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Genetic Evidence for the Role of the Vacuole in Supplying Secretory Organelles with Ca2+ in Hansenula polymorpha

  • Anastasia V. Fokina,

    Affiliation A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia

  • Maria B. Chechenova,

    Current address: Biology Department, University of New Mexico, Albuquerque, New Mexico, United States of America

    Affiliation A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia

  • Azamat V. Karginov,

    Affiliation A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia

  • Michael D. Ter-Avanesyan,

    Affiliation A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia

  • Michael O. Agaphonov

    agaphonov@inbi.ras.ru

    Affiliation A.N. Bach Institute of Biochemistry, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, Russia

Genetic Evidence for the Role of the Vacuole in Supplying Secretory Organelles with Ca2+ in Hansenula polymorpha

  • Anastasia V. Fokina, 
  • Maria B. Chechenova, 
  • Azamat V. Karginov, 
  • Michael D. Ter-Avanesyan, 
  • Michael O. Agaphonov
PLOS
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Abstract

Processes taking place in the secretory organelles require Ca2+ and Mn2+, which in yeast are supplied by the Pmr1 ion pump. Here we observed that in the yeast Hansenula polymorpha Ca2+ deficiency in the secretory pathway caused by Pmr1 inactivation is exacerbated by (i) the ret1-27 mutation affecting COPI-mediated vesicular transport, (ii) inactivation of the vacuolar Ca2+ ATPase Pmc1 and (iii) inactivation of Vps35, which is a component of the retromer complex responsible for protein transport between the vacuole and secretory organelles. The ret1-27 mutation also exerted phenotypes indicating alterations in transport between the vacuole and secretory organelles. These data indicate that ret1-27, pmc1 and vps35 affect a previously unknown Pmr1-independent route of the Ca2+ delivery to the secretory pathway. We also observed that the vacuolar protein carboxypeptidase Y receives additional modifications of its glycoside chains if it escapes the Vps10-dependent sorting to the vacuole.

Introduction

A number of processes in the secretory pathway of eukaryotes require the presence of Ca2+ and Mn2+. The Golgi apparatus is supplied with these ions by means of the secretory pathway calcium ATPase. In yeast this protein is encoded by the PMR1 gene [1]. The endoplasmic reticulum (ER) of animal cells is a Ca2+ storage organelle, which participates in signal transduction by releasing Ca2+ to the cytosol. Its Ca2+ pool is replenished by a special ion pump, the sarco/endoplasmic reticulum Ca2+ ATPase. In yeast, the ER is probably not involved in the Ca2+ signaling and no ER Ca2+ pump has been identified. However the processes taking place in the ER lumen, which are related to protein secretion, still require Ca2+. In yeast, the Golgi apparatus Ca2+ ATPase is a major contributor to the ER supply of Ca2+, since inactivation of the Saccharomyces cerevisiae PMR1 gene leads to a 50% decrease in the ER Ca2+ level [2]. However the source of the ER Ca2+ in the absence of Pmr1 remains unknown.

The main yeast Ca2+ storage/sink organelle is the vacuole, which possesses its own Ca2+ATPase Pmc1 and the H+/Ca2+ antiporter Vcx1. These proteins serve to replenish the vacuolar Ca2+ pool and to maintain cytosolic Ca2+ concentration at low levels [3, 4, 5]. In S. cerevisiae inactivation of the PMC1 gene leads to sensitivity to high concentrations of Ca2+ in culture medium and is lethal in absence of the PMR1 gene. Both these mutant phenotypes are suppressed by inactivation of the Ca2+/calmodulin-dependent protein phosphatase calcineurin, indicating that increased Ca2+ concentration blocks cell growth due to calcineurin activation, while Pmr1 acts together with Pmc1 in maintaining the cytosolic Ca2+ concentration at a low level [3].

Many vacuolar proteins are synthesized in the ER together with secretory proteins. Then they are sorted at the late Golgi to be delivered to the vacuole by vesicular transport via different routes [6]. For example, carboxypeptidase Y (CPY) is sorted to the vacuole by the Vps10 receptor, which cycles between the late Golgi and the endosomal or prevacuolar compartments [7, 8]. Proteins endocytosed from the cell surface are also transported to the vacuole [9, 10, 11, 12, 13, 14], while some secretory proteins pass through the endosomal compartments prior to exocytosis [15]. These pathways require intensive anterograde and retrograde traffic of proteins and lipids between the vacuole, endosomes and secretory compartments. Thus it is possible that this traffic connects the vacuolar Ca2+ pool to the secretory organelles.

Retrograde transport of proteins and lipids through the secretory organelles is mediated by COPI coated vesicles. The COPI coat consists of α, β, ε, β', γ, δ, and ζ subunits. The former three subunits comprise subcomplex B; the latter four comprise subcomplex F [16, 17]. Previously we have shown that in the methylotrophic yeast Hansenula polymorpha C-terminal truncation of α-COP leads to sensitivity to Ca2+ shortage in the culture medium [18]. This indicates involvement of the COPI coated vesicles in Ca2+ trafficking in the cell. Besides, COPI subunits were shown to be involved in membrane traffic in the endocytic pathway in animal cells [19, 20, 21]. In yeast, mutations in the components of the COPI subcomplex B were shown to affect protein sorting from late endosomes to the vacuole [22].

In this work we have obtained data indicating involvement of the vacuole in supply of the secretory pathway with Ca2+, which can be mediated by COPI-dependent vesicular transport.

