Organelle-specific phosphorylation. Identification of unique membrane phosphoproteins of the endoplasmic reticulum and endosomal apparatus.

Rindress, D.; Xia, Liangyu; Ahluwalia, Jatinder; Cameron, Pamela H.; Fazel, Ali; Posner, Barry I.; Bergeron, John J.M.

doi:10.1016/s0021-9258(18)53512-2

Cited by 20 publications

(5 citation statements)

References 50 publications

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“…Several protein kinases have been shown to be present in endosomes [3,4], and to play a role in cell-free endosome fusion [13,48]. In the present experiments, the protein kinase inhibitor genistein had little effect on its own on ASGP degradation (in contrast to previous observations, cf.…”

Section: Discussioncontrasting

confidence: 98%

“…Surface receptors can be phosphorylated both at serine/threonine [1] and tyrosine groups [2], resulting in inhibition or stimulation respectively of endocytosis. Endosomes contain sever-ai protein kinase activities [3,4] that may be involved in signal transduction [5] as well as in the control of endosomal function [6].…”

Section: Introductionmentioning

confidence: 99%

“…Figure4 Antagonistic effect of genistein on the inhibifton of 2a1iTCWAOM uptake and degradation by protein phosphatase inhibitors Hepatocytes were incubated with 1251-TC-AOM for 3 h at 37 0C in the absence (0) or presence of okadaic acid, 60 nM (0); calyculin A, 100 nM (A); or microcystin-LR, 100 nM (A), at the concentration of genistein indicated. The cells were then washed and precipitated with 10% TCA, and the total amount of cell-associated radioactivity (a) as well as the acidsoluble, i.e.…”

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confidence: 99%

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Inhibition of asialoglycoprotein endocytosis and degradation in rat hepatocytes by protein phosphatase inhibitors

Holen

Gordon

Strømhaug

et al. 1995

Biochemical Journal

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In isolated rat hepatocytes, a radiolabelled tyramine-cellobiose conjugate of asialo-orosomucoid, 125I-TC-AOM, was rapidly taken up by receptor-mediated endocytosis and proteolytically degraded in the lysosomes, where radioactive degradation products accumulated. Okadaic acid and other protein phosphatase inhibitors (microcystin-LR, calyculin A) strongly reduced the fraction of asialoglycoprotein (ASGP) receptors localized to the cell surface, and correspondingly inhibited the uptake of 125I-TC-AOM. In addition, the inhibitors suppressed 125I-TC-AOM degradation strongly (90% at 150 nM) and potently (half-maximal effect at 20 nM okadaic acid), indicating an involvement of protein phosphorylation, and of a protein phosphatase of type 2A, in the regulation of intracellular endocytic flux. The effects of okadaic acid on 125I-TC-AOM accumulation, as well as on degradation, could be eliminated by the protein kinase inhibitor genistein. Okadaic acid prevented the transfer of 125I-TC-AOM to a non-recycling endocytic compartment, causing its retention in a recycling compartment from which about one-third of the endocytosed 125I-TC-AOM could be returned to the cell surface and detached from its receptor in the presence of EGTA. ASGP receptors recycled extensively both in the presence and absence of okadaic acid, as indicated by a sustained uptake of 125I-TC-AOM. Sucrose density gradient analysis and sedimentation studies indicated that okadaic acid caused accumulation of 125I-TC-AOM in light endosomes (1.11 g/ml), preventing its transfer to dense endosomes (1.14 g/ml) and lysosomes (1.18 g/ml). The lysosomes could be identified in density gradients by their contents of lysosomal marker enzymes and acid-soluble radioactivity, and by their sensitivity towards the lysosome-disrupting agent glycyl-L-phenylalanine-2-naphthylamide. By using endocytosed AOM-gold particles as an ultrastructural endocytic marker, it could be shown that the light endosomes accumulating ASGP in the presence of okadaic acid had the morphological appearance of small endocytic vesicles/tubules and multivesicular endosomes. Whereas in control cells 4% of the AOM-gold was in small vesicles/tubules, 55% in multivesicular endosomes and 41% in lysosomes, the corresponding figures for okadaic acid-treated cells were 17%, 73% and 11%. Our results thus indicate that protein phosphatase inhibitors have two effects on ASGP endocytosis: (1) an early inhibition of ligand uptake, due to a reduction in the fraction of ASGP receptors at the cell surface, and (2) an inhibition of ASGP transfer from a recycling compartment consisting of light, small endocytic vesicles and multivesicular endosomes, to a non-recycling compartment consisting of dense multivesicular endosomes.(ABSTRACT TRUNCATED AT 400 WORDS)

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Section: Discussioncontrasting

confidence: 98%

Section: Introductionmentioning

confidence: 99%

mentioning

confidence: 99%

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Inhibition of asialoglycoprotein endocytosis and degradation in rat hepatocytes by protein phosphatase inhibitors

Holen

Gordon

Strømhaug

et al. 1995

Biochemical Journal

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“…by cell volume is due to alterations of intracellular Cl-, which is required to dissipate the membrane potential generated by the HI-pump in order to augment the acidification process. Evidence for a role of protein kinase A-dependent protein phosphorylation in the regulation of chloride channel activity and acidification in endosomes prepared from calf brain or rabbit proximal tubule has been given [32,33] and phosphoproteins in the endosomal membrane have been described recently [34]. However, protein kinase A is apparently not involved in the regulation of pHves.…”

