Characterization of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase from Acanthamoeba castellanii.
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The kinetic properties of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) partially purified from the soil amoeba Acanthamoeba castellanii have been studied. The transferase phosphorylated the lysosomal enzymes uteroferrin and cathepsin D 3-90-fold better than nonlysosomal glycoproteins and 16-83-fold better than a Man9GlcNAc oligosaccharide. Deglycosylated uteroferrin was a potent competitive inhibitor of the phosphorylation of intact uteroferrin (Ki of 48 microM) but did not inhibit the phosphorylation of RNase B or the simple sugar alpha-methylmannoside. Deglycosylated RNase (RNase A) did not inhibit the phosphorylation of RNase B or uteroferrin. These results indicate that purified amoeba GlcNAc-phosphotransferase recognizes a protein domain present on lysosomal enzymes but absent in most nonlysosomal glycoproteins. The transferase also exhibited a marked preference for oligosaccharides containing mannose alpha 1,2-mannose sequences, but this cannot account for the high affinity binding to lysosomal enzymes. A. castellanii extracts do not contain detectable levels of N-acetylglucosamine-1-phosphodiester alpha-N-acetylglucosaminidase, the second enzyme in the biosynthetic pathway for the mannose 6-phosphate recognition marker. We conclude that A. castellanii does not utilize the phosphomannosyl sorting pathway despite expression of very high levels of GlcNAc-phosphotransferase. (+info)
Interaction of mannose-6-phosphate with the hysteretic transition in glucose-6-phosphate hydrolysis in intact liver microsomes.
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We showed previously that glucose-6-phosphatase activity was characterised in intact liver microsomes by a hysteretic transition between a rapid and a slower catalytic form of the enzyme. We have now further investigated the substrate specificity of these two kinetic forms. It was found that the pre-incubation of intact microsomes with mannose-6-phosphate or glucose-6-phosphate (50 microM for 30 s) suppressed the burst in glucose-6-phosphatase activity, that the hysteretic transition was reversible and that mannose-6-phosphate inhibited glucose-6-phosphate hydrolysis during the first seconds of incubation, but not anymore after the burst. Our results indicate (i) that mannose-6-phosphate is recognised by the enzyme and can promote the hysteretic transition and (ii) that the transient phase is part of the catalytic mechanism itself. (+info)
Lumenal labeling of rat hepatocyte endocytic compartments. Distribution of several acid hydrolases and membrane receptors.
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We used a combination of subcellular fractionation and lactoperoxidase-mediated iodination to examine the polypeptide compositions of three hepatocyte endocytic compartments: early endosomes, late endosomes, and lysosomes. A chemical conjugate of asialoorosomucoid and lactoperoxidase which binds specifically to asialoglycoprotein receptors was perfused through isolated rat livers at 37 degrees C. Subcellular fractions enriched in various endocytic compartments were then isolated by differential and isopycnic centrifugation, and the lactoperoxidase moiety of the internalized conjugate was used to catalyze the iodination of lumenal-facing proteins. The 125I profiles of early and late endosomes were strikingly similar after gel electrophoresis. Using immunoprecipitation, we directly identified and compared the relative amounts of the Na+,K(+)-ATPase and several different acid hydrolases and membrane receptors in all three fractions. The asialoglycoprotein receptor and the low density lipoprotein related protein were approximately nine times more abundant in early endosomes than late endosomes, suggesting that they recycle from early endosomes. In addition, cathepsin D, but not cathepsin L, beta-glucuronidase, and lgp 120, was detected in early endosomes; however, all of these molecules were detected in lysosomes. Our findings provide strong evidence that early endosomes mature into late endosomes and that there is either selective delivery or selective retention of hydrolases at discrete points in the endocytic pathway. (+info)
Expression of the two mannose 6-phosphate receptors is spatially and temporally different during mouse embryogenesis.
