∆ cells, an approximately

22

ten-fold increase in phosphorylated Slt2-6xHA level was observed; moreover,

23

Dox-treated tetO2-MNN2 sur1∆ csh1∆ cells exhibited a much higher phosphorylated

24

Slt2-6xHA level than Dox-untreated ones (Fig. 8A and B).

25

phosphorylated Slt2 level were also confirmed when Slt2 was not tagged with 6xHA

26

(data not shown). Also, it should be noted that the total expression level of Slt2-6xHA in 16 This article is protected by copyright. All rights reserved.

The increases in the

Accepted Article 1

sur1∆ csh1∆, tetO2-MNN2 and tetO2-MNN2 sur1∆ csh1∆ cells was higher than that in

2

wild-type cells (Fig. 8A). When the level of phosphorylated Slt2-6xHA was normalized

3

to the total expression level of Slt2-6xHA, increase in the phosphorylation level was

4

observed in each mutant cells; however, the rate of increase was lower than when the

5

level of phosphorylated Slt2-6xHA was normalized to that of Pgk1 (Fig. 8B and Fig.

6

S5A). These results indicated that phosphorylated Slt2 is increased on deletion of SUR1

7

and CSH1, or repression of MNN2 expression, and magnitude of the increase is

8

dramatically potentiated when all the genes are mutated. Next, we examined the effects

9

of deletion of SLT2 on cell growth of sur1∆ csh1∆, tetO2-MNN2, and tetO2-MNN2 sur1∆

10

csh1∆ cells. As shown in Fig. 8C, deletion of SLT2 did not affect the cell growth of sur1∆

11

csh1∆ and Dox-treated tetO2-MNN2 cells; however, it significantly enhanced the growth

12

defect of Dox-treated tetO2-MNN2 sur1∆ csh1∆ cells. Rlm1, a transcription factor, is a

13

primary target of Slt2 in maintenance of cell integrity (Levin, 2005). The deletion of

14

RLM1 also caused enhancement of the growth defect of Dox-treated tetO2-MNN2 sur1∆

15

csh1∆ cells (Fig. 8D). Thus, these results indicated that defect of CWI pathway enhances

16

the growth defect of Dox-treated tetO2-MNN2 sur1∆ csh1∆ cells.

17

Deletion of SLT2 causes CFW high sensitivity in sur1∆ csh1∆ cells

18

Although MIPC synthesis was suggested to be important for the maintenance of cell

19

integrity, sur1∆ csh1∆ cells exhibited resistance to CFW (Fig. 6A). CFW causes rapid

20

activation of Slt2, and the activation is important for acquisition of resistance to CFW (de

21

Nobel et al., 2000). Since sur1∆ csh1∆ cells exhibited slight but significant increase in

22

the phosphorylated Slt2 level (Fig. 8A and B), the effect of deletion of SLT2 on the CFW

23

sensitivity of sur1∆ csh1∆ cells was examined (Fig. 9A). As reported previously (de

24

Nobel et al., 2000), slt2∆ cells exhibited high sensitivity to CFW; however, sur1∆ csh1∆

25

slt2∆ cells were much more sensitive to CFW than wild-type and slt2∆ cells. Next, we

26

investigated the activation of Slt2 by CFW in wild-type and sur1∆ csh1∆ cells (Fig. 9B 17 This article is protected by copyright. All rights reserved.

Accepted Article 1

and C). As reported previously (de Nobel et al., 2000), CFW caused an increase in the

2

phosphorylated Slt2 level in a dose-dependent manner; however, the rate of the increase

3

in the level in sur1∆ csh1∆ cells was higher than that in wild-type cells (Fig. 9B and C).

4

On the other hand, the enhancement of Slt2 phosphorylation in sur1∆ csh1∆ cells was not

5

remarkable when the level of phosphorylated Slt2-6xHA was normalized to the total

6

expression level of Slt2-6xHA (Fig. S5B), suggesting that the enhancement is at least

7

partly due to the increase in expression level of Slt2 by the deletion of SUR1 and CSH1.

