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.

scholarly journals Association of protein kinase-C-alpha with cytoplasmic vesicles in melanoma cells.

1996 ◽  
Vol 44 (2) ◽  
pp. 177-182 ◽  
Author(s):  
J Timar ◽  
B Liu ◽  
R Bazaz ◽  
K V Honn
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Fluid Phase ◽  
Subcellular Fractionation ◽  
Melanoma Cells ◽  
Kinase C ◽  
Cytoplasmic Vesicles ◽  
Protein Kinase C Alpha ◽  
Pkc Alpha

In B16a melanoma cells, protein kinase-C-alpha (PKC alpha) is immunomorphologically associated with cytoplasmic vesicles in addition to the previously observed locations (plasma membrane, cytoskeleton, nucleus), as detected with monoclonal antibody (MAb) MC3a. Subcellular fractionation indicated that the authentic 80-KD protein as well as PKC activity can be detected in several particulate fractions except for L2, which contains dense lysosomes. The highest PKC activity is associated with the cytosol-ultralight vesicles and the L1 fraction (containing plasma membrane, endosomes, and the Golgi apparatus). Both of these fractions contained the fluid-phase endocytosis marker peroxidase, indicating that PKC alpha, in addition to other subcellular structures, is most probably associated with endosomal membranes in B16a melanoma cells.

scholarly journals Sugar uptake by fluid-phase pinocytosis and diffusion in isolated rat hepatocytes

1987 ◽  
Vol 243 (3) ◽  
pp. 655-660 ◽  
Author(s):  
P B Gordon ◽  
H Høyvik ◽  
P O Seglen
Keyword(s):  
Plasma Membrane ◽  
Fluid Phase ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Free Sugar ◽  
Sugar Uptake ◽  
Isolated Rat Hepatocytes ◽  
Fluid Phase Endocytosis ◽  
And Diffusion ◽  
Isolated Rat

Measurements of sugar pinocytosis (fluid-phase endocytosis of radiolabelled sucrose, lactose and raffinose) in freshly isolated rat hepatocytes are disturbed by sugar diffusing into the cells through plasma-membrane blebs. Non-pinocytic entry may be even more pronounced at 0 degrees C, and is a major contributor to ‘background’ radioactivity. By electrodisruption of the plasma membrane, a distinction can be made between pinocytotically sequestered sugar and free sugar that has entered the cytosol by diffusion. Pinocytosis proceeds at a rate of 2%/h (relative to the intracellular fluid volume), whereas the rate of sucrose entry by diffusion is more than twice as high. Three pinocytotic compartments are distinguishable in isolated hepatocytes: (1) a rapidly recycling compartment, which is completely destroyed by electrodisruption, and which may represent pinocytic channels continuous with the plasma membrane; (2) a non-recycling (or very slowly recycling) electrodisruption-resistant compartment, which allows accumulation of the lysosomally hydrolysable sugar lactose, and which therefore must represent non-lysosomal vacuoles (endosomes?); (3) a lysosomal compartment (non-recycling, electrodisruption-resistant), which accumulates raffinose and sucrose, but which hydrolyses lactose. The last two compartments can be partially resolved in metrizamide/sucrose density gradients by the use of different sugar probes.

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.

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.

The Aminophospholipid Translocase: A Transmembrane Lipid Pump - Physiological Significance

Physiology ◽  
1990 ◽  
Vol 5 (2) ◽  
pp. 53-58
Author(s):  
PF Devaus
Keyword(s):  
Plasma Membrane ◽  
Fluid Phase ◽  
Physiological Significance ◽  
Lipid Asymmetry ◽  
Aminophospholipid Translocase ◽  
Fluid Phase Endocytosis

Aminophospholipid translocase selectively pumps phosphatidylserine and phosphatidylethanolamine from the outer to the inner plasma membrane monolayer. The function of this translocase activity may be to establish and maintain lipid asymmetry and to trigger fluid-phase endocytosis in nucleated cells.

