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

doi:10.1371/journal.pone.0145915

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.

Citation: Fokina AV, Chechenova MB, Karginov AV, Ter-Avanesyan MD, Agaphonov MO (2015) Genetic Evidence for the Role of the Vacuole in Supplying Secretory Organelles with Ca²⁺ in Hansenula polymorpha. PLoS ONE 10(12): e0145915. https://doi.org/10.1371/journal.pone.0145915

Editor: Juan Mata, University of Cambridge, UNITED KINGDOM

Received: August 5, 2015; Accepted: December 10, 2015; Published: December 30, 2015

Copyright: © 2015 Fokina et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: AVF, AVK, MDT, and MOA were supported by Russian Science Foundation grant 14-14-00361.

Competing interests: The authors have declared that no competing interests exist.

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.

Download:

Table 1. H. polymorpha strains.

https://doi.org/10.1371/journal.pone.0145915.t001

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::G418^r 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.

Download:

Table 2. Plasmids.

https://doi.org/10.1371/journal.pone.0145915.t002

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 H_f (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).

Download:

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.

https://doi.org/10.1371/journal.pone.0145915.g001

Download:

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.

https://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⁺.

Download:

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.

https://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.

Download:

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.

https://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.

Download:

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.

https://doi.org/10.1371/journal.pone.0145915.g005

Download:

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.

https://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).

Download:

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.

https://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).

Download:

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.

https://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).

Download:

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.

https://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).

Download:

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.

https://doi.org/10.1371/journal.pone.0145915.g010

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⁺ [40–43]. 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.

https://doi.org/10.1371/journal.pone.0145915.s001

(PDF)

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.

https://doi.org/10.1371/journal.pone.0145915.s002

(PDF)

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.

https://doi.org/10.1371/journal.pone.0145915.s003

(PDF)

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.

https://doi.org/10.1371/journal.pone.0145915.s004

(PDF)

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.

https://doi.org/10.1371/journal.pone.0145915.s005

(PDF)

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.

https://doi.org/10.1371/journal.pone.0145915.s006

(PDF)

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.

https://doi.org/10.1371/journal.pone.0145915.s007

(PDF)

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.

