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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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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.
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pone.0145915.g008: 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.

Mentions: 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).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145915.g008: 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.
Mentions: 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).

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