8

Collectively, these results suggested that the resistance to CFW in sur1∆ csh1∆ cells is

9

due to activation of Slt2, and that sur1∆ csh1∆ cells are very sensitive to CFW in the

10

absence of Slt2. In addition, the deletion of RLM1 attenuated the resistance to CFW in

11

sur1∆ csh1∆ cells (Fig. 9D), supporting the notion that the CFW resistance of sur1∆

12

csh1∆ cells is acquired through activation of the CWI pathway.

13

18 This article is protected by copyright. All rights reserved.

Accepted Article 1

DISCUSSION

2

In the present study, we showed that MIPC synthases, Sur1 and Csh1, are essential for

3

growth of S. cerevisiae with impaired synthesis of mannan-type N-glycans. Random

4

spore analysis of heterozygous diploid strains revealed that the deletion of MNN2, but not

5

MNN1 and MNN5, caused a synthetic lethal phenotype with sur1∆ csh1∆ (Fig. 1),

6

indicating that addition of the first alpha-1,2-linked mannose to form the branches on the

7

polymannoses of mannan-type N-glycans is essential for sur1∆ csh1∆ cells, but

8

elongation of the branches with additional mannoses is not. Deletion of SUR1, which

9

causes a partial reduction in MIPC synthesis (Uemura et al., 2003, Tani & Kuge, 2012),

10

or IPT1 encoding M(IP)2C synthase did not cause a growth defect in mnn2∆ cells (Fig.

11

2B). Thus, it was indicated that the synthetic lethal phenotype with mnn2∆ is caused

12

when the synthesis of MIPC, but not M(IP)2C, is completely lost. sur1∆ csh1∆ cells were

13

slightly more sensitive to zymolyase treatment than wild-type cells, and the sensitivity

14

was potentiated by the repression of MNN2 expression (Fig. 5). Furthermore, treatment

15

with SDS or caffeine caused a significant growth defect of sur1∆ csh1∆ cells (Fig. 6B and

16

C).

17

impairment of cell integrity, and that this defect is exacerbated by impairment of

18

synthesis of mannan-type N-glycans. The fact that SUR1 and CSH1 also genetically

19

interacted with TOS1 encoding a cell wall protein of unknown function supports the

20

notion of impairment of cell integrity caused by the loss of MIPC synthesis (Table 2).

Thus, collectively, it was suggested that the loss of MIPC synthesis causes

21

The phosphorylated Slt2 level was slightly but significantly increased in sur1∆ csh1∆

22

cells, and the magnitude of the increase was dramatically potentiated in tetO2-MNN2

23

sur1∆ csh1∆ cells (Fig. 8A and B). Furthermore, the deletion of SLT2 and RLM1 resulted

24

in enhancement of the growth defect of Dox-treated tetO2-MNN2 sur1∆ csh1∆ cells (Fig.

25

8C and D), indicating the growth defect is enhanced by defect of CWI pathway. It should

26

be noted that the enhancement of the growth defect on the deletion of SLT2 was more 19 This article is protected by copyright. All rights reserved.

Accepted Article 1

severe than on that of RLM1 (Fig. 8C and D), implying that other downstream

2

transcriptional factors (Swi4, Swi6, etc.) of Slt2 are also involved. In addition, sur1∆

3

csh1∆ cells exhibited CFW resistance; however, they were much more sensitive to CFW

4

than wild-type cells in the absence of Slt2 (Fig. 9). Thus, it is suggested that the CWI

5

pathway is involved in suppression of the growth defect by CFW in sur1∆ csh1∆ cells.

6

These results also suggested that impaired cell integrity caused by the loss of MIPC

7

synthesis is not caused by impairment of the CWI signaling cascade.

8

In many cases, growth defects caused by impaired cell integrity are suppressed by the

9

addition of osmostabilizer sorbitol (Levin, 2005); however, the growth defect of

10

Dox-treated tetO2-MNN2 sur1∆ csh1∆ cells was not rescued in the presence of 1 M

11

sorbitol (Fig. 6D and Fig. S3). This discrepancy may be explained by the increased

12

sensitivity to hyper-osmotic stress in Dox-treated tetO2-MNN2 sur1∆ csh1∆ cells,

13

because the growth defect of Dox-treated tetO2-MNN2 sur1∆ csh1∆ cells is enhanced in

14

the presence of 1 M KCl, which causes hyper-osmotic stress (Fig. S4) (Hohmann, 2002).