References

1. Rudolph HK, Antebi A, Fink GR, Buckley CM, Dorman TE, LeVitre J, et al. The yeast secretory pathway is perturbed by mutations in PMR1, a member of a Ca²⁺ ATPase family. Cell. 1989;58: 133–145. pmid:2526682
- View Article
- PubMed/NCBI
- Google Scholar
2. Strayle J, Pozzan T, Rudolph HK. Steady-state free Ca²⁺ in the yeast endoplasmic reticulum reaches only 10 μM and is mainly controlled by the secretory pathway pump Pmr1. EMBO J. 1999;18: 4733–4743. pmid:10469652
- View Article
- PubMed/NCBI
- Google Scholar
3. Cunningham KW, Fink GR. Calcineurin-dependent growth control in Saccharomyces cerevisiae mutants lacking PMC1, a homolog of plasma membrane Ca²⁺ ATPases. J Cell Biol. 1994;124: 351–363. pmid:7507493
- View Article
- PubMed/NCBI
- Google Scholar
4. Pozos TC, Sekler I, Cyert MS. The product of HUM1, a novel yeast gene, is required for vacuolar Ca²⁺/H⁺ exchange and is related to mammalian Na⁺/Ca²⁺ exchangers. Mol Cell Biol. 1996;16: 3730–3741. pmid:8668190
- View Article
- PubMed/NCBI
- Google Scholar
5. Cunningham KW. Acidic calcium stores of Saccharomyces cerevisiae. Cell Calcium. 2011;50: 129–138. pmid:21377728
- View Article
- PubMed/NCBI
- Google Scholar
6. Bryant NJ, Stevens TH. Vacuole biogenesis in Saccharomyces cerevisiae: protein transport pathways to the yeast vacuole. Microbiol Mol Biol Rev. 1998;62: 230–247. pmid:9529893
- View Article
- PubMed/NCBI
- Google Scholar
7. Marcusson EG, Horazdovsky BF, Cereghino JL, Gharakhanian E, Emr SD. The sorting receptor for yeast vacuolar carboxypeptidase Y is encoded by the VPS10 gene. Cell. 1994;77: 579–586. pmid:8187177
- View Article
- PubMed/NCBI
- Google Scholar
8. Cooper AA, Stevens TH. Vps10p cycles between the late-Golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases. J Cell Biol. 1996;133: 529–541. pmid:8636229
- View Article
- PubMed/NCBI
- Google Scholar
9. Galan JM, Moreau V, Andre B, Volland C, Haguenauer-Tsapis R. Ubiquitination mediated by the Npi1p/Rsp5p ubiquitin-protein ligase is required for endocytosis of the yeast uracil permease. J Biol Chem. 1996;271: 10946–10952. pmid:8631913
- View Article
- PubMed/NCBI
- Google Scholar
10. Gitan RS, Luo H, Rodgers J, Broderius M, Eide D. Zinc-induced inactivation of the yeast ZRT1 zinc transporter occurs through endocytosis and vacuolar degradation. J Biol Chem. 1998;273: 28617–28624. pmid:9786854
- View Article
- PubMed/NCBI
- Google Scholar
11. Jensen LT, Carroll MC, Hall MD, Harvey CJ, Beese SE, Culotta VC. Down-regulation of a manganese transporter in the face of metal toxicity. Mol Biol Cell. 2009;20: 2810–2819. pmid:19369420
- View Article
- PubMed/NCBI
- Google Scholar
12. Liu J, Sitaram A, Burd CG. Regulation of copper-dependent endocytosis and vacuolar degradation of the yeast copper transporter, Ctr1p, by the Rsp5 ubiquitin ligase. Traffic. 2007;8: 1375–1384. pmid:17645432
- View Article
- PubMed/NCBI
- Google Scholar
13. Roy A, Kim J-H. Endocytosis and vacuolar degradation of the yeast cell surface glucose sensors Rgt2 and Snf3. J Biol Chem. 2014;289: 7247–7256. pmid:24451370
- View Article
- PubMed/NCBI
- Google Scholar
14. Springael JY, André B. Nitrogen-regulated ubiquitination of the Gap1 permease of Saccharomyces cerevisiae. Mol Biol Cell. 1998;9: 1253–1263. pmid:9614172
- View Article
- PubMed/NCBI
- Google Scholar
15. Harsay E, Schekman R. A subset of yeast vacuolar protein sorting mutants is blocked in one branch of the exocytic pathway. J Cell Biol. 2002;156: 271–285. pmid:11807092
- View Article
- PubMed/NCBI
- Google Scholar
16. Eugster A, Frigerio G, Dale M, Duden R. COP I domains required for coatomer integrity, and novel interactions with ARF and ARF-GAP. EMBO J. 2000;19: 3905–3917. pmid:10921873
- View Article
- PubMed/NCBI
- Google Scholar
17. McMahon HT, Mills IG. COP and clathrin-coated vesicle budding: different pathways, common approaches. Curr Opin Cell Biol. 2004;16: 379–391. pmid:15261670
- View Article
- PubMed/NCBI
- Google Scholar
