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

Fokina AV, Chechenova MB, Karginov AV, Ter-Avanesyan MD, Agaphonov MO - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

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

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.

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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 Figpmr1-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.
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pone.0145915.g002: 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 Figpmr1-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.

Mentions: 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).


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

Fokina AV, Chechenova MB, Karginov AV, Ter-Avanesyan MD, Agaphonov MO - PLoS ONE (2015)

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 Figpmr1-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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696657&req=5

pone.0145915.g002: 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 Figpmr1-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.
Mentions: 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).

Bottom Line: 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.

View Article: PubMed Central - PubMed

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

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.

Show MeSH
Related in: MedlinePlus