15

In addition, it has been reported that a double mutant of CSG2 and GAS1 encoding a

16

GPI-anchored protein can not grow in the presence of 1 M sorbitol, supporting the notion

17

of the importance of MIPC synthesis for the hyper-osmotic stress response (Tomishige et

18

al., 2003). In S. cerevisiae, the hyper-osmotic response is mediated by the Hog1 MAP

19

kinase pathway (Hohmann, 2002). We found that there was no difference in the increase

20

in phosphorylation of the Hog1 caused by the addition of sorbitol between wild-type and

21

Dox-treated tetO2-MNN2 sur1∆ csh1∆ cells, suggesting that the Hog1 pathway is not

22

affected by the deletion of SUR1 and CSH1, and the repression of MNN2 (Morimoto Y

23

and Tani M, unpublished results). Further detailed investigation is required as to the

24

molecular mechanisms underlying the high sensitivity to hyper-osmotic stress in

25

MNN2-repressed sur1∆ csh1∆ cells.

20 This article is protected by copyright. All rights reserved.

Accepted Article 1

Several lines of evidences suggested that abnormal phenotypes in MIPC

2

synthesis-deficient mutants are caused by accumulation of an upstream product of MIPC,

3

but not by loss of MIPC itself. Supersensitivity to Ca2+, one of the typical phenotypes of

4

MIPC synthesis-deficient mutants, is caused by the accumulation of IPC-C, which

5

comprises a phytosphingosine-type long chain base and a hydroxylated very long chain

6

fatty acid (Zhao et al., 1994). That is, the Ca2+-sensitive phenotype of csg2∆ cells is

7

suppressed by several mutations, which causes a reduction in the IPC-C level. These

8

mutations include genes encoding sphingolipid hydroxylase (Scs7 and Sur2), serine

9

palmitoyltransferase (Lcb1, Lcb2, and Tsc3), and 3-ketosphinganine reductase (Tsc10)

10

(Dickson et al., 2006) (Fig. S1). Furthermore, the rapid cell death under nitrogen

11

starvation due to the loss of MIPC synthesis is also rescued by the deletion of SUR2 or an

12

inhibitor of serine palmitoyltransferase, myriocin (Yamagata et al., 2013). However, the

13

growth defect of Dox-treated tetO2-MNN2 sur1∆ csh1∆ cells was not suppressed on the

14

deletion of SCS7 or SUR2 (Fig. S6A). In addition, sur1∆ csh1∆ scs7∆ and sur1∆ csh1∆

15

sur2∆ cells exhibited high sensitivity to SDS (Fig. S6B). Moreover, mnn2∆ cells

16

exhibited high sensitivity to myriocin, which causes reductions in all sphingolipid levels

17

including that of MIPC (Fig. 2A). These results suggested that the impaired cell integrity

18

caused by the loss of MIPC synthesis is caused by loss of MIPC, but not by accumulation

19

of IPC-C.

20

Glycosylphosphatidylinositol (GPI)-anchored proteins are essential components of the

21

cell wall, and sphingolipids play important roles in the synthesis, trafficking, and

22

localization of GPI-anchored proteins.

23

ergostrol are important for correct intracellular trafficking of GPI-anchored proteins

24

through the formation of lipid microdomains (Fujita & Jigami, 2008). On the other hand,

25

the membrane anchors of many GPI-anchored proteins in S. cerevisiae are remodeled

26

from a diacylglycerol to a ceramide, and the remodeling is involved in cell integrity

For example, complex sphingolipids and

21 This article is protected by copyright. All rights reserved.

Accepted Article 1

(Fujita & Jigami, 2008). Thus, there is the possibility that the loss of MIPC synthesis

2

and/or repression of MNN2 cause aberrant trafficking and remodeling of GPI-anchored

3

proteins. However, we observed a normal cell-surface distribution of GFP-tagged Cwp2,

4

a major GPI-anchored protein in the cell wall, in Dox-treated tetO2-MNN2 sur1∆ csh1∆

5

cells (Morimoto Y and Tani M, unpublished results), suggesting that intracellular

6

trafficking of Cwp2 was not impaired. In addition, it should be noted that post-Golgi

7

trafficking of several proteins including Gas1 does not depend on the synthesis of MIPC

