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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 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.
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pone.0145915.g007: 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.

Mentions: 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).


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145915.g007: 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.
Mentions: 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).

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