scholarly journals Apparent endocytosis of fluorescein isothiocyanate-conjugated dextran by Saccharomyces cerevisiae reflects uptake of low molecular weight impurities, not dextran.

1987 ◽  
Vol 105 (5) ◽  
pp. 1981-1987 ◽  
Author(s):  
R A Preston ◽  
R F Murphy ◽  
E W Jones
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Lucifer Yellow ◽  
Ph Dependence ◽  
Fluid Phase ◽  
Fluorescein Isothiocyanate ◽  
Low Molecular Weight ◽  
Yeast Saccharomyces Cerevisiae ◽  
Fluid Phase Endocytosis ◽  
Rapid Kinetics

Concurrent with Riezman's report (Riezman, H. 1985, Cell. 40:1001-1009) that fluid-phase endocytosis of the small molecule Lucifer yellow occurs in the yeast Saccharomyces cerevisiae, Makarow (Makarow, M. 1985. EMBO [Eur. Mol. Biol. Organ.] J. 4:1861-1866) reported the endocytotic uptake of 70-kD FITC-dextran (FD) and its subsequent compartmentation into the yeast vacuole. Samples of FD synthesized and purified here failed to label yeast vacuoles under conditions that allowed labeling using commercial FD. Chromatography revealed that the commercial FD was heavily contaminated with at least three low molecular weight fluorescent compounds. Dialysis was ineffective for removing the contaminants. After purification (Sephadex G25, ethanol extraction), commercial FD was incapable of labeling vacuoles. Extracts of cells labeled with partially purified FD contained FITC, not FD, based on Sephadex and thin layer chromatography. In either the presence or absence of unlabeled 70-kD dextran, authentic FITC (10 micrograms/ml) was an effective labeling agent for vacuoles. The rapid kinetics (0.28 pmol/min per 10(6) cells at pH 5.5) and the pH dependence of FITC uptake suggest that the mechanism of FITC uptake involves diffusion rather than endocytosis. In view of these results, labeling experiments that use unpurified commercial FD should be interpreted with caution.

scholarly journals end3 and end4: two mutants defective in receptor-mediated and fluid-phase endocytosis in Saccharomyces cerevisiae.

1993 ◽  
Vol 120 (1) ◽  
pp. 55-65 ◽  
Author(s):  
S Raths ◽  
J Rohrer ◽  
F Crausaz ◽  
H Riezman
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Lucifer Yellow ◽  
Fluid Phase ◽  
Cell Surface Receptor ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Dependent Manner ◽  
Fluid Phase Endocytosis ◽  
Alpha Factor

alpha-factor, one of two peptide hormones responsible for synchronized mating between MATa and MAT alpha-cell types in Saccharomyces cerevisiae, binds to its cell surface receptor and is internalized in a time-, temperature-, and energy-dependent manner (Chvatchko, Y., I. Howald, and H. Riezman. 1986. Cell. 46:355-364). After internalization, alpha-factor is delivered to the vacuole via vesicular intermediates and degraded there consistent with an endocytic mechanism (Singer, B., and H. Riezman. 1990. J. Cell Biol. 110:1911-1922; Chvatchko, Y., I. Howald, and H. Riezman. 1986. Cell. 46:355-364). We have isolated two mutants that are defective in the internalization process. Both mutations confer a recessive, temperature-sensitive growth phenotype upon cells that cosegregates with their endocytosis defect. Lucifer yellow, a marker for fluid-phase endocytosis, shows accumulation characteristics in the mutants that are similar to the uptake characteristics of 35S-alpha-factor. The endocytic defect in end4 cells appears immediately upon shift to restrictive temperature and is reversible at permissive temperature if new protein synthesis is allowed. Furthermore, the end4 mutation only affects alpha-factor internalization and not the later delivery of alpha-factor to the vacuole. Other vesicle-mediated processes seem to be normal in end3 and end4 mutants. END3 and END4 are the first genes shown to be necessary for the internalization step of receptor-borne and fluid-phase markers in yeast.