8

(Lisman et al., 2004). Furthermore, deletion of CWH43, which encodes a protein

9

essential for the remodeling of the lipid anchors of GPI-anchored proteins (Umemura et

10

al., 2007, Ghugtyal et al., 2007), did not cause a synthetic growth defect with the

11

repression of MNN2 or the deletion of SUR1 and CSH1 (Fig. S7). Thus, collectively, it is

12

likely that the growth defect caused by mutation of the MNN2 and MIPC synthase genes

13

is not caused by impaired remodeling or intracellular trafficking of GPI-anchored

14

proteins. However, the possibility that the loss of MIPC synthesis and/or repression of

15

MNN2 may cause a functional defect of some specific GPI-anchored proteins should also

16

be considered. More detailed studies of the relationship between GPI-anchored proteins

17

and the impaired cell integrity in sur1∆ csh1∆ and MNN2-repressed sur1∆ csh1∆ cells is

18

required in the future.

19

It is possible that the cell lysis on zymolyase treatment, and the growth inhibition by

20

SDS and caffeine treatment can be enhanced by the impaired integrity of not only the cell

21

wall but also plasma membranes. Since MIPC is a component of plasma membranes

22

(Hechtberger et al., 1994), there is a possibility that the defect of cell integrity caused by

23

the loss of MIPC synthesis is the result of impaired integrity of plasma membranes or the

24

cell wall, or both. Very recently, it was reported that deletion of SUR1 and CSH1 in S.

25

cerevisiae suppresses the lateral diffusion of Hxt1, a plasma membrane glucose

26

transporter, suggesting that loss of MIPC synthesis can affect the physical properties of 22 This article is protected by copyright. All rights reserved.

Accepted Article 1

plasma membranes (Uemura et al., 2014). Further detailed investigation is required as to

2

impact of the loss of MIPC synthesis on the properties of cell membranes as well as the

3

cell wall.

4

In summary, the present study indicated that MIPC synthesis is involved in the

5

maintenance of cell integrity, and that the impaired synthesis of mannan-type N-glycans

6

causes enhancement of the defect of cell integrity induced by the loss of MIPC synthesis.

7

Further detailed elucidation of the molecular mechanism underlying the maintenance of

8

cell integrity by MIPC synthesis will provide a new insight into the physiological

9

functions of complex sphingolipids.

10 11

ACKNOWLEDGEMENTS

12

We wish to thank Drs. O. Kuge and T. Ogishima (Kyushu University) for the valuable

13

suggestions regarding this study. This study was funded by a KAKENHI (26450127)

14

from the Ministry of Education, Culture, Sports, Science, and Technology, Japan, The

15

Nagase Science and Technology Foundation, and The Noda Institute for Scientific

16

Research, Japan.

17

23 This article is protected by copyright. All rights reserved.

Accepted Article 1

FIGURE LEGENDS

2 3

Fig. 1. Genetic interaction between genes encoding MIPC synthases and

4

glycosyltransferases involved in synthesis of mannan-type N-glycans.

5

A, mannan-type N-glycan synthesis pathway in yeast Saccharomyces cerevisiae. B,

6

sur1∆::URA3 csh1∆::LEU2 cells were mated with och1∆::kanMX4, mnn9∆::kanMX4,

7

mnn2∆::kanMX4, mnn5∆::kanMX4, and mnn1∆::kanMX4 cells, and the resulting diploids

8

were allowed to sporulate and then subjected to random spore analysis. Cells were

9

selected in haploid selection medium, or haploid selection medium lacking uracil and

10

leucine (sur1∆ csh1∆ selection), and/or containing G418 (och1∆, mnn9∆, mnn2∆, mnn5∆,

11

and mnn1∆ (xxx∆) selection). The plates were incubated at 30°C for 3 days. The details

12

are given under “EXPERIMENTAL PROCEDURES”.

13 14

Fig.

2.

