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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 uPA from culture supernatants.uPA, the strains 64MA70UAL (ret1-27), 64MA70UA-RET (WT), 64MA70U-RET-Δvps10 (vps10-Δ), and 64MA70U-RET-Δvps35 (vps35-Δ), expressing the wild-type uPA; uPA-Q302, the strains 64MA70QAL (ret1-27), 64MA70QA-RET (WT), and 64MA70Q-RET-Δvps10 (vps10-Δ), expressing the unglycosylated uPA-Q302 mutant protein. Samples with the wild-type uPA were treated with EndoH prior to electrophoresis.
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pone.0145915.g009: Immunoblot analysis of uPA from culture supernatants.uPA, the strains 64MA70UAL (ret1-27), 64MA70UA-RET (WT), 64MA70U-RET-Δvps10 (vps10-Δ), and 64MA70U-RET-Δvps35 (vps35-Δ), expressing the wild-type uPA; uPA-Q302, the strains 64MA70QAL (ret1-27), 64MA70QA-RET (WT), and 64MA70Q-RET-Δvps10 (vps10-Δ), expressing the unglycosylated uPA-Q302 mutant protein. Samples with the wild-type uPA were treated with EndoH prior to electrophoresis.

Mentions: Analysis of CPY glycosylation and proteolytic processing revealed that the ret1-27 mutation affects processes taking place downstream of the Vps10 Golgi compartment. Additional evidence for this was obtained by analyzing the proteolysis of human uPA during secretion. This protein is synthesized as a zymogen (molecular weight of polypeptide chain 46 kDa), which is activated by proteolytic cleavage of the K158-I159 peptide bond. After this cleavage uPA migrates during SDS PAGE as ~30 kDa protein. Previously we have observed that this cleavage occurs during uPA secretion by yeast cells and that defects of vacuolar protein sorting enhance the efficiency of this cleavage [30]. The ret1-27 mutation also stimulated uPA proteolysis. This effect was even more evident for the unglycosylated mutant uPA-Q302. Though, in contrast to the vps mutants, which secreted only the 30 kDa fragment of this protein, a large portion of uPA and uPA-Q302 in the culture supernatant of the ret1-27 mutant remained uncleaved and, in addition to the 30 kDa form, two slightly larger forms were also revealed (Fig 9).


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 uPA from culture supernatants.uPA, the strains 64MA70UAL (ret1-27), 64MA70UA-RET (WT), 64MA70U-RET-Δvps10 (vps10-Δ), and 64MA70U-RET-Δvps35 (vps35-Δ), expressing the wild-type uPA; uPA-Q302, the strains 64MA70QAL (ret1-27), 64MA70QA-RET (WT), and 64MA70Q-RET-Δvps10 (vps10-Δ), expressing the unglycosylated uPA-Q302 mutant protein. Samples with the wild-type uPA were treated with EndoH prior to electrophoresis.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145915.g009: Immunoblot analysis of uPA from culture supernatants.uPA, the strains 64MA70UAL (ret1-27), 64MA70UA-RET (WT), 64MA70U-RET-Δvps10 (vps10-Δ), and 64MA70U-RET-Δvps35 (vps35-Δ), expressing the wild-type uPA; uPA-Q302, the strains 64MA70QAL (ret1-27), 64MA70QA-RET (WT), and 64MA70Q-RET-Δvps10 (vps10-Δ), expressing the unglycosylated uPA-Q302 mutant protein. Samples with the wild-type uPA were treated with EndoH prior to electrophoresis.
Mentions: Analysis of CPY glycosylation and proteolytic processing revealed that the ret1-27 mutation affects processes taking place downstream of the Vps10 Golgi compartment. Additional evidence for this was obtained by analyzing the proteolysis of human uPA during secretion. This protein is synthesized as a zymogen (molecular weight of polypeptide chain 46 kDa), which is activated by proteolytic cleavage of the K158-I159 peptide bond. After this cleavage uPA migrates during SDS PAGE as ~30 kDa protein. Previously we have observed that this cleavage occurs during uPA secretion by yeast cells and that defects of vacuolar protein sorting enhance the efficiency of this cleavage [30]. The ret1-27 mutation also stimulated uPA proteolysis. This effect was even more evident for the unglycosylated mutant uPA-Q302. Though, in contrast to the vps mutants, which secreted only the 30 kDa fragment of this protein, a large portion of uPA and uPA-Q302 in the culture supernatant of the ret1-27 mutant remained uncleaved and, in addition to the 30 kDa form, two slightly larger forms were also revealed (Fig 9).

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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