Materials and Methods

Culture conditions and genetic methods

H. polymorpha cells were cultivated at 37°C in complex YPD (1% yeast extract, 2% peptone, 2% glucose) and YPM (1% yeast extract, 2% peptone and 1% methanol) media or in synthetic SD medium (0.67% Yeast Nitrogene Base (Difco), 2% glucose) and Ca2+-deficient medium [1] SD* (Ca2+-deficient Yeast Nitrogen Base, 2% glucose). Solid media contained 2,5% agar. The Ca2+-deficient Yeast Nitrogen Base was made from 5 separately prepared solutions: A (ammonium sulfate 500 g/L), B (biotin 0.2 mg/L, folic acid 0.2 mg/L, inositol 2 g/L, niacin 0.4 g/L, pyridoxine hydrochloride 0.4 g/L, riboflavin 0.2 g/L, thiamine hydrochloride 0.4 g/L, calcium pantothenate 0.4 g/L, p-aminobenzoic acid 0.2 g/L), C (KH2PO4 20 g/L, MgSO4 10 g/L, NaCl 2 g/L), D (boric acid 5 g/L, CuSO4·5H2O 0.4 g/L, KI 0.5 g/L, FeCl3 2 g/L, MnCl2·4H2O 4 g/L, ZnSO4 4 g/L), D' (Na2MoO4·2H2O 2 g/L). To obtain the Ca2+-deficient Yeast Nitrogen Base 10X solution, components A, B, C, D, D' and deionized H2O were mixed in proportion 10:10:50:0.01:0.01:30. Additional depletion of Ca2+ was achieved by supplementing the SD* with ethylene glycol tetraacetic acid (EGTA). Where required, leucine, adenine, or uracil were added. H. polymorpha strains were crossed, and hybrids were sporulated on maltose-containing medium (2% maltose, 3% agar). For induction of expression of the unglycosylated mutant of urokinase-type plasminogen activator (uPA-Q302), overnight cultures grown in liquid YPD containing 0.1 M NaCl were diluted six-fold with induction medium containing 1% yeast extract, 3% peptone 25 mM NH4H2PO4, 25 mM (NH4)2HPO4, 0,1 M NaCl, 0.05% glycerol, 0.8% MeOH and incubated at 37°C for 70 h. For the analysis of CPY secretion cells were grown in liquid YPD for 40 h. H. polymorpha was transformed according to the modified lithium acetate method [23].

Strains

Strains used in this study are listed in Table 1. All the strains originated from the H. polymorpha CBS4732 (Ogataea polymorpha). The 64MA70 strain was obtained as a haploid segregant from the cross between the strains 1B (leu2 ade2) [23] and 2dMA56 (leu2 ade2 ret1-27 mox::uPA) [18]. To obtain 64MA70U and 64MA70Q, the MOX gene in the 64MA70 strain was replaced with either uPA or uPA-Q302 expression cassettes, respectively, as described [24]. The derivatives of these strains, which were disrupted for VPS10, VPS35, or PMC1, were obtained by transformation with disruption cassettes excised from the pU15, pCAF11, or pKAF2 plasmids, respectively. The derivatives with the wild type RET1 allele were obtained by integration of the p2CHA6-27OPU plasmid into genome. If the derivative strains possessed the leu2 or ade2 mutations, plasmids pCHLX or pCHAD3 bearing LEU2 or ADE2, respectively, were introduced into their genomes to obtain prototrophic strains.

The leu2 auxotrophic marker in the pmr1-Δ strain 1MA77/12 (leu2 ade2 mox::uPA pmr1::LEU2 [PMR1 ADE2]) was restored by replacing the pmr1::LEU2 allele with the pmr1::G418r allele from the pAF14 plasmid as described [25]. The resulting 1MA77/12/GP1 strain was disrupted for the VPS35 gene using the pCAF11-derived disruption cassette to obtain the 1MA77/12/GP1-Δvps35 strain. The 1MA77/12/GAP2 strain was constructed by disruption of the URA3 gene with the ADE2 selectable marker in the 1MA77/12/GP1 strain and by subsequent introduction of the autonomously replicating plasmid pAF18 bearing the URA3 selectable marker and the PMR1 gene. The 1MA77/12/GAP2-Δpmc1 strain was obtained by disruption of the PMC1 gene in 1MA77/12/GAP2 using the pAM655 plasmid. The MC39 strain (leu2 ade2 mox::u-PAQ302 ret1-27 pmr1::LEU2 [PMR1, ADE2]) is a segregant from the cross between the ret1-27 mutant 2dMA56 and the pmr1-Δ mutant 1MA77/12. Its derivatives MC39-MOX and MC39-RET-MOX were obtained by introduction of the pMOX-H36 or pRET1-MOX plasmid, respectively.

Plasmids

Plasmids used are listed in Table 2. The p2CHA6-27OPU plasmid was constructed by insertion of the BamHI-EheI fragment of p27OPU8 [18], which carries the RET1 gene with its native promoter, into the p2CHA6 vector [25]. Although this plasmid is capable of autonomous replication in H. polymorpha cells, it was always digested with BamHI and EcoRI prior to transformation to achieve genomic integration of its fragment bearing the ADE2 and RET1 genes. pAF18 was constructed by insertion of the SacI-EcoRI fragment of pE1, which carries the PMR1 gene with its native promoter, between the SacI and EcoRI restriction sites of the pAM459 vector. The latter vector was obtained from the pRS426 S. cerevisiae shuttle vector [26] by in vivo capturing of DNA fragment improving its autonomous maintenance in H. polymorpha cells as follows. The H. polymorpha ura3 mutant was transformed with pRS426 digested with NdeI and SnaBI. The digestion was aimed to induce random capturing of a DNA fragment, which would improve the plasmid replication and the selectable marker expression like it was described previously [23]. Finally, the plasmid, which was designated as pAM459, was isolated from one of the obtained transformants. The pCHAD3 plasmid was obtained by cloning of the H. polymorpha ADE2 gene into the pBC-SK(+) vector. To construct pCAF11, the VPS35 gene was amplified by PCR and its Eco47III-SalI fragment was cloned in the pBC-KS(+) vector. Then the BamHI-EcoRI fragment within the VPS35 ORF was replaced with the LEU2-carrying BamHI-EcoRI fragment of the pCHLX plasmid. Prior to yeast transformation, pCAF11 was digested with BglII and SalI.

The pMOX-H36 plasmid contained the SphI-NgoMI fragment of H. polymorpha genomic DNA bearing the MOX gene and the HindIII-SphI fragment of AMIpSL1, possessing HARS36 [31]. These two fragments were inserted between the HindIII and XmaI sites of the pTZ19R vector. pRET1-MOX was obtained by replacement of the HARS36-bearing fragment of the pMOX-H36 plasmid with the EheI-SphI fragment of p27OPU8, bearing the RET1 gene with its native promoter.

