The yeast secretory pathway is perturbed by mutations in PMR1, a member of a Ca2+ ATPase family

Rudolph, Hans; Antebi, Adam; Fink, Gerald R.; Buckley, Catherine M.; Dorman, Thomas E.; LeVitre, JoAnn; Davidow, Lance S.; Mao, Jen-i; Moir, Donald T.

doi:10.1016/0092-8674(89)90410-8

Cited by 529 publications

(512 citation statements)

References 62 publications

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“…The growth rate of the pmrl mutant cells was slightly stimulated by the addition of 20 mM Ca ~+ to the medium, markedly inhibited by reducing external Ca 2+ by EGTA, as previously reported [1,4], but was reduced by only 12% (which is no more than the decrease in the growth rate of the WT cells) by increasing strains. Cells of the indicated strains were grown in YPD medium labeled with 4~Cae+ to the stationary phase.…”

Section: '; Measurements Of Ca 2+ Influxsupporting

confidence: 85%

“…It was suggested that PMR1 encodes a Ca2+-ATPase. This suggestion was based on sequence homology to mammalian Ca2+-ATPase, inhibition of the growth of the prnrl mutant in very low Ca 2+ medium, and reversal of the defects in Golgi function in the pmrl mutant by the addition of extracellular Ca 2+ [1,4]. In the present study we showed that deletion of PMR1 but not of PMR2 causes elevation of [Ca2+]i.…”

Section: Discussionsupporting

confidence: 58%

“…Cls2p/ Csg2p appears to regulate Ca 2+ sequestration into the ER, since cls2 null mutant cells do not grow in high Ca 2+ media and when grown in low Ca 2+ medium sequester huge amounts of Ca 2+ into a non-vacuolar Ca 2+ pool, probably the ER [20,21]. The third Ca 2+ transporter is Pmrlp [1] which was localized in Golgi-like particles by immunofluorescence staining of yeast cells expressing epitope tagged PMR1 [4]. It was shown in the present study that Pmrlp participates in lowering [Ca2+]i in cells exposed to moderately high [Ca~+]out, probably by pumping Ca 2+ into the Golgi.…”

Section: Discussionmentioning

confidence: 99%

“…Two genes for P-type ATPase, PMR1 and PMR2, were cloned by Rudolph et al [1] PMR2 was found later to encode a plasma membrane Na+-ATPase responsible for resistance to high concentrations of Na ÷ and Li + [12][13][14]. It was suggested that PMR1 encodes a Ca2+-ATPase.…”

Section: Discussionmentioning

confidence: 99%

“…]?he cells of the pmrl mutant and the WT strain were harvested from the growth medium, washed 4 times with distilled water and resuspended (2x 108 cells/ml) in buffer-glucose solution containing MES/DMG buffer (25 mM, pH 5.0) and glucose (20 mM [1,4], the isogenic pmr2 mutant cells, lacking a cell-membrane Na+-ATPase [12][13][14] and the isogenic WT cells grew well in YPD medium containing 0.3 mM Ca 2+. The growth rate of the pmrl mutant cells was slightly stimulated by the addition of 20 mM Ca ~+ to the medium, markedly inhibited by reducing external Ca 2+ by EGTA, as previously reported [1,4], but was reduced by only 12% (which is no more than the decrease in the growth rate of the WT cells) by increasing strains.…”

Section: '; Measurements Of Ca 2+ Influxmentioning

confidence: 99%

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Elevated cytosolic free Ca²⁺ concentrations and massive Ca²⁺ accumulation within vacuoles, in yeast mutant lacking PMR1, a homolog of Ca²⁺‐ATPase

Halachmi

Eilam

1996

FEBS Letters

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Section: '; Measurements Of Ca 2+ Influxsupporting

confidence: 85%

Section: Discussionsupporting

confidence: 58%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: '; Measurements Of Ca 2+ Influxmentioning

confidence: 99%

See 3 more Smart Citations

Elevated cytosolic free Ca²⁺ concentrations and massive Ca²⁺ accumulation within vacuoles, in yeast mutant lacking PMR1, a homolog of Ca²⁺‐ATPase

Halachmi

Eilam

1996

FEBS Letters

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Secretion of a variant of human single-chain urokinase-type plasminogen activator without an N-glycosylation site in the methylotrophic yeast, Pichia pastoris and characterization of the secreted product

