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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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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.
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pone.0145915.g001: 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.

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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145915.g001: 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.
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