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Mammalian cells express two mannose 6-phosphate receptors, MPR46 and MPR300, both of which mediate the targeting of lysosomal enzymes to lysosomes. Additionally the receptors mediate the secretion (MPR46) and the endocytosis (MPR300) of lysosomal enzymes and the binding of IGFII (MPR300). We have analyzed the distribution of MPR46 and MPR300 transcripts during mouse embryogenesis by in situ hybridization. Up to day 15.5 of embryonic development we found a non-overlapping distribution of the transcripts for the two receptors. High expression of MPR46 was observed at sites of hemopoiesis and in the thymus while MPR300 was highly expressed in the cardiovascular system. Late in embryogenesis (day 17.5) a wide variety of tissues expressed the receptors, but still the expression pattern was almost non-overlapping. This unexpected complementary expression pattern points to specific functions of the two mannose 6-phosphate receptors during mouse embryogenesis. (+info)
Polymyxin B inhibits insulin-induced glucose transporter and IGF II receptor translocation in isolated adipocytes.
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In isolated adipocytes, polymyxin B inhibited insulin-induced glucose incorporation into lipids in a dose-dependent manner, while polymyxin E, a structurally related antibiotic, was ineffective. To approach the mechanism of this effect, the subcellular distribution of the glucose transporter Glut 4 was investigated. Adipocytes were pretreated without or with polymyxin B before insulin stimulation, subcellular fractionation was performed and Glut 4 was detected by immunodetection. Incubation of adipocytes with polymyxin B prevented the insulin-induced appearance of Glut 4 in the plasma membranes, but did not prevent their decrease from the low-density microsomal fraction. A lower purity of the plasma membrane fractions, a detergent effect of polymyxin B on the membranes or an interference of the substance with the immunodetection of the Glut 4 molecules were excluded. These results suggest that polymyxin B was interfering with the Glut 4 translocation process stimulated by insulin in adipocytes. In a similar fashion, polymyxin B inhibited the insulin-induced increase in IGF II binding to adipocytes. This resulted from a blockade of the appearance of IGF II receptors in the plasma membranes. Since low-molecular-mass GTP-binding proteins have been implicated in the regulation of vesicular trafficking, we have used [alpha-32P]GTP binding to analyze such proteins in adipocyte fractions, after SDS/PAGE and transfer to nitrocellulose. Specific and distinct subsets of GTP-binding proteins were revealed in plasma membrane and low-density microsomal fractions of control adipocytes, whether they were stimulated or not with insulin. Polymyxin B treatment of adipocytes markedly modified the profile of the low-molecular-mass GTP-binding proteins in plasma membranes, but not in low-density microsomal fractions. Our results suggest that polymyxin B was interfering with the exocytotic process of the Glut 4 and IGF II receptor-containing vesicles, perhaps at the fusion step between vesicles and plasma membranes. (+info)
A His-Leu-Leu sequence near the carboxyl terminus of the cytoplasmic domain of the cation-dependent mannose 6-phosphate receptor is necessary for the lysosomal enzyme sorting function.
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The determinants on the cytoplasmic tail of the cation-dependent mannose 6-phosphate receptor (CD-MPR) required for lysosomal enzyme sorting have been analyzed. Mouse L cells deficient in the mannose 6-phosphate/insulin-like growth factor-II receptor were transfected with normal bovine CD-MPR cDNA or cDNAs containing mutations in the 67-amino acid cytoplasmic tail and assayed for their ability to target the lysosomal enzyme cathepsin D to lysosomes. Cells expressing the wild-type bovine CD-MPR sorted 67 +/- 2% of newly synthesized cathepsin D compared with the base-line value of 47 +/- 1%. The presence of mannose 6-phosphate in the medium did not affect the efficiency of cathepsin D sorting, indicating that the routing of the ligand-receptor complex is completely intracellular. Mutant receptors with the carboxyl-terminal His-Leu-Leu-Pro-Met67 residues deleted or replaced with alanines sorted cathepsin D below the base-line value. A mutant receptor with the outermost Pro-Met residues replaced with alanines sorted cathepsin D better than the wild-type receptor, indicating that the essential residues for sorting are the His-Leu-Leu sequence. Disruption of a putative casein kinase II phosphorylation site at Ser57 had no detectable effect on sorting. The mutant receptor with the five-amino acid deletion was able to bind to a phosphopentamannose affinity column, proving that its ligand binding site was grossly intact. Resialylation experiments showed that this mutant receptor recycled from the cell surface to the Golgi at a rate similar to the normal CD-MPR, indicating that the defect in sorting is at the level of the Golgi. (+info)
Role of protein phosphatases in insulin-like growth factor II (IGF II)-stimulated mannose 6-phosphate/IGF II receptor redistribution.