Tsujikawa¹,

Okabayashi²,

Morita³

et al. 1996

Yeast

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Human single‐chain urokinase‐type plasminogen activator without an N‐glycosylation site (scu‐PA‐Q302) was produced in the methylotrophic yeast, Pichia pastoris using the shortened prepeptide sequence of a fungal aspartic proteinase, Mucor pusillus rennin (MPR). The level of urokinase‐type plasminogen activator (u‐PA) immunoreactive material in YPM medium was 0·47 mg/l; however, most of the secreted product had been processed to smaller polypeptides. The N‐terminal amino acid sequence of major species was identical to that of the low molecular weight two‐chain u‐PA. Some approaches to minimizing the proteolysis of scu‐PA‐Q302 were attempted. Addition of Triton X‐100, l‐arginine and ammonium phosphate to the YPM medium minimized the proteolysis of scu‐PA‐Q302 and increased the yield of immunoreactive material to approximately 5 mg/l. Use of proteinase A‐ or proteinase B‐deficient strains of yeast did not reduce the degradation. Co‐expression of scu‐PA‐Q302 and urinary trypsin inhibitor resulted in partial reduction of the major species of proteolysis. Scu‐PA‐Q302 was purified from the culture supernatant of the improved medium by two successive chromatographies on Phenyl‐Sepharose and S‐Sepharose. The purified protein had a molecular weight of 47 kDa. It did not contain detectable N‐linked oligosaccharides, but contained O‐linked oligosaccharides attached to the light chain. N‐terminal amino acid sequencing of the purified preparation showed that the shortened prepeptide sequence of MPR was correctly processed by the Pichia yeast. Scu‐PA‐Q302 closely resembles natural scu‐PA with respect to its enzymatic activity against the chromogenic substrate S‐2444 and its in vitro fibrinolytic properties.

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Secretion of a Variant of Human Single‐Chain Urokinase‐Type Plasminogen Activator without an N‐Glycosylation Site in the Methylotrophic Yeast, Pichia pastoris and Characterization of the Secreted Product

TSUJIKAWA¹,

OKABAYASHI²,

MORITA³

et al. 1996

Yeast

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Human single-chain urokinase-type plasminogen activator without an N-glycosylation site (scu-PA-Q302) was produced in the methylotrophic yeast, Pichia pastoris using the shortened prepeptide sequence of a fungal aspartic proteinase, Mucor pusillus rennin (MPR). The level of urokinase-type plasminogen activator (u-PA) immunoreactive material in YPM medium was 0.47 mg/l; however, most of the secreted product had been processed to smaller polypeptides. The N-terminal amino acid sequence of major species was identical to that of the low molecular weight two-chain u-PA. Some approaches to minimizing the proteolysis of scu-PA-Q302 were attempted. Addition of Triton X-100, L-arginine and ammonium phosphate to the YPM medium minimized the proteolysis of scu-PA-Q302 and increased the yield of immunoreactive material to approximately 5 mg/l. Use of proteinase A- or proteinase B-deficient strains of yeast did not reduce the degradation. Co-expression of scu-PA-Q302 and urinary trypsin inhibitor resulted in partial reduction of the major species of proteolysis. Scu-PA-Q302 was purified from the culture supernatant of the improved medium by two successive chromatographies on Phenyl-Sepharose and S-Sepharose. The purified protein had a molecular weight of 47 kDa. It did not contain detectable N-linked oligosaccharides, but contained O-linked oligosaccharides attached to the light chain. N-terminal amino acid sequencing of the purified preparation showed that the shortened prepeptide sequence of MPR was correctly processed by the Pichia yeast. Scu-PA-Q302 closely resembles natural scu-PA with respect to its enzymatic activity against the chromogenic substrate S-2444 and its in vitro fibrinolytic properties.

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The yeast secretory pathway is perturbed by mutations in PMR1, a member of a Ca2+ ATPase family

Cited by 529 publications

References 62 publications

Elevated cytosolic free Ca²⁺ concentrations and massive Ca²⁺ accumulation within vacuoles, in yeast mutant lacking PMR1, a homolog of Ca²⁺‐ATPase

Elevated cytosolic free Ca²⁺ concentrations and massive Ca²⁺ accumulation within vacuoles, in yeast mutant lacking PMR1, a homolog of Ca²⁺‐ATPase

Secretion of a variant of human single-chain urokinase-type plasminogen activator without an N-glycosylation site in the methylotrophic yeast, Pichia pastoris and characterization of the secreted product

Secretion of a Variant of Human Single‐Chain Urokinase‐Type Plasminogen Activator without an N‐Glycosylation Site in the Methylotrophic Yeast, Pichia pastoris and Characterization of the Secreted Product

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The yeast secretory pathway is perturbed by mutations in PMR1, a member of a Ca2+ ATPase family

Cited by 529 publications

References 62 publications

Elevated cytosolic free Ca2+ concentrations and massive Ca2+ accumulation within vacuoles, in yeast mutant lacking PMR1, a homolog of Ca2+‐ATPase

Elevated cytosolic free Ca2+ concentrations and massive Ca2+ accumulation within vacuoles, in yeast mutant lacking PMR1, a homolog of Ca2+‐ATPase

Secretion of a variant of human single-chain urokinase-type plasminogen activator without an N-glycosylation site in the methylotrophic yeast, Pichia pastoris and characterization of the secreted product

Secretion of a Variant of Human Single‐Chain Urokinase‐Type Plasminogen Activator without an N‐Glycosylation Site in the Methylotrophic Yeast, Pichia pastoris and Characterization of the Secreted Product

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Elevated cytosolic free Ca²⁺ concentrations and massive Ca²⁺ accumulation within vacuoles, in yeast mutant lacking PMR1, a homolog of Ca²⁺‐ATPase

Elevated cytosolic free Ca²⁺ concentrations and massive Ca²⁺ accumulation within vacuoles, in yeast mutant lacking PMR1, a homolog of Ca²⁺‐ATPase