18. Chechenova MB, Romanova NV, Deev AV, Packeiser AN, Smirnov VN, Agaphonov MO, et al. C-terminal truncation of α-COP affects functioning of secretory organelles and calcium homeostasis in Hansenula polymorpha. Eukaryot Cell. 2004;3: 52–60. pmid:14871936
- View Article
- PubMed/NCBI
- Google Scholar
19. Daro E, Sheff D, Gomez M, Kreis T, Mellman I. Inhibition of endosome function in CHO cells bearing a temperature-sensitive defect in the coatomer (COPI) component epsilon-COP. J Cell Biol. 1997;139: 1747–1759. pmid:9412469
- View Article
- PubMed/NCBI
- Google Scholar
20. Gu F, Aniento F, Parton RG, Gruenberg J. Functional dissection of COP-I subunits in the biogenesis of multivesicular endosomes. J Cell Biol. 1997;139: 1183–1195. pmid:9382865
- View Article
- PubMed/NCBI
- Google Scholar
21. Whitney JA, Gomez M, Sheff D, Kreis TE, Mellman I. Cytoplasmic coat proteins involved in endosome function. Cell. 1995;83: 703–713. pmid:8521487
- View Article
- PubMed/NCBI
- Google Scholar
22. Gabriely G, Kama R, Gerst JE. Involvement of specific COPI subunits in protein sorting from the late endosome to the vacuole in yeast. Mol Cell Biol. 2007;27: 526–540. pmid:17101773
- View Article
- PubMed/NCBI
- Google Scholar
23. Bogdanova AI, Agaphonov MO, Ter-Avanesyan MD. Plasmid reorganization during integrative transformation in Hansenula polymorpha. Yeast. 1995;11: 343–353. pmid:7785335
- View Article
- PubMed/NCBI
- Google Scholar
24. Agaphonov MO, Beburov MYu, Ter-Avanesyan MD, Smirnov VN. A disruption-replacement approach for the targeted integration of foreign genes in Hansenula polymorpha. Yeast. 1995;11: 1241–1247. pmid:8553695
- View Article
- PubMed/NCBI
- Google Scholar
25. Agaphonov MO, Plotnikova TA, Fokina AV, Romanova NV, Packeiser AN, Kang HA, et al. Inactivation of the Hansenula polymorpha PMR1 gene affects cell viability and functioning of the secretory pathway. FEMS Yeast Res. 2007;7: 1145–1152. pmid:17498212
- View Article
- PubMed/NCBI
- Google Scholar
26. Sikorski RS, Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989;122: 19–27. pmid:2659436
- View Article
- PubMed/NCBI
- Google Scholar
27. Agaphonov M, Alexandrov A. Self-excising integrative yeast plasmid vectors containing an intronated recombinase gene. FEMS Yeast Res. 2014;14: 1048–1054. pmid:25124534
- View Article
- PubMed/NCBI
- Google Scholar
28. Sohn JH, Choi ES, Kim CH, Agaphonov MO, Ter-Avanesyan MD, Rhee JS, et al. A novel autonomously replicating sequence (ARS) for multiple integration in the yeast Hansenula polymorpha DL-1. J Bacteriol. 1996;178: 4420–4428. pmid:8755868
- View Article
- PubMed/NCBI
- Google Scholar
29. Fokina AV, Sokolov SS, Kang HA, Kalebina TS, Ter-Avanesyan MD, Agaphonov MO. Inactivation of Pmc1 vacuolar Ca²⁺ ATPase causes G₂ cell cycle delay in Hansenula polymorpha. Cell Cycle. 2012;11: 778–784. pmid:22374675
- View Article
- PubMed/NCBI
- Google Scholar
30. Agaphonov M, Romanova N, Sokolov S, Iline A, Kalebina T, Gellissen G, et al. Defect of vacuolar protein sorting stimulates proteolytic processing of human urokinase-type plasminogen activator in the yeast Hansenula polymorpha. FEMS Yeast Res. 2005;5: 1029–1035. pmid:16181812
- View Article
- PubMed/NCBI
- Google Scholar
31. Agaphonov MO, Trushkina PM, Sohn JH, Choi ES, Rhee SK, Ter-Avanesyan MD. Vectors for rapid selection of integrants with different plasmid copy numbers in the yeast Hansenula polymorpha DL1. Yeast. 1999;15: 541–551. pmid:10341417
- View Article
- PubMed/NCBI
- Google Scholar
32. Agaphonov MO, Romanova NV, Trushkina PM, Smirnov VN, Ter-Avanesyan MD. Aggregation and retention of human urokinase type plasminogen activator in the yeast endoplasmic reticulum. BMC Mol Biol. 2002;3: 15. pmid:12366865
- View Article
- PubMed/NCBI
- Google Scholar
33. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227: 680–685. pmid:5432063
- View Article
- PubMed/NCBI
- Google Scholar