Electrophoresis and immunoblotting

Proteins from culture supernatants were concentrated 3-fold in case of CPY analysis and 20-fold in the case of uPA and uPA-Q302 analysis by precipitation with trichloroacetic acid. The amounts of uPA, uPA-Q302 and CPY from culture medium and in cell lysates were normalized to the levels of total cellular protein as described in [32] and subsequently resolved by electrophoresis in 10% polyacrylamide gel as described in [33]. Rabbit antisera against E. coli expressed H. polymorpha CPY [30] and Pmr1 [18], tubulin beta antibody (PA5-16863, ThermoFisher Scientific) and monoclonal mouse antibody specific to the uPA protease domain (#MGH U11, IMTEK, Moscow, Russia) were used to detect corresponding proteins. The immunoblots were developed using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific, Illinois, U.S.A.).

Analysis of carboxypeptidase Y proteolytic fragments

Two major forms of CPY were revealed in cell lysates and culture supernatants of the H. polymorpha vps35-Δ and vps10-Δ mutants. After treatment with endoglycosidase Hf (EndoH, New England BioLabs, Ipswich, Massachusetts, U.S.A.), one of them migrated close to the 46 kDa marker band, the other—between the 46 kDa and 30 kDa marker bands. CPY from culture supernatants was partially purified by ion exchange chromatography, treated with EndoH and separated by SDS PAGE. Gel fragments containing CPY were prepared, incubated with sequencing grade trypsin (Promega, Madison, Wisconsin, U.S.A.), and the peptides were analyzed using an Ultraflextreme MALDI-TOF/TOF mass spectrometer (Bruker Daltonics, Billerica, Massachusetts, U.S.A.) equipped with an Nd laser (354 nm) as described elsewhere [34]. According to this analysis both CPY forms possessed an intact C-terminus and were shortened at the N-terminus. The larger form lacked only the pro-region (up to K122), which was predicted based on homology to the S. cerevisiae ortholog [35]. Calculated molecular weight of the H. polymorpha CPY polypeptide chain starting from K123 is 47 kDa. In the shorter form, the most N-terminal peptide, which was revealed by mass spectrometric analysis, started from S227.

Analysis of vacuolar morphology

Cells grown in liquid YPD were collected at exponential phase, washed with H2O, and then with 10 mM HEPES buffer pH 7.4, containing 5% D-glucose, resuspended at a density of 106 cells/ml in the same HEPES buffer, containing glucose and 200 μM CellTracker Blue CMAC (Thermo Scientific). Cells were incubated for 30–45 min at room temperature and visualized by fluorescence microscopy using an Axioskop 40 (Zeiss, Oberkochen, Germany) with a cooled CCD camera (Olympus Corporation, Tokyo, Japan). Images were assembled in Photoshop (Adobe) with only linear adjustments.

Results

A defect of COPI-mediated vesicular transport exacerbates the requirement of H. polymorpha pmr1-Δ mutant for Ca2+ and Mn2+

The mutant ret1-27 allele encodes α-COP lacking more than 300 C-terminal amino acids. Its manifestations, e.g. inability to grow on Ca2+-depleted medium and enhanced ability to secrete human uPA, resemble those of the pmr1-Δ mutation, which inactivates the Golgi apparatus Ca2+ ATPase [18, 25]. It was previously suggested that these phenotypes of ret1-27 are due to the decreased function of Pmr1 [18]. Here, to ascertain whether the effect of ret1-27 on the Ca2+ homeostasis is indeed mediated by Pmr1, we studied interaction between the pmr1-Δ and ret1-27 mutations. To do this we used the strain MC39 carrying these mutations and the autonomously-replicating plasmid with the PMR1 gene (see Materials and Methods and Table 1). Cells of this strain were able to lose the PMR1-carrying plasmid only after introduction of the second plasmid bearing the wild type RET1 gene, which indicated that the ret1-27 and pmr1-Δ mutations were synthetically lethal. Remarkably, cells of the MC39 strain grown on medium supplemented with 10 mM CaCl2 could lose the PMR1-containing plasmid even in the absence of the RET1 plasmid, indicating that synthetic lethality was caused by insufficient supply of the secretory organelles with Ca2+. Transferring cells of the pmr1-Δ ret1-27 double mutant to regular YPD medium led to rapid cell death accompanied by DNA fragmentation (S1 Fig), resembling what was previously observed in a strain carrying the pmr1-Δ mutation alone upon incubation in medium with phosphate buffer and methanol as a sole carbon source [25]. Thus the ret1-27 mutation exacerbated this effect, since in the pmr1-Δ ret1-27 double mutant, cell death and DNA fragmentation occurred in regular YPD medium.

In S. cerevisiae Mn2+ was shown to have a dual effect: its cytosolic accumulation is toxic, while it can functionally replace Ca2+ in some life essential process(es) and supports cell growth upon Ca2+ shortage [36]. Cytosolic accumulation of Mn2+ apparently is also toxic in H. polymorpha, since this yeast was sensitive to elevation of Mn2+ concentration in culture medium (Fig 1). Notably, the sensitivity to Mn2+ greatly depended on the medium used. The highest sensitivity was observed in SD*, which contains Ca2+ in low concentration and is not favorable for growth of the pmr1-Δ mutant (S2 Fig). In contrast to the wild-type control strain, the pmr1-Δ mutant was almost unable to grow on SD* supplemented with 3 mM MnCl2, while 0.5 mM and 1 mM MnCl2 noticeably improved its growth (Fig 1). This agrees with the role of Pmr1 in sequestration of Mn2+ into the secretory organelles [37]. Remarkably, the ret1-27 mutation alone also conferred hypersensitivity to Mn2+ (Fig 1). At the same time it exacerbated the requirement in external Mn2+ caused by the pmr1-Δ mutation, since growth of the ret1-27 pmr1-Δ double mutant on YPD (but not on SD) could be rescued by elevation of Mn2+ concentration and this strain could grow without the PMR1-containing plasmid not only in excess of Ca2+, but also in the presence of 1 mM MnCl2 (Fig 2).

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Fig 1. Growth of the pmr1-Δ and ret1-27 mutants on SD* medium supplemented with different concentrations of MnCl2.