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The regulated expression of mannose 6-phosphate/insulin-like growth factor II (M6P/IGF II) receptors in plasma membranes has previously been shown to be accompanied by marked changes in the phosphorylation state of the receptors (Corvera, S., Folander, K., Clairmont, K. B., and Czech, M. P. (1988) Proc. Natl. Acad. Sci. U. S. A. 85, 7567-7571). In the present study we show that protein phosphatase 2A dephosphorylates the human M6P/IGF II receptor in vitro. Incubation of human fibroblasts with okadaic acid, a specific inhibitor of this phosphatase, resulted in a depletion of M6P/IGF II receptors at the cell surface without affecting their internalization kinetics. The phosphorylation state of the remaining cell surface receptors was 3-fold increased. Thus, the endocytosis rate of M6P/IGF II receptors appears to be unaltered by increased phosphorylation. While the decreased cell surface expression of receptors was reversible upon removal of okadaic acid the IGF II-induced redistribution of M6P/IGF II receptors to the plasma membrane (Braulke, T., Tippmer, S., Neher, E., and von Figura, K. (1989) EMBO J. 8, 681-686) was irreversibly inhibited by the phosphatase inhibitor. Receptor redistribution in response to protein kinase C activation was not affected by okadaic acid. These results suggest that the cell surface expression of M6P/IGF II receptor can be regulated by phosphatase-dependent and -independent pathways. In addition, the phosphorylation state and the steady-state cell surface number of transferrin receptors were not affected by okadaic acid, whereas it impaired the IGF II-stimulated receptor redistribution similarly as for M6P/IGF II receptors. The data indicate that okadaic acid-sensitive protein phosphatases may play a general role in terms of IGF II-modulated receptor recycling. (+info)
Divalent cation-dependent stimulation of ligand binding to the 46-kDa mannose 6-phosphate receptor correlates with divalent cation-dependent tetramerization.
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The quaternary structure and binding activity of the murine 46-kDa mannose 6-phosphate receptor (46MPR) were studied in semi-intact murine cells that overexpress the murine receptor. Chemical cross-linking studies showed that the murine 46MPR exists in monomer, dimer, and tetramer forms in membranes of overexpressing murine cells. Treatment of permeabilized cells with Mn2+ increased the tetramer form of 46MPR, and this tetramerization was reversed by removal of Mn2+. Thus, the divalent cations affected the distribution of receptor among the three forms, favoring tetramerization at the expense of dimer and monomer. Low temperature (4 degrees C) also increases the fraction present as tetramer. The binding assay results show that Mn2+ is required for the 46MPR to achieve and retain the ability to bind ligand at 37 degrees C but not at 4 degrees C. Preincubation with Mn2+ produced a 3-fold increase in Man-6-P-specific binding of beta-glucuronidase which paralleled the 3-fold increase in tetramer seen during preincubation with Mn2+. The similarity of the effects of addition and removal of Mn2+ on enzyme binding to the effects of Mn2+ on favoring tetramer formation suggests that divalent cation-dependent tetramerization of the 46MPR contributes to the stimulation of ligand binding to the 46MPR by divalent cations. (+info)