34. Nizhnikov AA, Alexandrov AI, Ryzhova TA, Mitkevich OV, Dergalev AA, Ter-Avanesyan MD, et al. Proteomic screening for amyloid proteins. PloS One. 2014;9: e116003. pmid:25549323
- View Article
- PubMed/NCBI
- Google Scholar
35. Bellu AR, van der Klei IJ, Rechinger KB, Yavuz M, Veenhuis M, Kiel JA. Characterization of the Hansenula polymorpha CPY gene encoding carboxypeptidase Y. Yeast. 1999;15: 181–189. pmid:10077185
- View Article
- PubMed/NCBI
- Google Scholar
36. Loukin S, Kung C. Manganese effectively supports yeast cell-cycle progression in place of calcium. J Cell Biol. 1995;131: 1025–1037. pmid:7490280
- View Article
- PubMed/NCBI
- Google Scholar
37. Dürr G, Strayle J, Plemper R, Elbs S, Klee SK, Catty P, et al. The medial-Golgi ion pump Pmr1 supplies the yeast secretory pathway with Ca²⁺ and Mn²⁺ required for glycosylation, sorting, and endoplasmic reticulum-associated protein degradation. Mol Biol Cell. 1998;9: 1149–1162. pmid:9571246
- View Article
- PubMed/NCBI
- Google Scholar
38. Ballou L, Hernandez LM, Alvarado E, Ballou CE. Revision of the oligosaccharide structures of yeast carboxypeptidase Y. Proc Natl Acad Sci U S A. 1990;87: 3368–3372. pmid:2185468
- View Article
- PubMed/NCBI
- Google Scholar
39. Raymond CK, Howald-Stevenson I, Vater CA, Stevens TH. Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mol Biol Cell. 1992;3: 1389–1402. pmid:1493335
- View Article
- PubMed/NCBI
- Google Scholar
40. Haselbeck A, Schekman R. Interorganelle transfer and glycosylation of yeast invertase in vitro. Proc Natl Acad Sci U S A. 1986;83: 2017–2021. pmid:2421286
- View Article
- PubMed/NCBI
- Google Scholar
41. Nakajima T, Ballou CE. Yeast manno-protein biosynthesis: solubilization and selective assay of four mannosyltransferases. Proc Natl Acad Sci U S A. 1975;72: 3912–3916. pmid:1105568
- View Article
- PubMed/NCBI
- Google Scholar
42. Parodi AJ. Biosynthesis of yeast mannoproteins. Synthesis of mannan outer chain and of dolichol derivatives. J Biol Chem. 1979;254: 8343–8352. pmid:381304
- View Article
- PubMed/NCBI
- Google Scholar
43. Sharma CB, Babczinski P, Lehle L, Tanner W. The role of dolicholmonophosphate in glycoprotein biosynthesis in Saccharomyces cerevisiae. Eur J Biochem. 1974;46: 35–41. pmid:4605330
- View Article
- PubMed/NCBI
- Google Scholar
44. Ramsay LM, Gadd GM. Mutants of Saccharomyces cerevisiae defective in vacuolar function confirm a role for the vacuole in toxic metal ion detoxification. FEMS Microbiol Lett. 1997;152: 293–298. pmid:9231423
- View Article
- PubMed/NCBI
- Google Scholar
45. Yang M, Jensen LT, Gardner AJ, Culotta VC. Manganese toxicity and Saccharomyces cerevisiae Mam3p, a member of the ACDP (ancient conserved domain protein) family. Biochem J. 2005;386: 479–487. pmid:15498024
- View Article
- PubMed/NCBI
- Google Scholar
46. Glick BS, Elston T, Oster G. A cisternal maturation mechanism can explain the asymmetry of the Golgi stack. FEBS Lett. 1997;414: 177–181. pmid:9315681
- View Article
- PubMed/NCBI
- Google Scholar
47. Patterson GH, Hirschberg K, Polishchuk RS, Gerlich D, Phair RD, Lippincott-Schwartz J. Transport through the Golgi apparatus by rapid partitioning within a two-phase membrane system. Cell. 2008;133: 1055–1067. pmid:18555781
- View Article
- PubMed/NCBI
- Google Scholar
48. Cohen Y, Megyeri M, Chen OCW, Condomitti G, Riezman I, Loizides-Mangold U, et al. The yeast P5 type ATPase, Spf1, regulates manganese transport into the endoplasmic reticulum. Michnick SW, editor. PLoS ONE. 2013;8: e85519. pmid:24392018
- View Article
- PubMed/NCBI
- Google Scholar
49. Vashist S. Two distinctly localized P-Type ATPases collaborate to maintain organelle homeostasis required for glycoprotein processing and quality control. Mol Biol Cell. 2002;13: 3955–3966. pmid:12429838
- View Article
- PubMed/NCBI
- Google Scholar
50. Paidhungat M, Garrett S. Cdc1 is required for growth and Mn²⁺ regulation in Saccharomyces cerevisiae. Genetics. 1998;148: 1777–1786. pmid:9560392
- View Article
- PubMed/NCBI
- Google Scholar