Cell suspensions with equal densities were spotted onto corresponding media and grown for 2 days. The experiment was performed using serial dilutions of cell suspensions (S3 Fig) and a representative dilution is shown in this figure. pmr1-Δ, a subclone of the 1MA27/12/GP1 strain lacking the PMR1 containing plasmid; the PMR1, 1MA27/12/GP1 strain, ret1-27, the 64MA70QAL strain; RET1, the 64MA70QA-RET strain.

http://dx.doi.org/10.1371/journal.pone.0145915.g001

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Fig 2. Rescue of growth of the pmr1-Δ ret1-27 double mutant by CaCl2 and MnCl2.

Since the ret1-27 pmr1-Δ double mutant was inviable on regular media, it was obtained from the MC39-MOX strain, which carried a PMR1-contaning plasmid. To allow the MC39-MOX and MC39-RET-MOX strains to lose the PMR1-contaning plasmid, they were streaked on YPD plate supplemented with 10 mM CaCl2. Equal amounts of cells from single colonies obtained on this medium were suspended in sterile water and spotted onto test plates. Growth of only one subclone for each case is shown in the figure. Growth of three additional subclones is shown in S4 Fig pmr1-Δ ret1-27, a MC39-MOX subclone lacking the plasmid; PMR1 ret1-27, a MC39-MOX subclone retaining the plasmid; pmr1-Δ RET1, a MC39-RET-MOX subclone lacking the plasmid; PMR1 RET1, a MC39-RET-MOX subclone retaining the plasmid.

http://dx.doi.org/10.1371/journal.pone.0145915.g002

Thus, the ret1-27 mutation essentially increased the requirement of the pmr1-Δ mutant for Ca2+ and Mn2+. The obtained data demonstrate that the ret1-27 mutation is able to affect Ca2+ homeostasis independently of Pmr1.

Inactivation of PMC1 or VPS35 exacerbates deficiency of Ca2+ in the secretory pathway of the pmr1-Δ mutant

In S. cerevisiae inactivation of the vacuolar Ca2+ ATPase Pmc1 disturbs the control of Ca2+ concentration in the cytosol, thus leading to inability of mutant cells to grow at high levels of Ca2+ in the culture medium. The Pmr1 pump of S. cerevisiae is also involved in the maintenance of low level of cytosolic Ca2+. Inactivation of both ion pumps is lethal due to increased cytosolic Ca2+ concentration [3]. Inactivation of Pmc1 in H. polymorpha also leads to sensitivity to high Ca2+ concentrations in culture medium [29]. Although manifestations of the pmc1-Δ and pmr1-Δ mutations in H. polymorpha are similar to those in S. cerevisiae, the role of Pmr1 in the cytosolic Ca2+ control and the role of Pmc1 in the secretory pathway Ca2+ supply in H. polymorpha remained uncertain. To resolve this uncertainty, we studied interaction of the pmc1-Δ and pmr1-Δ mutations with each other. We inactivated the PMC1 gene in the strain disrupted for PMR1, which carried an autonomous PMR1-containing plasmid. Surprisingly, the pmc1-Δ pmr1-Δ double mutant was able to lose the PMR1-containing plasmid during growth on YPD medium, though the clones lacking the plasmid were unable to grow on SD medium (Fig 3). The effect of elevated concentration of external Mn2+ on growth of the pmc1-Δ pmr1-Δ double mutant resembled that observed in the ret1-27 pmr1-Δ strain, since 1–3 mM MnCl2 allowed the pmc1-Δ pmr1-Δ double mutant to grow on SD medium (Fig 3). Notably, as in the case of the pmr1-Δ ret1-27 strain, supplementing the culture medium with 5mM CaCl2 also rescued growth of the pmc1-Δ pmr1-Δ double mutant (Fig 3). The observations that the pmc1-Δ pmr1-Δ double mutant is viable and that its growth defect can be rescued by elevation of Ca2+ concentration in culture medium indicate that, in contrast to S. cerevisiae, in H. polymorpha Pmr1 is not essentially involved in the control of concentration of cytosolic Ca2+. The exacerbation of dependence of the pmr1-Δ mutant on external Ca2+ and Mn2+ by the PMC1 inactivation suggests a role of the vacuole in supply of the secretory organelles with Ca2+.

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Fig 3. Rescue of growth of the pmr1-Δ pmc1-Δ double mutant by CaCl2 and MnCl2.

Cell suspensions with equal densities were spotted onto corresponding media and grown for 2 days. The experiment was performed using serial dilutions of cell suspensions (S5 Fig) and a representative dilution is shown in this figure. PMR1 pmc1-Δ and pmr1-Δ pmc1-Δ, the 1MA77/12/GAP2-Δpmc strain with or without the PMR1-containing plasmid, respectively; PMR1 PMC1 and pmr1-Δ PMC1, the 1MA77/12/GAP2 strain with or without the PMR1-containing plasmid, respectively.

http://dx.doi.org/10.1371/journal.pone.0145915.g003

The role of the vacuole in supplying the secretory organelles with Ca2+ also followed from effects of the inactivation of Vps35, which is a component of the retromer complex responsible for the retrograde trafficking from the prevacuolar compartments to the Golgi apparatus. Similarly to ret1-27 ([18] and Fig 2), the vps35-Δ mutation led to hypersensitivity to Ca2+ shortage in culture medium and exacerbated Ca2+ dependence of the pmr1-Δ mutant (Fig 4). Indeed, the growth of the vps35-Δ mutant was abolished by supplementing of SD* with 20 mM EGTA, while the strain with the wild-type VPS35 allele still could grow. The vps35-Δ effect on Ca2+ dependence of the pmr1-Δ mutant was less pronounced then the effect of ret1-27, since the vps35-Δ pmr1-Δ double mutant was able to grow on regular SD and YPD media. The exacerbation of the pmr1-Δ dependence on Ca2+ by the vps35-Δ mutation indicated involvement of pre-vacuolar compartments in a Pmr1-independent supply of the secretory organelles with Ca2+. At the same time the vps35-Δ mutation exerted hypersensitivity to Mn2+, which masked the ability of Mn2+ to suppress the growth defect caused by the pmr1-Δ mutation. We speculate that Vps35 is involved in degradation of the plasma membrane Mn2+ transporter and thus the loss of Vps35 may increase Mn2+ uptake.