Genetic

interaction

15

sphingolipid-metabolizing enzymes.

between

MNN2

and

genes

encoding

16

A, wild-type and mnn2∆ cells were cultured overnight in YPD medium, and then

17

spotted onto YPD plates with or without the indicated concentrations of myriocin, in

18

10-fold serial dilutions starting with a density of 0.7 A600 units/ml. All plates were

19

incubated at 30°C and photographed after 2 days. B, mnn2∆::kanMX4 cells were mated

20

with sur1∆::URA3, csh1∆::URA3, csg2∆::URA3, sur1∆::URA3 csh1∆::LEU2, and

21

ipt1∆::URA3 cells, and the resulting diploids were allowed to sporulate and then

22

subjected to random spore analysis. Cells were selected in haploid selection medium, or

23

haploid selection medium containing G418 (mnn2∆ selection), and/or lacking uracil

24

(sur1∆, csh1∆, csg2∆, and ipt1∆ (xxx∆) selection), or uracil and leucine (sur1∆ csh1∆

25

(xxx∆) selection). The plates were incubated at 30°C for 3 days. The details are given

26

under “EXPERIMENTAL PROCEDURES”. 24 This article is protected by copyright. All rights reserved.

Accepted Article 1 2

Fig. 3. Synthetic growth defects caused by deletion of SUR1 and CSH1, and repression of

3

MNN2 expression by a tetracycline-regulatable system (tetO2-MNN2).

4

A,

Western

blotting

analysis

of

expression

of

Mnn2-6xHA

with

a

5

tetracycline-regulatable system. MNN2-6xHA, tetO2-MNN2-6xHA, tetO2-MNN2-6xHA

6

sur1∆ csh1∆ cells were cultured overnight in YPD medium, diluted (0.1 A600 units/ml) in

7

fresh YPD with or without 10 µg/ml doxycycline (Dox), and then incubated for 6 h at

8

30°C. Yeast cell extracts were immunoblotted using anti-HA or anti-Pgk1. The details

9

are given under “EXPERIMENTAL PROCEDURES”. B, cells were cultured overnight

10

in YPD medium, and then spotted onto YPD plates with or without 10 µg/ml Dox in

11

10-fold serial dilutions starting with a density of 0.7 A600 units/ml. All plates were

12

incubated at 30°C and photographed after 1 day or 2 days. C, time-courses of cell growth.

13

Cells were cultured overnight in YPD medium and then diluted (0.06 A600 units/ml) in

14

fresh YPD medium with or without 10 µg/ml Dox, and aliquots of cell suspensions were

15

subjected to cell density measurements (A600) at the indicated times. Data represent

16

means ± SD for at least three independent experiments.

17 18

Fig. 4. TLC analysis of complex sphingolipids.

19

A, cells were cultured overnight in YPD medium with or without 10 µg/ml Dox, diluted

20

(0.1 A600 units/ml) in fresh YPD medium with or without 10 µg/ml Dox, and then

21

incubated for 8 h. Lipids (3 A600 U) were extracted, treated with MMA, and then

22

separated by TLC.

23

orthophosphoric acid reagent.

24

PROCEDURES”. B, Complex sphingolipids (IPC, MIPC, and M(IP)2C) were quantified

25

with ImageJ software (NIH). The amount of IPC in wild-type cells was taken as 1. Data

26

represent means ± SD from three independent experiments.

The lipids were visualized with a copper sulphate and The details are given under

25 This article is protected by copyright. All rights reserved.

“EXPERIMENTAL

Accepted Article 1 2

Fig. 5. Zymolyase sensitivity.

3

Cells were cultured 12 h in YPD medium with or without 10 µg/ml Dox. They were

4

then washed with 20 mM HEPES buffer (pH7.5), resuspended (2 A600 units/ml) in the

5

same buffer containing 15 µg/ml zymolyase-20T (Nacalai Tesque, Kyoto, Japan), and

6

then incubated at 30°C. Aliquots of cell suspensions were subjected to cell density

7

measurements (A600) at the indicated times. The values of wild type, tetO2-MNN2, and

8

Dox-treated tetO2-MNN2-6xHA cells almost totally overlapped. Data represent means ±

9

SD for at least three independent experiments.

10 11

Fig. 6. Calcofluor white, SDS, and caffeine sensitivities.