51. García-Rodríguez N, Manzano-López J, Muñoz-Bravo M, Fernández-García E, Muñiz M, Wellinger RE. Manganese redistribution by calcium-stimulated vesicle trafficking bypasses the need for P-type ATPase function. J Biol Chem. 2015;290: 9335–9347. pmid:25713143
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Rudolph HK, Antebi A, Fink GR, Buckley CM, Dorman TE, LeVitre J, et al. The yeast secretory pathway is perturbed by mutations in PMR1, a member of a Ca²⁺ ATPase family. Cell. 1989;58: 133–145. pmid:2526682
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Strayle J, Pozzan T, Rudolph HK. Steady-state free Ca²⁺ in the yeast endoplasmic reticulum reaches only 10 μM and is mainly controlled by the secretory pathway pump Pmr1. EMBO J. 1999;18: 4733–4743. pmid:10469652
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Cunningham KW, Fink GR. Calcineurin-dependent growth control in Saccharomyces cerevisiae mutants lacking PMC1, a homolog of plasma membrane Ca²⁺ ATPases. J Cell Biol. 1994;124: 351–363. pmid:7507493
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Pozos TC, Sekler I, Cyert MS. The product of HUM1, a novel yeast gene, is required for vacuolar Ca²⁺/H⁺ exchange and is related to mammalian Na⁺/Ca²⁺ exchangers. Mol Cell Biol. 1996;16: 3730–3741. pmid:8668190
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Cunningham KW. Acidic calcium stores of Saccharomyces cerevisiae. Cell Calcium. 2011;50: 129–138. pmid:21377728
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Bryant NJ, Stevens TH. Vacuole biogenesis in Saccharomyces cerevisiae: protein transport pathways to the yeast vacuole. Microbiol Mol Biol Rev. 1998;62: 230–247. pmid:9529893
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Marcusson EG, Horazdovsky BF, Cereghino JL, Gharakhanian E, Emr SD. The sorting receptor for yeast vacuolar carboxypeptidase Y is encoded by the VPS10 gene. Cell. 1994;77: 579–586. pmid:8187177
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Cooper AA, Stevens TH. Vps10p cycles between the late-Golgi and prevacuolar compartments in its function as the sorting receptor for multiple yeast vacuolar hydrolases. J Cell Biol. 1996;133: 529–541. pmid:8636229
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Galan JM, Moreau V, Andre B, Volland C, Haguenauer-Tsapis R. Ubiquitination mediated by the Npi1p/Rsp5p ubiquitin-protein ligase is required for endocytosis of the yeast uracil permease. J Biol Chem. 1996;271: 10946–10952. pmid:8631913
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Gitan RS, Luo H, Rodgers J, Broderius M, Eide D. Zinc-induced inactivation of the yeast ZRT1 zinc transporter occurs through endocytosis and vacuolar degradation. J Biol Chem. 1998;273: 28617–28624. pmid:9786854
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Jensen LT, Carroll MC, Hall MD, Harvey CJ, Beese SE, Culotta VC. Down-regulation of a manganese transporter in the face of metal toxicity. Mol Biol Cell. 2009;20: 2810–2819. pmid:19369420
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Liu J, Sitaram A, Burd CG. Regulation of copper-dependent endocytosis and vacuolar degradation of the yeast copper transporter, Ctr1p, by the Rsp5 ubiquitin ligase. Traffic. 2007;8: 1375–1384. pmid:17645432
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref13] 13. Roy A, Kim J-H. Endocytosis and vacuolar degradation of the yeast cell surface glucose sensors Rgt2 and Snf3. J Biol Chem. 2014;289: 7247–7256. pmid:24451370
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref14] 14. Springael JY, André B. Nitrogen-regulated ubiquitination of the Gap1 permease of Saccharomyces cerevisiae. Mol Biol Cell. 1998;9: 1253–1263. pmid:9614172
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref15] 15. Harsay E, Schekman R. A subset of yeast vacuolar protein sorting mutants is blocked in one branch of the exocytic pathway. J Cell Biol. 2002;156: 271–285. pmid:11807092
View Article
PubMed/NCBI
Google Scholar