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Fig 4. Effect of the vps35-Δ mutation on growth of strains with or without the PMR1 gene.

Cell suspensions with equal densities were spotted onto corresponding media and grown for 2 days. The experiment was performed serial dilutions of cell suspensions (S6 Fig) and a representative dilution is shown in this figure. pmr1-Δ vps35-Δ, the 1MA27/12/GP1-Δvps35 strain lacking the plasmid with PMR1; vps35-Δ, the 1MA27/12/GP1-Δvps35 strain, pmr1-Δ VPS35, the 1MA27/12/GP1 strain lacking the plasmid with PMR1; PMR1 VPS35, the 1MA27/12/GP1 strain.

http://dx.doi.org/10.1371/journal.pone.0145915.g004

Defect of COPI-dependent supply of the secretory organelles with Ca2+ is not mediated by decreasing function of the Golgi Ca2+/Mn2+ ATPase Pmr1

Previously we have shown that the ret1-27 mutation noticeably decreases the amount of the Pmr1 protein [18]. This implies that some manifestations of the ret1-27 mutation may result from insufficient Pmr1-dependent supply of the secretory pathway with Ca2+ ions. Since, as it was shown above, pmc1-Δ exacerbates dependence of the pmr1-Δ mutant on external Ca2+ and Mn2+ and leads to inability to grow on synthetic medium, we expected the pmc1-Δ mutation to inhibit growth on synthetic medium and exacerbate dependence on external Ca2+ in the ret1-27 mutant. However, this was proved to be incorrect. Specifically, PMC1 could easily be disrupted in the ret1-27 mutant, even though the disruptants were selected on synthetic medium. Also, the sensitivity of the pmc1 ret1-27 double mutant to a shortage of Ca2+ did not differ from that of the strain bearing the ret1-27 mutation alone, while sensitivity of this double mutant to an increased concentration of external Ca2+ was approximately the same as of the pmc1 mutant (Fig 5). This indicates that the Ca2+ dependence of the ret1-27 mutant is not related to insufficient function of Pmr1, since otherwise pmc1-Δ would exacerbate Ca2+ dependence of the ret1-27 mutant. One could expect that the lack of Pmc1 should increase cytosolic Ca2+ concentration, which in turn can enhance expression levels of genes coding for proteins involved in the control of cytosolic Ca2+ concentration including Pmr1. If the PMR1 expression was increased in response to the loss of the Pmc1 Ca2+ pump, it might compensate the negative effect of ret1-27 mutation on Pmr1 level and mask exacerbation of Ca2+ dependence. However, this suggestion was ruled out, since no increase in the Pmr1 level in response to PMC1 inactivation in the ret1-27 mutant was observed (Fig 6). Moreover, the Pmr1 level was even decreased in this case, which still did not noticeably affect the ret1-27 Ca2+ dependence.

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Fig 5. Sensitivity of the ret1-27 pmc1-Δ double mutant to a shortage or excess of Ca2+ in culture medium.

Cell suspensions with equal densities were spotted onto corresponding media and grown for 2 days. Ca2+ shortage was achieved by addition of EGTA to SD* medium. Excess of Ca2+ was achieved by supplementing YPD with CaCl2. The experiment was repeated with serially diluted cell suspensions (S7 Fig). ret1-27 pmc1-Δ, the 64MA70QA-Δpmc strain; pmc1-Δ, the 64MA70Q-RET-Δpmc strain; ret1-27, the 64MA70QAL strain; WT, the 64MA70QL-RET strain.

http://dx.doi.org/10.1371/journal.pone.0145915.g005

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Fig 6. Effect of inactivation of PMC1 on the level of Pmr1 in the ret1-27 mutant strain and in the strain bearing the RET1 wild-type allele.

Proteins from cell lysates were resolved by SDS PAGE and transferred to nitrocellulose membrane, which was then divided in two parts at the level of the 80 kDa marker band. The upper part was stained with antiserum against H. polymorpha Pmr1, while the lower part was stained with antibody against tubulin used as a loading control. ret1-27 pmc1-Δ, the 64MA70QA-Δpmc strain; pmc1-Δ, the 64MA70Q-RET-Δpmc strain; ret1-27, the 64MA70QAL strain; WT and WT 1/2, undiluted and two-fold diluted sample of the 64MA70QL-RET strain, respectively.

http://dx.doi.org/10.1371/journal.pone.0145915.g006

The ret1-27 mutation causes phenotypes indicating defects in the Golgi-to-vacuole transport

In S. cerevisiae, some mutations in the COPI subcomplex B affect transport between the Golgi apparatus and the vacuole [22]. We suggested that H. polymorpha ret1-27 also affects this pathway and leads to secretion of some vacuolar proteins, e. g. CPY. However, we did not observe any noticeable increase in the amount of CPY in the culture supernatant of the ret1-27 mutant, while testing the vps35-Δ and vps10-Δ mutants, which have been previously shown to be defective in CPY sorting [30] showed that corresponding mutations led to accumulation of extracellular CPY and reduced its intracellular amount (Fig 7).

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Fig 7. Immunoblot analysis of CPY from culture supernatants (A) and cell lysates (B).

ret1-27 vps35-Δ, the 64MA70UA-Δvps35 strain; ret1-27 vps10-Δ, the 64MA70UA-Δvps10 strain; vps35-Δ, the 64MA70U-RET-Δvps35 strain; vps10-Δ, the 64MA70U-RET-Δvps10 strain; ret1-27, the 64MA70UAL strain; WT, the 64MA70UA-RET strain. +EndoH, samples treated with endoglycosidase H. X3, an overexposed image (~3-fold longer time) of the "vps35-Δ" lane. 1/8, an underexposed (~8-fold shorter time) image of the “ret1-27” and “WT” lanes.

http://dx.doi.org/10.1371/journal.pone.0145915.g007

The H. polymorpha CPY sequence [35] contains four consensus N-glycosylation sites, one of which is located within the pro-region. In the vps mutants the most abundant form of extracellular CPY (Fig 7A) migrated during SDS PAGE as a dispersed band or as a smear above the 46 kDa marker band due to glycosylation, since treatment with EndoH converted it into a form migrating as a sharp ~46 kDa band. This band corresponded to CPY, which lacks only the pro-region (see Materials and Methods). We designated this form as m1CPY. Importantly, in contrast to H. polymorpha, CPY of S. cerevisiae migrates as a compact band since it possesses mature core-type uniform N-glycosides [38]. The electrophoretic pattern of H. polymorpha CPY indicated that at least one of its N-glycoside chains underwent outer chain elongation by attachment of an irregular number of mannose residues.