12

A, B, and C, cells were cultured overnight in YPD medium, and then spotted onto YPD

13

plates with or without 10 µg/ml Dox and the indicated concentrations of calcofluor white

14

(CFW), SDS, or caffeine in 10-fold serial dilutions starting with a density of 0.7 A600

15

units/ml. All plates were incubated at 30°C and photographed after 2 days. D, cells were

16

cultured overnight in YPD medium, and then spotted onto YPD plates containing 1 M

17

sorbitol with or without 10 µg/ml Dox in 10-fold serial dilutions starting with a density of

18

0.7 A600 units/ml. All plates were incubated at 30°C and photographed after 2 days.

19 20

Fig. 7. Calcofluor white staining.

21

A, cells were cultured overnight in YPD medium, diluted (0.1 A600 units/ml) in fresh

22

YPD with or without 10 µg/ml Dox, and then incubated for 9 h at 30°C. They were then

23

fixed, stained with CFW, and observed by fluorescence microscopy. B, frequency

24

distribution of CFW fluorescence intensity in individual cells. The fluorescence intensity

25

was quantified with ImageJ software. Data represent the value for 100 cells for each

26

strain. The details are given under “EXPERIMENTAL PROCEDURES”. 26 This article is protected by copyright. All rights reserved.

Accepted Article 1 2

Fig. 8. Phosphorylation of Slt2, and effect of deletion of SLT2 and RLM1 on cell growth.

3

A, Western blotting analysis of phospho-Slt2-6xHA. Cells expressing Slt2-6xHA were

4

cultured overnight in YPD medium, diluted (0.1 A600 units/ml) in fresh YPD with or

5

without 10 µg/ml Dox, and then incubated for 9 h at 30°C. Yeast cell extracts were

6

immunoblotted using anti-phospho-p44/42 MAPK, anti-HA, or anti-Pgk1. The details

7

are given under “EXPERIMENTAL PROCEDURES”.

8

phospho-Slt2-6xHA were determined with ImageJ software.

9

phospho-Slt2-6xHA/Pgk1 in wild-type cells was taken as 1. Data represent means ± SD

10

for at least three independent experiments. C and D, effects of deletion of SLT2 (C) and

11

RLM1 (D) on the growth of wild-type, sur1∆ csh1∆, tetO2-MNN2, and tetO2-MNN2

12

sur1∆ csh1∆ cells. Cells were cultured overnight in YPD medium, and then spotted onto

13

YPD plates with or without 10 µg/ml Dox in 10-fold serial dilutions starting with a

14

density of 0.7 A600 units/ml. All plates were incubated at 30°C and photographed after 2

15

days.

B, the relative amounts of The amount of

16 17

Fig. 9. Effects of deletion of SLT2 and RLM1 on CFW sensitivity of sur1∆ csh1∆ cells.

18

A, cells were cultured overnight in YPD medium, and then spotted onto YPD plates

19

containing the indicated concentrations of CFW in 10-fold serial dilutions starting with a

20

density of 0.7 A600 units/ml. All plates were incubated at 30°C and photographed after 2

21

days.

22

Wild-type and sur1∆ csh1∆ cells expressing Slt2-6xHA were cultured overnight in YPD

23

medium, diluted (0.1 A600 units/ml) in fresh YPD, and then incubated for 5 h at 30°C. The

24

cell suspensions were treated with the indicated concentrations of CFW for 30 min at

25

30°C.

26

anti-HA, or anti-Pgk1. The details are given under “EXPERIMENTAL PROCEDURES”.

B, Western blotting analysis of phospho-Slt2-6xHA of CFW-treated cells.

Yeast cell extracts were immunoblotted using anti-phospho-p44/42 MAPK,

27 This article is protected by copyright. All rights reserved.

Accepted Article 1

C, the relative amounts of phospho-Slt2-6xHA were determined with ImageJ software.

2

The amount of phospho-Slt2-6xHA/Pgk1 in wild-type cells was taken as 1. Data

3

represent means ± SD for at least three independent experiments. D, cells were cultured

4

overnight in YPD medium, and then spotted onto YPD plates containing the indicated

5

concentrations of CFW in 10-fold serial dilutions starting with a density of 0.7 A600

6

units/ml.

All plates were incubated at 30°C and photographed after 2 days.