[58] View Article

[59] PubMed/NCBI

[60] Google Scholar

[ref16] 16. Eugster A, Frigerio G, Dale M, Duden R. COP I domains required for coatomer integrity, and novel interactions with ARF and ARF-GAP. EMBO J. 2000;19: 3905–3917. pmid:10921873
View Article
PubMed/NCBI
Google Scholar

[62] View Article

[63] PubMed/NCBI

[64] Google Scholar

[ref17] 17. McMahon HT, Mills IG. COP and clathrin-coated vesicle budding: different pathways, common approaches. Curr Opin Cell Biol. 2004;16: 379–391. pmid:15261670
View Article
PubMed/NCBI
Google Scholar

[66] View Article

[67] PubMed/NCBI

[68] Google Scholar

[ref18] 18. Chechenova MB, Romanova NV, Deev AV, Packeiser AN, Smirnov VN, Agaphonov MO, et al. C-terminal truncation of α-COP affects functioning of secretory organelles and calcium homeostasis in Hansenula polymorpha. Eukaryot Cell. 2004;3: 52–60. pmid:14871936
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref19] 19. Daro E, Sheff D, Gomez M, Kreis T, Mellman I. Inhibition of endosome function in CHO cells bearing a temperature-sensitive defect in the coatomer (COPI) component epsilon-COP. J Cell Biol. 1997;139: 1747–1759. pmid:9412469
View Article
PubMed/NCBI
Google Scholar

[74] View Article

[75] PubMed/NCBI

[76] Google Scholar

[ref20] 20. Gu F, Aniento F, Parton RG, Gruenberg J. Functional dissection of COP-I subunits in the biogenesis of multivesicular endosomes. J Cell Biol. 1997;139: 1183–1195. pmid:9382865
View Article
PubMed/NCBI
Google Scholar

[78] View Article

[79] PubMed/NCBI

[80] Google Scholar

[ref21] 21. Whitney JA, Gomez M, Sheff D, Kreis TE, Mellman I. Cytoplasmic coat proteins involved in endosome function. Cell. 1995;83: 703–713. pmid:8521487
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

[84] Google Scholar

[ref22] 22. Gabriely G, Kama R, Gerst JE. Involvement of specific COPI subunits in protein sorting from the late endosome to the vacuole in yeast. Mol Cell Biol. 2007;27: 526–540. pmid:17101773
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref23] 23. Bogdanova AI, Agaphonov MO, Ter-Avanesyan MD. Plasmid reorganization during integrative transformation in Hansenula polymorpha. Yeast. 1995;11: 343–353. pmid:7785335
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref24] 24. Agaphonov MO, Beburov MYu, Ter-Avanesyan MD, Smirnov VN. A disruption-replacement approach for the targeted integration of foreign genes in Hansenula polymorpha. Yeast. 1995;11: 1241–1247. pmid:8553695
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref25] 25. Agaphonov MO, Plotnikova TA, Fokina AV, Romanova NV, Packeiser AN, Kang HA, et al. Inactivation of the Hansenula polymorpha PMR1 gene affects cell viability and functioning of the secretory pathway. FEMS Yeast Res. 2007;7: 1145–1152. pmid:17498212
View Article
PubMed/NCBI
Google Scholar

[98] View Article

[99] PubMed/NCBI

[100] Google Scholar

[ref26] 26. Sikorski RS, Hieter P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989;122: 19–27. pmid:2659436
View Article
PubMed/NCBI
Google Scholar

[102] View Article

[103] PubMed/NCBI

[104] Google Scholar

[ref27] 27. Agaphonov M, Alexandrov A. Self-excising integrative yeast plasmid vectors containing an intronated recombinase gene. FEMS Yeast Res. 2014;14: 1048–1054. pmid:25124534
View Article
PubMed/NCBI
Google Scholar

[106] View Article

[107] PubMed/NCBI

[108] Google Scholar

[ref28] 28. Sohn JH, Choi ES, Kim CH, Agaphonov MO, Ter-Avanesyan MD, Rhee JS, et al. A novel autonomously replicating sequence (ARS) for multiple integration in the yeast Hansenula polymorpha DL-1. J Bacteriol. 1996;178: 4420–4428. pmid:8755868
View Article
PubMed/NCBI
Google Scholar

[110] View Article

[111] PubMed/NCBI

[112] Google Scholar

[ref29] 29. Fokina AV, Sokolov SS, Kang HA, Kalebina TS, Ter-Avanesyan MD, Agaphonov MO. Inactivation of Pmc1 vacuolar Ca²⁺ ATPase causes G₂ cell cycle delay in Hansenula polymorpha. Cell Cycle. 2012;11: 778–784. pmid:22374675
View Article
PubMed/NCBI
Google Scholar

[114] View Article

[115] PubMed/NCBI

[116] Google Scholar

[ref30] 30. Agaphonov M, Romanova N, Sokolov S, Iline A, Kalebina T, Gellissen G, et al. Defect of vacuolar protein sorting stimulates proteolytic processing of human urokinase-type plasminogen activator in the yeast Hansenula polymorpha. FEMS Yeast Res. 2005;5: 1029–1035. pmid:16181812
View Article
PubMed/NCBI
Google Scholar