Heparin-induced Thrombocytopenia: IgG Binding to PF4-Heparin Complexes in the Fluid Phase and Cross-reactivity with Low Molecular Weight Heparin and Heparinoid

1998 ◽  
Vol 80 (08) ◽  
pp. 292-297 ◽  
Author(s):  
Peter Newman ◽  
Rebecca Swanson ◽  
Beng Chong
Keyword(s):  
Molecular Weight ◽  
Fluid Phase ◽  
Cross Reactivity ◽  
Low Molecular Weight ◽  
Absolute Contraindication ◽  
Low Molecular Weight Heparins ◽  
Heparin Induced Thrombocytopenia ◽  
Igg Binding ◽  
Heparin Complexes ◽  
Low Levels

SummaryEarly diagnosis of heparin-induced thrombocytopenia (HIT) is essential to reduce morbidity and mortality. We report an enzyme immunoassay which detects the binding of HIT IgG to PF4-heparin in the fluid phase. Our fluid phase assay produces consistently low background and can detect low levels of anti-PF4-heparin. It is suited to testing alternative anticoagulants because, unlike in an ELISA, a clearly defined amount of antigen is available for antibody binding. We were able to detect anti-PF4-heparin IgG in 26/28 (93%) HIT patients. We investigated cross-reactivity of anti-PF4-heparin antibodies with PF4 complexed to alternative heparin-like anticoagulants. Low molecular weight heparins cross-reacted with 23/26 (88%) of the sera from HIT patients while half of the HIT sera weakly cross-reacted with PF4-danaparoid (Orgaran). The thrombocytopenia and thrombosis of most of these patients resolved during danaparoid therapy, indicating that detection of low affinity antibodies to PF4-danaparoid by immunoassay may not be an absolute contraindication for danaparoid administration.

scholarly journals TOR Complex 2-Regulated Protein Kinase Fpk1 Stimulates Endocytosis via Inhibition of Ark1/Prk1-Related Protein Kinase Akl1 in Saccharomyces cerevisiae

2017 ◽  
Vol 37 (7) ◽  
Author(s):  
Françoise M. Roelants ◽  
Kristin L. Leskoske ◽  
Ross T. A. Pedersen ◽  
Alexander Muir ◽  
Jeffrey M.-H. Liu ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase ◽  
Lucifer Yellow ◽  
Fluid Phase ◽  
Effector Protein ◽  
Multiple Substrates ◽  
Downstream Effector ◽  
Fluid Phase Endocytosis ◽  
Actin Patch ◽  
Resistance To Doxorubicin

ABSTRACT Depending on the stress, plasma membrane alterations activate or inhibit yeast target of rapamycin (TOR) complex 2, which, in turn, upregulates or downregulates the activity of its essential downstream effector, protein kinase Ypk1. Through phosphorylation of multiple substrates, Ypk1 controls many processes that restore homeostasis. One such substrate is protein kinase Fpk1, which is negatively regulated by Ypk1. Fpk1 phosphorylates and stimulates flippases that translocate aminoglycerophospholipids from the outer to the inner leaflet of the plasma membrane. Fpk1 has additional roles, but other substrates were uncharacterized. We show that Fpk1 phosphorylates and inhibits protein kinase Akl1, related to protein kinases Ark1 and Prk1, which modulate the dynamics of actin patch-mediated endocytosis. Akl1 has two Fpk1 phosphorylation sites (Ark1 and Prk1 have none) and is hypophosphorylated when Fpk1 is absent. Conversely, under conditions that inactivate TORC2-Ypk1 signaling, which alleviates Fpk1 inhibition, Akl1 is hyperphosphorylated. Monitoring phosphorylation of known Akl1 substrates (Sla1 and Ent2) confirmed that Akl1 is hyperactive when not phosphorylated by Fpk1. Fpk1-mediated negative regulation of Akl1 enhances endocytosis, because an Akl1 mutant immune to Fpk1 phosphorylation causes faster dissociation of Sla1 from actin patches, confers elevated resistance to doxorubicin (a toxic compound whose entry requires endocytosis), and impedes Lucifer yellow uptake (a marker of fluid phase endocytosis). Thus, TORC2-Ypk1, by regulating Fpk1-mediated phosphorylation of Akl1, adjusts the rate of endocytosis.

