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Query: UMLS:C0038362 (stomatitis)
 8,852 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This paper describes a rapid and efficient two-step procedure for the isolation of mutant cells with defects in receptor-mediated endocytosis. The procedure takes advantage of two fungal metabolites, compactin (ML236B), a potent inhibitor of cholesterol biosynthesis, and amphotericin B, a polyene antibiotic that forms toxic complexes with sterols in membranes. Mutagen-treated Chinese hamster ovary cells were preincubated overnight in a medium containing mevalonate, low density lipoprotein (LDL), and compactin (Mev/LDL/Com). At the end of the preincubation period, wild-type cells were cholesterol replete while mutant cells that could not utilize the cholesterol in LDL were cholesterol deficient. Subsequent incubation with amphotericin B for 6 hr killed most of the wild-type cells. After a second round of Mev/LDL/Com-amphotericin B selection, endocytosis-defective clones appeared at a frequency of approximately equal to 2.6 X 10(-5). Some of these clones expressed LDL receptor-defective phenotypes and fell into one of two previously defined classes of mutation. Sensitivity of the mutants to infection by vesicular stomatitis virus suggested that the mutations do not disrupt the coated pit-coated vesicle pathway of endocytosis. Minor modifications in the Mev/LDL/Com-amphotericin B selection permit the isolation of cholesterol auxotrophs and might allow the isolation of conditional-lethal mutations. Because LDL can be coupled to ligands that bind to receptors other than the LDL receptor, Mev/LDL/Com-amphotericin B selection may permit the isolation of mutant cells with defects that specifically disrupt other endocytic pathways.
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PMID:Amphotericin B selection of mutant Chinese hamster cells with defects in the receptor-mediated endocytosis of low density lipoprotein and cholesterol biosynthesis. 631 May 83

Fluorescence-activated cell sorting was used to isolate 19 independent, temperature-sensitive, low density lipoprotein (LDL) receptor-deficient Chinese hamster ovary cell mutants that define three recessive complementation groups, ldlE, ldlF, ldlG. LDL receptor activity, essentially normal at the permissive temperature (34 degrees C), was virtually abolished in the mutants after incubation for 8-12 h at the nonpermissive temperature (39-40.5 degrees C). The mutants died after incubation for > 24 h at 39.5 degrees C. These mutants exhibited two striking and unexpected abnormalities that suggest that they define three genes important for general vesicular membrane traffic. First, LDL receptors were degraded abnormally rapidly at the nonpermissive temperature (chloroquine inhibited this degradation in ldlE and ldlG, but not in ldlF). In ldlE cells, the rapid degradation did not require efficient receptor clustering into coated pits and was not observed for all cell surface proteins. This selective degradation may be due to endocytic missorting. Second, the mutants exhibited temperature-sensitive defects in the posttranslational processing and intracellular transport of many membrane-associated and secreted proteins, including the LDL, mannose 6-phosphate/insulin-like growth factor II, and scavenger receptors, the vesicular stomatitis virus G protein and decay accelerating factor. Although the initial synthesis, folding, and processing of precursor forms of these proteins in the endoplasmic reticulum were apparently normal at the nonpermissive temperature, there was either a delay or a block in oligosaccharide processing associated with endoplasmic reticulum to medial Golgi transport at the nonpermissive temperature. This was accompanied by a dramatic inhibition of total soluble protein secretion. The posttranslational processing defects, the instability of cell surface LDL receptors, and the defective protein secretion exhibited by these mutants suggest that the ldlE-G gene products regulate or participate in reactions that are vital for a variety of secretory and endocytic membrane transport processes. This suggestion is strongly supported by our recent observation that a cDNA encoding a component of the coatomer, epsilon-COP, corrects the mutant phenotypes of ldlF cells (Guo, Q., Vasile, E., and Krieger, M. (1994) J. Cell Biol. 125, 1213-1224). Thus, these mutant cells should prove useful for further genetic and biochemical analysis of the mechanisms underlying intracellular membrane traffic.
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PMID:Isolation of three classes of conditional lethal Chinese hamster ovary cell mutants with temperature-dependent defects in low density lipoprotein receptor stability and intracellular membrane transport. 806 14

Overlapping mechanisms that function simultaneously in the intracellular sorting of mammalian membrane proteins often confound delineation of individual sorting pathways. By analyzing sorting in the evolutionarily simpler organism Toxoplasma gondii, we demonstrate a role for transmembrane domain (TMD) length in modulating the signal-dependent segregation of membrane proteins to distinct intracellular organelles. The dense granule localization of the single pass transmembrane protein GRA4 could be completely rerouted to the Golgi and cell surface simply by replacement of its TMD with that from either vesicular stomatitis virus G or the low density lipoprotein (LDL) receptor. Mutational and biochemical analyses suggested that this effect was not caused by any specific sequence motif or strength of membrane association of the GRA4 TMD. Instead, a property imparted by the vesicular stomatitis virus G or LDL receptor TMDs, both of which are longer than the GRA4 TMD, appeared to be a decisive factor. Indeed, shortening the LDL receptor TMD to a length similar to that of GRA4 resulted in dense granule localization, whereas lengthening the GRA4 TMD resulted in rerouting to the Golgi. From these data, we conclude that although the TMD may not necessarily be a sole determinant in membrane protein sorting, its properties can markedly modulate the utilization of more conventional signal-mediated sorting pathways.
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PMID:Transmembrane domain modulates sorting of membrane proteins in Toxoplasma gondii. 1505 59

The epithelial-specific adaptor AP1B sorts basolateral plasma membrane (PM) proteins in both biosynthetic and recycling routes, but the site where it carries out this function remains incompletely defined. Here, we have investigated this topic in Fischer rat thyroid (FRT) epithelial cells using an antibody against the medium subunit micro1B. This antibody was suitable for immunofluorescence and blocked the function of AP1B in these cells. The antibody blocked the basolateral recycling of two basolateral PM markers, Transferrin receptor (TfR) and LDL receptor (LDLR), in a perinuclear compartment with marker and functional characteristics of recycling endosomes (RE). Live imaging experiments demonstrated that in the presence of the antibody two newly synthesized GFP-tagged basolateral proteins (vesicular stomatitis virus G [VSVG] protein and TfR) exited the trans-Golgi network (TGN) normally but became blocked at the RE within 3-5 min. By contrast, the antibody did not block trafficking of green fluorescent protein (GFP)-LDLR from the TGN to the PM but stopped its recycling after internalization into RE in approximately 45 min. Our experiments conclusively demonstrate that 1) AP1B functions exclusively at RE; 2) TGN-to-RE transport is very fast and selective and is mediated by adaptors different from AP1B; and 3) the TGN and AP1B-containing RE cooperate in biosynthetic basolateral sorting.
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PMID:Antibody to AP1B adaptor blocks biosynthetic and recycling routes of basolateral proteins at recycling endosomes. 1788 25