Glycosylation patterns of extracellular CPY depended on the vps35-Δ, vps10-Δ, and ret1-27 mutations. Indeed, if the strains carried the wild type RET1 allele, the vps10-Δ mutant secreted more extensively glycosylated CPY than the vps35-Δ mutant, while the vps35-Δ mutant secreted more extensively glycosylated enzyme than the vps10-Δ mutant if they carried the ret1-27 allele (Fig 7A). The only CPY form, which was revealed in cell lysates of the strains bearing the VPS10 and VPS35 wild-type alleles, was the CPY fragment resulting from the additional cleavage of m1CPY (see Materials and Methods). After EndoH treatment it migrated between the 46 kDa and 30 kDa marker bands (Fig 7B). We designated this form as m2CPY. Notably, intracellular m2CPY was less glycosylated in the ret1-27 mutant than in the wild type strain. At the same time cell lysates of the vps10-Δ and vps35-Δ mutants contained approximately the same amounts of m1CPY and m2CPY. As one could expect, the total amount of intracellular CPY in these two mutants was drastically reduced. The glycosylation of these CPY forms in the vps35-Δ and vps10-Δ mutants followed a pattern resembling that of the extracellular protein. Surprisingly, similar effects on the glycosylation pattern were observed for the cell surface protein Gas1. Particularly, it was less glycosylated in the ret1-27 and vps35-Δ single mutants, while its glycosylation pattern in the strain bearing both these mutations was indistinguishable from that in the wild-type control strain. At the same time the vps10-Δ mutation did not noticeably affect the glycosylation pattern of Gas1 (Fig 8).

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Fig 8. Immunoblot analysis of Gas1 from cell lysates.

ret1-27 vps35-Δ, the 64MA70UA-Δvps35 strain; ret1-27 vps10-Δ, the 64MA70UA-Δvps10 strain; vps35-Δ, the 64MA70U-RET-Δvps35 strain; vps10-Δ, the 64MA70U-RET-Δvps10 strain; ret1-27, the 64MA70UAL strain; WT, the 64MA70UA-RET strain. +EndoH, samples treated with endoglycosidase H.

http://dx.doi.org/10.1371/journal.pone.0145915.g008

Analysis of CPY glycosylation and proteolytic processing revealed that the ret1-27 mutation affects processes taking place downstream of the Vps10 Golgi compartment. Additional evidence for this was obtained by analyzing the proteolysis of human uPA during secretion. This protein is synthesized as a zymogen (molecular weight of polypeptide chain 46 kDa), which is activated by proteolytic cleavage of the K158-I159 peptide bond. After this cleavage uPA migrates during SDS PAGE as ~30 kDa protein. Previously we have observed that this cleavage occurs during uPA secretion by yeast cells and that defects of vacuolar protein sorting enhance the efficiency of this cleavage [30]. The ret1-27 mutation also stimulated uPA proteolysis. This effect was even more evident for the unglycosylated mutant uPA-Q302. Though, in contrast to the vps mutants, which secreted only the 30 kDa fragment of this protein, a large portion of uPA and uPA-Q302 in the culture supernatant of the ret1-27 mutant remained uncleaved and, in addition to the 30 kDa form, two slightly larger forms were also revealed (Fig 9).

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Fig 9. Immunoblot analysis of uPA from culture supernatants.

uPA, the strains 64MA70UAL (ret1-27), 64MA70UA-RET (WT), 64MA70U-RET-Δvps10 (vps10-Δ), and 64MA70U-RET-Δvps35 (vps35-Δ), expressing the wild-type uPA; uPA-Q302, the strains 64MA70QAL (ret1-27), 64MA70QA-RET (WT), and 64MA70Q-RET-Δvps10 (vps10-Δ), expressing the unglycosylated uPA-Q302 mutant protein. Samples with the wild-type uPA were treated with EndoH prior to electrophoresis.

http://dx.doi.org/10.1371/journal.pone.0145915.g009

Finally we observed the effect of the ret1-27 mutation on the vacuole morphology in the vps35-Δ background. The S. cerevisiae vps35 mutation belongs to the A class of vps mutations, which do not affect morphology of the vacuole [39]. In the H. polymorpha vps35-Δ mutant, vacuole morphology was also unaltered (Fig 10). Based on the observation of increased proteolysis of uPA-Q302 we expected the ret1-27 mutation to affect traffic between the secretory organelles and the vacuole. Despite this, we did not reveal any effect of this mutation alone on the vacuole morphology. However cells of the vps35-Δ ret1-27 double mutant, in addition to the vacuole of regular morphology, possessed several smaller compartments, which were stained by a vacuole-specific dye (Fig 10).

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Fig 10. Cell Tracker Blue staining of vacuoles.

ret1-27 vps35-Δ, the 64MA70UA-Δvps35 strain; ret1-27 vps10-Δ, the 64MA70UA-Δvps10 strain; vps35-Δ, the 64MA70U-RET-Δvps35 strain; vps10-Δ, the 64MA70U-RET-Δvps10 strain; ret1-27, the 64MA70UAL strain; WT, the 64MA70UA-RET strain. The white bar corresponds to 5 μm. Arrows indicate the additional compartments stained like the vacuole.

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Thus, these data indicate that similarly to the COPI sub-complex B mutations in S. cerevisiae [22], the H. polymorpha ret1-27 mutation also affects traffic between the secretory organelles and the vacuole.