Section: Signalling Eventsmentioning

confidence: 99%

Characterization of the swelling-induced alkalinization of endocytotic vesicles in fluorescein isothiocyanate-dextran-loaded rat hepatocytes

Schreiber

Häussinger

1995

Biochemical Journal

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Short-term cultivated rat hepatocytes were allowed to endocytose fluorescein isothiocyanate (FITC)-coupled dextran and the apparent vesicular pH (pHves) was measured by single-cell fluorescence. After 2 h of exposure to FITC-dextran, the apparent pH in the vesicular compartments accessible to endocytosed FITC-dextran was 6.01 +/- 0.05 (n = 39) in normo-osmotic media. Hypo-osmotic exposure increased, whereas hyper-osmotic exposure decreased apparent pHves. by 0.18 +/- 0.02 (n = 26) and 0.12 +/- 0.01 (n = 23) respectively. Incubation of the cells with unlabelled dextran for 2h before a 2-h FITC-dextran exposure had no effect on apparent pHves and its osmosensitivity. When, however, hepatocytes were exposed to unlabelled dextran for 5 h after a 2 h exposure to FITC-dextran, in order to allow transport of endocytosed FITC-dextran to late endocytotic/lysosomal compartments, apparent pHves. decreased to 5.38 +/- 0.04 (n = 12) and the apparent pH in the vesicular compartment containing the dye was no longer sensitive to aniso-osmotic exposure. These findings indicate that the osomosensitivity of pHves. is apparently restricted to early endocytotic compartments. Aniso-osmotic regulation of apparent pHves. in freshly FITC-loaded hepatocytes was not accompanied by aniso-osmolarity-induced changes of the cytosolic free calcium concentration, and neither vasopressin nor extracellular ATP, which provoked a marked Ca2+ signal, affected apparent pHves. Dibutyryl-cyclic AMP (cAMP) or vanadate (0.5 mmol/l) were without effect on apparent pHves. and its osmosensitivity. However, pertussis toxin-treatment or genistein (but not daidzein) or the erbstatin analogue methyl 2,5-dihydroxycinnamate fully abolished the osmo-sensitivity of apparent pHves., but did not affect apparent pHves. It is concluded that regulation of pHves. by cell volume occurs in early endocytotic compartments, but probably not in lysosomes, and is mediated by a G-protein and tyrosine kinase-dependent, but Ca2+- and cAMP-independent mechanism.

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“…The W N G Golgi fraction, a highly enriched Golgi fraction largely depleted of endosomes (Rindress et al, 1993) revealed immunoreactive apoE at molecular masses of 29 and 36 kD (Fig. 5 A, lane 6) presumably representing the nonglycosylated and completely O-glycosylated forms of apoE, respectively (Reardon et al, 1986).…”

Section: Western Blotting Of Purified Organeues Of Rat Livermentioning

confidence: 99%

Concentration of intracellular hepatic apolipoprotein E in Golgi apparatus saccular distensions and endosomes.

Dahan

Ahluwalia

Wong

et al. 1994

The Journal of Cell Biology

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Abstract. The intrahepatic distribution of apolipoprotein E has been assessed by immunogold labeling of cryosections as well as by Western blotting of organelles isolated from liver homogenates. Both techniques supported the prior analytical fractionation studies of Wong (1989) who concluded that intrahepatic apoE was largely endosomal. All endosomal components decorated by gold particles indicative of apoE antigenicity in cryosections appeared filled with lipoprotein-like particles thereby accounting for this prominent morphological feature of isolated liver endosomes. The distribution of gold particles about the hepatic Golgi apparatus revealed a high content of apoE in closely apposed endosomes, ca. 400 nm in diameter, double labeled for apoE and internalized HRP. Remarkably, apoE (but not internalized HRP) was also observed within saccular distensions of all saccules of stacked Golgi cisternae but absent from the flattened saccular components as was also observed for apoB. This contrasted with albumin, the major secretory protein, which was uniformly distributed throughout the hepatic Golgi apparatus. These observations support a growing body of evidence for intra-Golgi sorting of secretory material in hepatic Golgi apparatus. The lack of any immunoreactive apoE or albumin in small 70-90 nm vesicles about the Golgi cisternae suggests limits to current models of vesicle-mediated intra-Golgi transport.

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Organelle-specific phosphorylation. Identification of unique membrane phosphoproteins of the endoplasmic reticulum and endosomal apparatus.

Cited by 20 publications

References 50 publications

Inhibition of asialoglycoprotein endocytosis and degradation in rat hepatocytes by protein phosphatase inhibitors

Inhibition of asialoglycoprotein endocytosis and degradation in rat hepatocytes by protein phosphatase inhibitors

Characterization of the swelling-induced alkalinization of endocytotic vesicles in fluorescein isothiocyanate-dextran-loaded rat hepatocytes

Concentration of intracellular hepatic apolipoprotein E in Golgi apparatus saccular distensions and endosomes.

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