[118] View Article

[119] PubMed/NCBI

[120] Google Scholar

[ref31] 31. Agaphonov MO, Trushkina PM, Sohn JH, Choi ES, Rhee SK, Ter-Avanesyan MD. Vectors for rapid selection of integrants with different plasmid copy numbers in the yeast Hansenula polymorpha DL1. Yeast. 1999;15: 541–551. pmid:10341417
View Article
PubMed/NCBI
Google Scholar

[122] View Article

[123] PubMed/NCBI

[124] Google Scholar

[ref32] 32. Agaphonov MO, Romanova NV, Trushkina PM, Smirnov VN, Ter-Avanesyan MD. Aggregation and retention of human urokinase type plasminogen activator in the yeast endoplasmic reticulum. BMC Mol Biol. 2002;3: 15. pmid:12366865
View Article
PubMed/NCBI
Google Scholar

[126] View Article

[127] PubMed/NCBI

[128] Google Scholar

[ref33] 33. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227: 680–685. pmid:5432063
View Article
PubMed/NCBI
Google Scholar

[130] View Article

[131] PubMed/NCBI

[132] Google Scholar

[ref34] 34. Nizhnikov AA, Alexandrov AI, Ryzhova TA, Mitkevich OV, Dergalev AA, Ter-Avanesyan MD, et al. Proteomic screening for amyloid proteins. PloS One. 2014;9: e116003. pmid:25549323
View Article
PubMed/NCBI
Google Scholar

[134] View Article

[135] PubMed/NCBI

[136] Google Scholar

[ref35] 35. Bellu AR, van der Klei IJ, Rechinger KB, Yavuz M, Veenhuis M, Kiel JA. Characterization of the Hansenula polymorpha CPY gene encoding carboxypeptidase Y. Yeast. 1999;15: 181–189. pmid:10077185
View Article
PubMed/NCBI
Google Scholar

[138] View Article

[139] PubMed/NCBI

[140] Google Scholar

[ref36] 36. Loukin S, Kung C. Manganese effectively supports yeast cell-cycle progression in place of calcium. J Cell Biol. 1995;131: 1025–1037. pmid:7490280
View Article
PubMed/NCBI
Google Scholar

[142] View Article

[143] PubMed/NCBI

[144] Google Scholar

[ref37] 37. Dürr G, Strayle J, Plemper R, Elbs S, Klee SK, Catty P, et al. The medial-Golgi ion pump Pmr1 supplies the yeast secretory pathway with Ca²⁺ and Mn²⁺ required for glycosylation, sorting, and endoplasmic reticulum-associated protein degradation. Mol Biol Cell. 1998;9: 1149–1162. pmid:9571246
View Article
PubMed/NCBI
Google Scholar

[146] View Article

[147] PubMed/NCBI

[148] Google Scholar

[ref38] 38. Ballou L, Hernandez LM, Alvarado E, Ballou CE. Revision of the oligosaccharide structures of yeast carboxypeptidase Y. Proc Natl Acad Sci U S A. 1990;87: 3368–3372. pmid:2185468
View Article
PubMed/NCBI
Google Scholar

[150] View Article

[151] PubMed/NCBI

[152] Google Scholar

[ref39] 39. Raymond CK, Howald-Stevenson I, Vater CA, Stevens TH. Morphological classification of the yeast vacuolar protein sorting mutants: evidence for a prevacuolar compartment in class E vps mutants. Mol Biol Cell. 1992;3: 1389–1402. pmid:1493335
View Article
PubMed/NCBI
Google Scholar

[154] View Article

[155] PubMed/NCBI

[156] Google Scholar

[ref40] 40. Haselbeck A, Schekman R. Interorganelle transfer and glycosylation of yeast invertase in vitro. Proc Natl Acad Sci U S A. 1986;83: 2017–2021. pmid:2421286
View Article
PubMed/NCBI
Google Scholar

[158] View Article

[159] PubMed/NCBI

[160] Google Scholar

[ref41] 41. Nakajima T, Ballou CE. Yeast manno-protein biosynthesis: solubilization and selective assay of four mannosyltransferases. Proc Natl Acad Sci U S A. 1975;72: 3912–3916. pmid:1105568
View Article
PubMed/NCBI
Google Scholar

[162] View Article

[163] PubMed/NCBI

[164] Google Scholar

[ref42] 42. Parodi AJ. Biosynthesis of yeast mannoproteins. Synthesis of mannan outer chain and of dolichol derivatives. J Biol Chem. 1979;254: 8343–8352. pmid:381304
View Article
PubMed/NCBI
Google Scholar