Discussion

Both Ca2+ and Mn2+ ions are required for a number of processes taking place in the secretory pathway. Although the sequestration of both these ions into the secretory organelles is performed by Pmr1, which is located in the medial Golgi, the roles, which these ions play, do not overlap in most cases. For example, while Ca2+ is required for folding of proteins in the ER and for their binding to the vacuolar sorting receptor Vps10 [37], the functioning of mannosyltransferases in the secretory pathway depends on Mn2+ [4043]. At the same time it was shown that Mn2+ can replace Ca2+ in supporting cell growth [36], though the essential cellular process, which is affected by the shortage of both Ca2+ and Mn2+ remains yet unidentified. Increased concentration of cytosolic Mn2+ was shown to be toxic in S. cerevisiae [11, 44, 45]. The same is true for H. polymorpha since its pmr1-Δ mutant is hypersensitive to an increase of Mn2+concentration in culture medium. Despite this, supplementing the culture medium with subtoxic concentrations of MnCl2 even improved growth of the pmr1-Δ mutant, as was observed for CaCl2 supplementation. This indicates that the process, in which Ca2+ and Mn2+ are interchangeable, takes place in the secretory pathway. Indeed, inactivation of Pmr1, which supplies the secretory organelles with Ca2+ and Mn2+, noticeably affects cell viability, while elevation of external concentration of either of these ions can equally alleviate the loss of Pmr1.

The H. polymorpha ret1-27 mutation causes a truncation of the C-terminal domain of α-COP, which is an essential component of the COPI coat complex involved in protein and lipid traffic between the secretory organelles. Earlier it was shown that ret1-27 causes sensitivity to Ca2+ shortage in culture medium and improves secretion of a heterologous protein. Since these phenotypes resemble the manifestations of the pmr1-Δ mutation, it was suggested that they arise due to insufficient function of Pmr1 in the ret1-27 mutant [18]. However, here we observed that the pmr1-Δ and ret1-27 mutations are synthetically lethal. Notably, the viability of this double mutant can be rescued by increasing Ca2+ or Mn2+ concentration in culture medium, which indicates that synthetic lethality was due to a shortage of these ions in the secretory organelles. Based on these data one can conclude that the ret1-27 mutation affects Pmr1-independent supply of the secretory organelles with Ca2+ and Mn2+. If the effect of ret1-27 on Ca2+ homeostasis was mediated exclusively through Pmr1, this mutation would not be able to exacerbate dependence of the pmr1-Δ mutant on Ca2+ and Mn2+.

In S. cerevisiae, simultaneous inactivation of the vacuolar and the Golgi Ca2+ ATPases is lethal due to increase in the level of cytosolic Ca2+ [3]. However, the H. polymorpha pmr1-Δ pmc1-Δ double mutant is able to grow on YPD medium and its growth on synthetic medium can be restored by increasing concentrations of external Ca2+ or Mn2+. Thus, the loss of the vacuolar Ca2+ ion pump exacerbates manifestations of the loss of the secretory pathway Ca2+/Mn2+ ATPase, but not vice versa. This distinguishes H. polymorpha from S. cerevisiae, in which the pmc1 pmr1 synthetic lethality is due to exacerbation of the pmc1 manifestation. The effect of pmc1-Δ on manifestations of the pmr1-Δ mutation observed in H. polymorpha indicates a role of the vacuole in supplying the secretory organelles with Ca2+ ions and agrees with the idea that the sensitivity of the ret1-27 mutant to Ca2+ shortage is due to disruption of the Pmr1-independent Ca2+ supply of the secretory organelles. Absence of the effect of PMC1 inactivation on manifestations of the ret1-27 mutation supports this conclusion since ret1-27 should block this Ca2+ transport pathway downstream of Pmc1, i.e. en route from the vacuole to the secretory organelles. The putative involvement of COPI dependent transport in supply of the secretory pathway with Ca2+ from the vacuole is supported by the implication of the COPI subcomplex B in protein transport between the Golgi apparatus and the vacuole in S. cerevisiae. Mutations in COPI subunits α, β', and ε cause defects in vacuolar protein sorting and alterations of the vacuolar morphology [22]. In the case of α-COP, only mutations in its N-terminal domain cause this effect. However the H. polymorpha ret1-27 mutation, which was studied here, causes a C-terminal truncation of α-COP. Nevertheless, we have observed effects of ret1-27 on (i) uPA-Q302 processing, which resemble the effect of mutations disturbing vacuolar protein sorting, on (ii) glycosylation of the vacuolar enzyme CPY, as well as (iii) on vacuolar morphology in the vps35-Δ background.

CPY is sorted out of the proteins, which are transported to the plasma membrane, by the Vps10 vacuolar sorting receptor. According to studies performed in S. cerevisiae, this occurs at the most trans-Golgi cisternae [8]. However in H. polymorpha we observed that CPY receives additional glycosylation when it is secreted in the absence of the Vps10 receptor, indicating that it becomes more exposed to glycosylation enzymes. This could be due to its passage through additional compartments where glycosylation occurs, or due to longer retention in the Golgi apparatus. Interestingly, inactivation of Vps35, which is responsible for retrieval of Vps10 from endosomes, leads to secretion of less glycosylated CPY than inactivation of Vps10 per se, while the ret1-27 mutation inverses effects of these mutations on CPY glycosylation. It is worth to note that the ret1-27 mutation decreases intracellular CPY glycosylation in the absence of vacuolar protein sorting defects as well. At the same time the effects of ret1-27 and vps35-Δ mutations on the glycosylation pattern of the vacuolar protein CPY and of the cell surface protein Gas1 were very similar. Individually, each of these mutations decreased Gas1 glycosylation, while their interaction returned it to the wild-type pattern. This indicates that ret1-27 and vps35-Δ affect protein glycosylation via different, though interacting, mechanisms. Importantly, the interaction of these mutations was also highlighted by their synthetic effect on the vacuolar morphology.

Involvement of the COPI components including α-COP in the membrane traffic from endosomes [22] can be the reason for the effect of ret1-27 on Ca2+ homeostasis. Endosomes may receive Ca2+ from the environment or from the vacuole and then it can be transported to the secretory organelles with assistance of the COPI subcomplex B. The classical "cisternae maturation" model of the Golgi apparatus suggests recycling of the Golgi enzymes by COPI vesicles transporting them gradually from the later compartment to the earlier one [46]. In this case Ca2+ transported to the latest Golgi compartment would be gradually diluted en route to the earlier compartments. However, this problem is abolished if Ca2+ is transported directly to the earliest secretory compartments. This does not contradict the later revision of the "cisternae maturation" model [47].

