Article

Aggregation and retention of human urokinase type plasminogen activator in the yeast endoplasmic reticulum

Institute of Experimental Cardiology, Cardiology Research Center, 3rd Cherepkovskaya Str, 15A, Moscow, 121552, Russia.
BMC Molecular Biology (Impact Factor: 2.19). 11/2002; 3(1):15. DOI: 10.1186/1471-2199-3-15
Source: PubMed

ABSTRACT

Secretion of recombinant proteins in yeast can be affected by their improper folding in the endoplasmic reticulum and subsequent elimination of the misfolded molecules via the endoplasmic reticulum associated protein degradation pathway. Recombinant proteins can also be degraded by the vacuolar protease complex. Human urokinase type plasminogen activator (uPA) is poorly secreted by yeast but the mechanisms interfering with its secretion are largely unknown.
We show that in Hansenula polymorpha overexpression worsens uPA secretion and stimulates its intracellular aggregation. The absence of the Golgi modifications in accumulated uPA suggests that aggregation occurs within the endoplasmic reticulum. Deletion analysis has shown that the N-terminal domains were responsible for poor uPA secretion and propensity to aggregate. Mutation abolishing N-glycosylation decreased the efficiency of uPA secretion and increased its aggregation degree. Retention of uPA in the endoplasmic reticulum stimulates its aggregation.
The data obtained demonstrate that defect of uPA secretion in yeast is related to its retention in the endoplasmic reticulum. Accumulation of uPA within the endoplasmic reticulum disturbs its proper folding and leads to formation of high molecular weight aggregates.

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Available from: Michael Agaphonov, Mar 31, 2015
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    • "Proteins from culture supernatants were concentrated 3-fold in case of CPY analysis and 20-fold in the case of uPA and uPA-Q 302 analysis by precipitation with trichloroacetic acid. The amounts of uPA, uPA-Q 302 and CPY from culture medium and in cell lysates were normalized to the levels of total cellular protein as described in [32] and subsequently resolved by electrophoresis in 10% polyacrylamide gel as described in [33]. Rabbit antisera against E. coli expressed H. polymorpha CPY [30] and Pmr1 [18] , tubulin beta antibody (PA5-16863, ThermoFisher Scientific) and monoclonal mouse antibody specific to the uPA protease domain (#MGH U11, IMTEK, Moscow, Russia) were used to detect corresponding proteins. "
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    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.
    Full-text · Article · Dec 2015 · PLoS ONE
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    • "The S. cerevisiae strain BY4741 (MATa his3-D1 leu2-D0 met15-D0 ura3-D0) [25] and its derivatives disrupted for the VPS8, VPS10, and VPS17 genes were obtained from the Euroscarf collection. To study uPA secretion, these strains were transformed with the EcoRV-digested pNR9 plasmid [23] "
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    ABSTRACT: Human urokinase-type plasminogen activator (uPA) is poorly secreted by yeast cells. Here, we have selected Hansenula polymorpha mutants with increased productivity of active extracellular uPA. Several of the obtained mutants also demonstrated a defect of sorting of carboxypeptidase Y to the vacuole and the mutant loci have been identified in six of them. All these mutations damaged genes involved in protein traffic between the Golgi apparatus and the vacuole, namely PEP3, VPS8, VPS10, VPS17, and VPS35. We have shown that inactivation of the VPS10 gene encoding the vacuolar protein sorting receptor does not increase uPA secretion but stimulates its proteolytic processing.
    Full-text · Article · Dec 2005 · FEMS Yeast Research
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    • "To distinguish the S. cerevisiae and H. polymorpha genes or proteins, they are designated when necessary by Sc and Hp, respectively. Culture conditions used for cultivation of H. polymorpha strains are described elsewhere (Agaphonov et al., 2002). "
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    ABSTRACT: Human urokinase-type plasminogen activator (uPA) is poorly secreted and aggregates in the endoplasmic reticulum of yeast cells due to inefficient folding. A screen for Hansenula polymorpha mutants with improved uPA secretion revealed a gene encoding a homologue of the Saccharomyces cerevisiae protein-O-mannosyltransferase Pmt1p. Expression of the H. polymorpha PMT1 gene (HpPMT1) abolished temperature sensitivity of the S. cerevisiae pmt1 pmt2 double mutant. As in S. cerevisiae, inactivation of the HpPMT1 gene affected electrophoretic mobility of the O-glycosylated protein, extracellular chitinase. In contrast to S. cerevisiae, disruption of HpPMT1 alone caused temperature sensitivity. Inactivation of the HpPMT1 gene decreased intracellular aggregation of uPA, suggesting that enhanced secretion of uPA was due to improvement of its folding in the endoplasmic reticulum. Unlike most of the endoplasmic reticulum membrane proteins, HpPmt1p possesses the C-terminal KDEL retention signal.
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