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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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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.
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pone.0145915.g005: 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.

Mentions: 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.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145915.g005: 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.
Mentions: 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.

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