Mn2+ does not necessarily follow the same route to the ER lumen as Ca2+, since it may be absorbed from the cytosol by Spf1, which is believed to be the ER Mn2+ ATPase [48]. Indeed, simultaneous inactivation of Pmr1 and Spf1 has much more pronounced defects of CPY* degradation and N-linked oligosaccharide trimming in the ER than individual inactivation of each of these proteins [49]. Inactivation of the plasma membrane high affinity Mn2+ transporter Smf1 is lethal in cells lacking Pmr1 [50]. This lethality can be overcome by increasing Ca2+ concentration in culture medium [51]. Similarly to the interactions of the H. polymorpha pmr1-Δ mutation with ret1-27, pmc1-Δ and vps35-Δ studied here, the interactions of pmr1 with spf1 and smf1 in S. cerevisiae can be explained in terms of a requirement for Ca2+ and Mn2+ in a life-essential process in the secretory organelles, in which these ions are interchangeable.

Supporting Information

S1 Fig. Electrophoresis of chromosomal DNA of the MC39 strain lacking the PMR1-containing plasmid (pmr1-Δ ret1-27).

The MC39 cells lacking the PMR1-containing plasmid were grown in YPD supplemented with 10 mM CaCl2, spun down, resuspended in regular YPD medium and incubated for the indicated time. Chromosomal DNA samples of the MC39 bearing the PMR1-containing plasmid (ret1-27) and 1MA77/12 (pmr1-Δ) strains grown in regular YPD were used as a control.

doi:10.1371/journal.pone.0145915.s001

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S2 Fig. Effect of media composition on growth of the pmr1-Δ mutant (A) and on sensitivity of H. polymorpha to MnCl2 (B).

Cell suspensions with equal densities were serially diluted (10-fold) and spotted onto corresponding media. pmr1-Δ, a subclone of the 1MA27/12/GP1 strain lacking the PMR1 containing plasmid; PMR1, 1MA27/12/GP1 strain. Only two representative dilutions are shown in the panel B.

doi:10.1371/journal.pone.0145915.s002

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S3 Fig. Growth of the pmr1-Δ and ret1-27 mutants on SD* medium supplemented with different concentrations of MnCl2.

Cell suspensions with equal densities were serially diluted (10-fold) and spotted onto corresponding media. Two subclones of each strain were analyzed. pmr1-Δ, subclone of the 1MA27/12/GP1 strain lacking the PMR1 containing plasmid; PMR1, 1MA27/12/GP1 strain; ret1-27, 64MA70QAL strain; RET1, 64MA70QA-RET strain.

doi:10.1371/journal.pone.0145915.s003

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S4 Fig. Rescue of growth of the pmr1-Δ ret1-27 double mutant by CaCl2 and MnCl2.

Since the ret1-27 pmr1-Δ double mutant was inviable on regular media, it was obtained from the MC39-MOX strain, which carried a PMR1-contaning plasmid. To allow the MC39-MOX and MC39-RET-MOX strains to lose the PMR1-contaning plasmid, they were streaked onto a YPD plate supplemented with 10 mM CaCl2. Equal amounts of cells from single colonies obtained on this medium were suspended in sterile water and spotted onto test plates. Growth of three subclones in each case is shown in this figure. Growth of the fourth subclone is shown in Fig 2. pmr1-Δ ret1-27, a MC39-MOX subclone lacking the plasmid; PMR1 ret1-27, a MC39-MOX subclone retaining the plasmid; pmr1-Δ RET1, a MC39-RET-MOX subclone lacking the plasmid; PMR1 RET1, a MC39-RET-MOX subclone retaining the plasmid.

doi:10.1371/journal.pone.0145915.s004

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S5 Fig. Rescue of growth of the pmr1-Δ pmc1-Δ double mutant by CaCl2 and MnCl2.

Cell suspensions with equal densities were serially diluted (10-fold) and spotted onto corresponding media. Two subclones of each strain were analyzed. PMR1 pmc1-Δ and pmr1-Δ pmc1-Δ, 1MA77/12/GAP2-Δpmc strain with or without the PMR1-containing plasmid, respectively; PMR1 PMC1 and pmr1-Δ PMC1, 1MA77/12/GAP2 strain with or without the PMR1-containing plasmid, respectively.

doi:10.1371/journal.pone.0145915.s005

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S6 Fig. Effect of the vps35-Δ mutation on growth of strains with or without the PMR1 gene.

Cell suspensions with equal densities were serially diluted (10-fold) and spotted onto corresponding media. Two subclones of each strain were analyzed. pmr1-Δ vps35-Δ, 1MA27/12/GP1-Δvps35 strain lacking the plasmid with PMR1; vps35-Δ, 1MA27/12/GP1-Δvps35 strain, pmr1-Δ VPS35, 1MA27/12/GP1 strain lacking the plasmid with PMR1; PMR1 VPS35, 1MA27/12/GP1 strain.

doi:10.1371/journal.pone.0145915.s006

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S7 Fig. Sensitivity of the ret1-27 pmc1-Δ double mutant to a shortage (achieved by addition of EGTA) or excess of Ca2+ in culture medium.

Cell suspensions with equal densities were serially diluted (10-fold) and spotted onto corresponding media. The replication of this experiment with additional concentrations of Ca2+ and EGTA is shown in Fig 5. ret1-27 pmc1-Δ, 64MA70QA-Δpmc strain; pmc1-Δ, 64MA70Q-RET-Δpmc strain; ret1-27, 64MA70QAL strain; RET1 PMC1, 64MA70QL-RET strain; #1 and #2, independently obtained clones.

doi:10.1371/journal.pone.0145915.s007

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Acknowledgments

We are grateful to Alexander Alexandrov for critical reading of the manuscript.

Author Contributions

Conceived and designed the experiments: MOA. Performed the experiments: AVF MBC AVK MOA MDT. Analyzed the data: MOA AVF MDT. Wrote the paper: MOA MDT AVF.

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