[166] View Article

[167] PubMed/NCBI

[168] Google Scholar

[ref43] 43. Sharma CB, Babczinski P, Lehle L, Tanner W. The role of dolicholmonophosphate in glycoprotein biosynthesis in Saccharomyces cerevisiae. Eur J Biochem. 1974;46: 35–41. pmid:4605330
View Article
PubMed/NCBI
Google Scholar

[170] View Article

[171] PubMed/NCBI

[172] Google Scholar

[ref44] 44. Ramsay LM, Gadd GM. Mutants of Saccharomyces cerevisiae defective in vacuolar function confirm a role for the vacuole in toxic metal ion detoxification. FEMS Microbiol Lett. 1997;152: 293–298. pmid:9231423
View Article
PubMed/NCBI
Google Scholar

[174] View Article

[175] PubMed/NCBI

[176] Google Scholar

[ref45] 45. Yang M, Jensen LT, Gardner AJ, Culotta VC. Manganese toxicity and Saccharomyces cerevisiae Mam3p, a member of the ACDP (ancient conserved domain protein) family. Biochem J. 2005;386: 479–487. pmid:15498024
View Article
PubMed/NCBI
Google Scholar

[178] View Article

[179] PubMed/NCBI

[180] Google Scholar

[ref46] 46. Glick BS, Elston T, Oster G. A cisternal maturation mechanism can explain the asymmetry of the Golgi stack. FEBS Lett. 1997;414: 177–181. pmid:9315681
View Article
PubMed/NCBI
Google Scholar

[182] View Article

[183] PubMed/NCBI

[184] Google Scholar

[ref47] 47. Patterson GH, Hirschberg K, Polishchuk RS, Gerlich D, Phair RD, Lippincott-Schwartz J. Transport through the Golgi apparatus by rapid partitioning within a two-phase membrane system. Cell. 2008;133: 1055–1067. pmid:18555781
View Article
PubMed/NCBI
Google Scholar

[186] View Article

[187] PubMed/NCBI

[188] Google Scholar

[ref48] 48. Cohen Y, Megyeri M, Chen OCW, Condomitti G, Riezman I, Loizides-Mangold U, et al. The yeast P5 type ATPase, Spf1, regulates manganese transport into the endoplasmic reticulum. Michnick SW, editor. PLoS ONE. 2013;8: e85519. pmid:24392018
View Article
PubMed/NCBI
Google Scholar

[190] View Article

[191] PubMed/NCBI

[192] Google Scholar

[ref49] 49. Vashist S. Two distinctly localized P-Type ATPases collaborate to maintain organelle homeostasis required for glycoprotein processing and quality control. Mol Biol Cell. 2002;13: 3955–3966. pmid:12429838
View Article
PubMed/NCBI
Google Scholar

[194] View Article

[195] PubMed/NCBI

[196] Google Scholar

[ref50] 50. Paidhungat M, Garrett S. Cdc1 is required for growth and Mn²⁺ regulation in Saccharomyces cerevisiae. Genetics. 1998;148: 1777–1786. pmid:9560392
View Article
PubMed/NCBI
Google Scholar

[198] View Article

[199] PubMed/NCBI

[200] Google Scholar

[ref51] 51. García-Rodríguez N, Manzano-López J, Muñoz-Bravo M, Fernández-García E, Muñiz M, Wellinger RE. Manganese redistribution by calcium-stimulated vesicle trafficking bypasses the need for P-type ATPase function. J Biol Chem. 2015;290: 9335–9347. pmid:25713143
View Article
PubMed/NCBI
Google Scholar

[202] View Article

[203] PubMed/NCBI

[204] Google Scholar

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

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

Figures

Abstract

Introduction

Materials and Methods

Culture conditions and genetic methods

Strains

Plasmids

Electrophoresis and immunoblotting

Analysis of carboxypeptidase Y proteolytic fragments

Analysis of vacuolar morphology

Results

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

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

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

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

Discussion

Supporting Information

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

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

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

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

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

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

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.

Acknowledgments

Author Contributions

References

Figures

Abstract

Introduction

Materials and Methods

Culture conditions and genetic methods

Strains

Plasmids

Electrophoresis and immunoblotting

Analysis of carboxypeptidase Y proteolytic fragments

Analysis of vacuolar morphology

Results

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

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

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

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

Discussion

Supporting Information

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

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

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

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

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

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

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.

Acknowledgments

Author Contributions

References

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

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

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

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

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

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

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

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.