PURIFICATION AND PROPERTIES OF AN INDUCIBLE β-GLUCOSIDASE OF BAKERS' YEAST

1966 ◽  
Vol 44 (8) ◽  
pp. 1099-1108 ◽  
Author(s):  
A. N. Inamdar ◽  
J. G. Kaplan
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Weight ◽  
Hydrolytic Activity ◽  
Chromogenic Substrate ◽  
Yeast Saccharomyces Cerevisiae ◽  
Intact Cells ◽  
Highly Active ◽  
Naturally Occurring ◽  
Purification And Properties ◽  
Competitive Inhibitors

The inducible β-glucosidase present in crude extracts of cellobiose-grown bakers' yeast (Saccharomyces cerevisiae C) was purified 50-fold and found to be homogeneous in the ultracentrifuge, with a molecular weight of 313,000. The enzyme was virtually identical in its properties with the internal, cryptic enzyme of the yeast cell, revealed by butanol treatment of the suspensions. It was unlike the membrane-localized enzyme found at the surface of intact cells in its low affinity for cellobiose and methyl-β-glucoside as substrates and inhibitors. The enzyme was specific for the β configuration and had no activity against substrates such as α-glucosides, β-galactosides, or β-xylosides. It was highly active against both naturally occurring and synthetic substrates with aromatic aglycones, and may thus be classed as an aryl-β-glucosidase. The enzyme had weak hydrolytic activity against methyl-β-glucoside and cellobiose, but these compounds, unlike all of the aryl-β-glucosides tested, were not competitive inhibitors of its activity against the chromogenic substrate pNPG. There were about 40,000 molecules of enzyme per cell in fully induced cultures and the enzyme represented about 3% of the total protein of these cells.

Is fluid-phase endocytosis conserved in hepatocytes of species acclimated and adapted to different temperatures?

2000 ◽  
Vol 278 (2) ◽  
pp. R529-R536 ◽  
Author(s):  
David Padrón ◽  
Michael E. Bizeau ◽  
Jeffrey R. Hazel
Keyword(s):  
Lucifer Yellow ◽  
Fluid Phase ◽  
Membrane Lipid ◽  
Primary Objective ◽  
Chain Polyunsaturated Fatty Acid ◽  
Chow Diet ◽  
Sprague Dawley ◽  
Fluid Phase Endocytosis ◽  
Different Temperatures ◽  
Long Chain

Our primary objective was to determine if rates of fluid-phase endocytosis (FPE) were conserved in hepatocytes from organisms acclimated and adapted to different temperatures. To this aim, the fluorescent dye Lucifer yellow was employed to measure FPE at different assay temperatures (AT) in hepatocytes from 5°C- and 20°C-acclimated trout, Oncorhynchus mykiss (at 5 and 20°C AT), 22°C- and 35°C-acclimated tilapia, Oreochromis nilotica (at 22 and 35°C AT), and the Sprague-Dawley rat (at 10, 20, and 37°C AT). FPE was also studied in rats fed a long-chain polyunsaturated fatty acid (PUFA)-enriched diet (at 10°C AT). Despite being temperature dependent, endocytic rates (values in pl ⋅ cell− 1 ⋅ h− 1) in both species of fish were compensated after a period of acclimation. For example, in 20°C-acclimated trout, the rate of endocytosis declined from 1.84 to 1.07 when the AT was reduced from 20 to 5°C; however, after a period of acclimation at 5°C, the rate (at 5°C AT) was largely restored (1.80) and almost perfectly compensated (95%). In tilapia, endocytic rates were also temperature compensated, although only partially (36%). Relatively similar rates obtained at 5°C in 5°C-acclimated trout (1.8), at 20°C in 20°C-acclimated trout (1.84), and at 22°C in 22°C-acclimated tilapia (2.2) suggest that endocytic rates are somewhat conserved in these two species of fish. In contrast, the rate in rat measured at 37°C (16.83) was severalfold greater than in fish at their respective body temperatures. A role for lipids in determining rates of endocytosis was supported by data obtained at 10°C in hepatocytes isolated from rats fed a long-chain PUFA-enriched diet: endocytic rates were higher (5.35 pl ⋅ cell− 1 ⋅ h− 1) than those of rats fed a standard chow diet (2.33 pl ⋅ cell− 1 ⋅ h− 1). The conservation of endocytic rates in fish may be related to their ability to conserve other membrane characteristics (i.e., order or phase behavior) by restructuring their membrane lipid composition or by modulating the activities of proteins that regulate endocytosis and membrane traffic, whereas the lack of conservation between fish and rat may be due to differences in metabolic rate.

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