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Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface.

Cell

Acid Phosphatase, analysis, Phosphatidylinositols, Membrane Proteins, Membrane Lipids, chemistry, Liposomes, Golgi Apparatus, Glycosylphosphatidylinositols, Glycolipids, Dogs, Cell Membrane, Cell Line, Cell Compartmentation, Animals

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      Abstract

      We show that a protein with a glycosylphosphatidyl inositol (GPI) anchor can be recovered from lysates of epithelial cells in a low density, detergent-insoluble form. Under these conditions, the protein is associated with detergent-resistant sheets and vesicles that contain other GPI-anchored proteins and are enriched in glycosphingolipids, but do not contain a basolateral marker protein. The protein is recovered in this complex only after it has been transported to the Golgi complex, suggesting that protein-sphingolipid microdomains form in the Golgi apparatus and plasma membrane and supporting the model proposed by Simons and colleagues for sorting of certain membrane proteins to the apical surface after intracellular association with glycosphingolipids.

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      Assembly of asparagine-linked oligosaccharides.

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        Isolation of intracellular membranes by means of sodium carbonate treatment: application to endoplasmic reticulum

        A rapid and simple method for the isolation of membranes from subcellular organelles is described. The procedure consists of diluting the organelles in ice-cold 100 mM Na2CO3 followed by centrifugation to pellet the membranes. Closed vesicles are converted to open membrane sheets, and content proteins and peripheral membrane proteins are released in soluble form. Here we document the method by applying it to various subfractions of a rat liver microsomal fraction, prepared by continuous density gradient centrifugation according to Beaufay et al. (1974, J. Cell Biol. 61:213-231). The results confirm and extend those of previous investigators on the distribution of enzymes and proteins among the membranes of the smooth and rough endoplasmic reticulum. In the accompanying paper (1982, J. Cell Biol. 93:103-110) the procedure is applied to peroxisomes and mitochondria.
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          The role of the cell adhesion molecule uvomorulin in the formation and maintenance of the epithelial junctional complex

          The role of the epithelial adhesion molecule uvomorulin in the formation of the epithelial junctional complex in the Madin-Darby canine kidney (MDCK) cell line was investigated. Experiments were carried out to determine whether specific inhibition of uvomorulin function would interfere selectively with the formation, stability, or function of the apical zonula adherens (ZA) and zonula occludens (ZO), or whether it would interfere with all forms of intercellular contact including the desmosomes. The effects of blocking antibodies and Fab fragments to uvomorulin on the formation of the junctional complex was examined with a Ca2+ switch assay for de novo junction assembly. The formation of the ZO, the ZA, and the desmosomes was assayed by fluorescence staining with an antibody to the tight junction-specific protein ZO-1, with rhodamine-phalloidin for ZA-associated actin filaments, and with an anti-desmoplakin antibody, respectively. Under different conditions and times of antibody treatment the extent of inhibition of the formation of each of the junctional elements was very similar. The ability of the cells to eventually overcome the inhibitory effect of the antibodies and form junctions correlated with the reappearance of uvomorulin at the regions of cell-cell contact. Therefore uvomorulin seems to mediate an early adhesion event between epithelial cells that is a prerequisite for the assembly of all elements of the junctional complex. In contrast, the transepithelial electrical resistance of confluent, well-established monolayers of MDCK cells grown on filters was not greatly affected by treatment with the various antibodies or Fab fragments. A small transient decrease in resistance observed with the polyclonal alpha-uvomorulin IgG may be due to a more subtle modulation of the junctional complex.
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            1531449

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