28 This article is protected by copyright. All rights reserved.

Accepted Article

Table 1. Strains used in this study. Strain

Genotype

Source

BY4741

MATa his3∆1 leu2∆0 met15∆0 ura3∆0

Brachmann et al. (1998)

Y7092

MATα can1∆::STE2pr-his5 lyp1∆ his3∆1 leu2∆0 met15∆0 ura3∆0

Tong and Boone (2006)

YMY12

Y7092, sur1∆::URA3 csh1∆::LEU2

This study

YMY51

BY4741, och1∆::kanMX4

This study

YMY53

BY4741, mnn9∆::kanMX4

This study

MTY1451

BY4741, mnn2∆::kanMX4

This study

YMY49

BY4741, mnn5∆::kanMX4

This study

YNY52

BY4741, ktr6∆::kanMX4

This study

MTY1445

BY4741, mnn1∆::kanMX4

This study

YMY21

Y7092, sur1∆::URA3

This study

YMY22

Y7092, csh1∆::URA3

This study

YMY23

Y7092, csg2∆::URA3

This study

YMY24

Y7092, ipt1∆::URA3

This study

YMY78

BY4741, sur1∆::kanMX4 csh1∆::LEU2

This study

YMY71

BY4741, tetO2-MNN2::kanMX4

This study

YMY73

BY4741, tetO2-MNN2::kanMX4 sur1∆::natMX4 csh1∆::LEU2

This study

YMY83

BY4741, MNN2-6xHA:: hphNT1

This study

YMY84

BY4741, tetO2-MNN2-6xHA::kanMX4, hphNT1

This study

YMY85

BY4741, tetO2-MNN2-6xHA::kanMX4, hphNT1 sur1∆::natMX4 csh1∆::LEU2

This study

YMY74

BY4741, slt2∆::hphMX4

This study

YMY75

BY4741, sur1∆::URA3 csh1∆::LEU2 slt2∆::hphMX4

This study

YMY76

BY4741, tetO2-MNN2::kanMX4 slt2∆::hphMX4

This study

YMY77

BY4741, tetO2-MNN2::kanMX4 sur1∆::natMX4 csh1∆::LEU2 slt2∆::hphMX4

This study

YMY99

BY4741, SLT2-6xHA::hphNT1

This study

YMY100

BY4741, sur1∆::URA3 csh1∆::LEU2 SLT2-6xHA::hphNT1

This study

YMY101

BY4741, tetO2-MNN2::kanMX4 SLT2-6xHA::hphNT1

This study

YMY102

BY4741, tetO2-MNN2::kanMX4 sur1∆::natMX4 csh1∆::LEU2 SLT2-6xHA::hphNT1

This study

MTY1457

BY4741, rlm1∆::kanMX4

This study

MTY1458

BY4741, sur1∆::URA3 csh1∆::LEU2 rlm1∆::kanMX4

This study

MTY1459

BY4741, tetO2-MNN2::hphMX4 sur1∆::natMX4 csh1∆::LEU2 rlm1∆::kanMX4

This study

29 This article is protected by copyright. All rights reserved.

Accepted Article

Table 2. Genes exhibiting synthetic lethality with sur1∆ csh1∆.

Gene

ORF

Function

MNN11

YJL183W

Subunit of mannosyltransferase

KTR6

YPL053C

Mannosylphosphate transferase

VPS74

YDR372C

Regulation of localization of Golgi glycosyltransferases

PDR12

YPL058C

Plasma membrane ATP-binding cassette transporter

VPS1

YKR001C

Dynamin-like GTPase

VPS9

YML097C

Guanine nucleotide exchange factor

PDB1

YBR221C

E1 beta subunit of pyruvate dehydrogenase complex

LAT1

YNL071W

Dihydrolipoamide acetyltransferase of pyruvate dehydrogenase complex

LPD1

YFL018C

Dihydrolipoamide dehydrogenase of pyruvate dehydrogenase complex

MPC1

YGL080W

Mitochondrial pyruvate carrier

TOS1

YBR162C

Cell wall protein

SAC1

YKL212W

Phosphoinositide phosphatase

GCV3

YAL044C

H subunit of mitochondrial glycine decarboxylase complex

MNL2

YLR057W

ER-associated protein degradation

SYS1

YJL004C

Regulation of Arl-like GTPase Arl3

YLR111W

Unknown

Synthetic lethal interactions between sur1∆ csh1∆ and deletions of the listed genes were all confirmed by random spore analysis.

30 This article is protected by copyright. All rights reserved.

Accepted Article

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