Uptake of Lucifer Yellow CH into intact barley roots: Evidence for fluid-phase endocytosis

Planta ◽  
1988 ◽  
Vol 176 (4) ◽  
pp. 541-547 ◽  
Author(s):  
K. J. Oparka ◽  
D. Robinson ◽  
D. A. M. Prior ◽  
P. Derrick ◽  
K. M. Wright
Keyword(s):  
Lucifer Yellow ◽  
Fluid Phase ◽  
Lucifer Yellow Ch ◽  
Fluid Phase Endocytosis

scholarly journals Angiotensin Peptide Regulation of Fluid-Phase Endocytosis in Brain Microvessel Endothelial Cell Monolayers

1990 ◽  
Vol 10 (6) ◽  
pp. 827-834 ◽  
Author(s):  
François L. Guillot ◽  
Kenneth L. Audus
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Lucifer Yellow ◽  
Primary Cultures ◽  
Fluid Phase ◽  
Ang Ii ◽  
Microvessel Endothelial Cell ◽  
Fluid Phase Endocytosis ◽  
Microvessel Endothelial Cells ◽  
Brain Microvessel

An in vitro model comprised of primary cultures of brain microvessel endothelial cells was used to investigate angiotensin II (Ang II) effects on blood–brain barrier fluid-phase endocytosis. The effects of Ang II, saralasin, sarathrin, bradykinin (BK), and phorbol myristate acetate (PMA) on brain microvessel endothelial cell fluid-phase endocytosis were determined using the fluorescent marker, Lucifer yellow. Nanomolar concentrations of saralasin (a partial Ang II agonist) stimulated brain microvessel endothelial cell endocytosis by 30% whereas Ang II treatment enhanced Lucifer yellow uptake by 20%. Sarathrin (an Ang II antagonist) had no effect on Lucifer yellow uptake. Nanomolar concentrations of BK and PMA also stimulated Lucifer yellow uptake by the brain microvessel endothelial cell by 40 and 95%, respectively. Stimulatory effects of Ang II and saralasin on Lucifer yellow uptake by brain microvessel endothelial cells could be completely blocked by pretreatment with either sarathrin or indomethacin (an inhibitor of prostaglandin synthesis). In contrast, the effects of neither BK nor PMA on brain microvessel endothelial cell uptake of Lucifer yellow were altered by indomethacin pretreatment. Results indicated that Ang II, saralasin, BK, and PMA produce similar stimulatory effects on brain microvessel endothelial cell fluid-phase endocytosis with only Ang II and saralasin, producing increases in brain microvessel endothelial cell fluid-phase endocytosis that appeared to be mediated by prostaglandins.

The organic anion transport inhibitor, probenecid, inhibits the transport of Lucifer Yellow at the plasma membrane and the tonoplast in suspensioncultured plant cells

1991 ◽  
Vol 99 (3) ◽  
pp. 545-555 ◽  
Author(s):  
LOUISE COLE ◽  
JULIAN COLEMAN ◽  
ANNE KEARNS ◽  
GARETH MORGAN ◽  
CHRIS HAWES
Keyword(s):  
Plasma Membrane ◽  
Lucifer Yellow ◽  
Organic Anion ◽  
Plant Cells ◽  
Fluid Phase ◽  
Cell Suspension Cultures ◽  
Carrot Cells ◽  
Anion Transporters ◽  
Plant Cell Suspension Cultures ◽  
Plant Membranes

In this paper we report on the uptake of the membrane-impermeant fluorescent probe Lucifer Yellow CH (LY-CH) into the vacuolar system of plant cell suspension cultures. LY-CH is internalised into vacuoles of maize cells at a faster ‘rate’ than carrot cells and in each case, the probe is also trapped at the cell wall. In the presence of the uricosuric drug probenecid, the vacuolar uptake of LY-CH by carrot and maize cells is inhibited and in some cells internalisation of probe is blocked at the plasma membrane. In electroporated carrot cells, LY-CH is sequestered slowly from the cytoplasm into vacuoles by a probenecid-inhibitable transport process. These results are compared with the effects of probenecid on the sequestration of LY-CH from the cytoplasm into the lysosomal system of fibroblasts. In view of the above findings and recent evidence for the putative uptake of LY-CH by fluid-phase endocytosis in plant cells, the possibility that LY-CH is transported across plant membranes via probenecidinhibitable organic anion transporters is discussed

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