O-Linked N-acetylglucosamine (O-GlcNAc O-Linked N-acetylglucosamine (O-GlcNAc)1 is a major form of post-translational modification found on nuclear and cytoplasmic proteins (1-3). It consists of the monosaccharide Nacetylglucosamine linked to the hydroxyl group of either serine or threonine. Well over 50 proteins have been identified to date with the modification (1-3), including RNA polymerase II (4), several RNA polymerase II transcription factors (5), nuclear pore proteins (6), the tumor suppressor protein p53 (7), and c-Myc (8). Sites of glycosylation have been identified on a number of O-GlcNAc-modified proteins, and although a strict consensus sequence cannot be discerned, most sites contain a proline residue amino-terminal to the modified serine or threonine (1). About half of the sites contain the motif PX(S/T), where X is valine, serine, or threonine and (S/T) is the modified serine or threonine.Accumulating evidence suggests that O-GlcNAc is a regulated modification much like phosphorylation (1-3). In several instances it has been shown that the sugar turns over more rapidly than the protein it modifies (9, 10), implying that OGlcNAc is dynamically added to and removed from proteins. In addition, conditions of growth stimulation (11) and of growth arrest (12) have been demonstrated to alter the level of OGlcNAc in cells. Enzymes for the addition (O-GlcNAc transferase) (13-16) and removal (O-GlcNAcase) (17) of the sugar are known to exist in the cytoplasm of most eukaryotic cells. Thus, a system capable of dynamic regulated addition and removal of the sugar analogous to a kinase/phosphatase system is present in cells.A major function of O-GlcNAc may be to compete with phosphorylation for sites on proteins (1-3). Since GlcNAc is neutral, it is possible that it would have different effects on protein function than a strongly negatively charged phosphate group. Thus, competition between GlcNAc and phosphate for similar sites would add an extra level of control to signal transduction cascades in cells. Alternatively, O-GlcNAc or phosphate could affect protein activity in a similar manner, although the modifications would be the result of different signaling cascades. In either case, only one modification could occur at an individual site at any one time. Evidence indicating that this type of competition occurs has been obtained by demonstration that the sites of glycosylation on both RNA polymerase II (4) and c-Myc (8) coincide with known phosphorylation sites. Even so, direct modulation of the level of phosphate on a protein by changing the level of glycosylation on the same protein has not yet been demonstrated in vivo.Selective inhibitors of protein kinases and phosphatases have been used extensively to modulate the level of phosphate on proteins as an approach to determine the functional implications of phosphorylation. By analogy to the phosphorylation system, specific inhibitors of either the O-GlcNAcase or OGlcNAc transferase would be valuable tools in the study of the function of the O-GlcNAc modifi...

Recent data suggest that membrane microdomains or rafts that are rich in sphingolipids and cholesterol are important in signal transduction and membrane trafficking. Two models of raft structure have been proposed. One proposes a unique role for glycosphingolipids (GSL), suggesting that GSL-head-group interactions are essential in raft formation. The other model suggests that close packing of the long saturated acyl chains found on both GSL and sphingomyelin plays a key role and helps these lipids form liquid-ordered phase domains in the presence of cholesterol. To distinguish between these models, we compared rafts in the MEB-4 melanoma cell line and its GSL-deficient derivative, GM-95. Rafts were isolated from cell lysates as detergentresistant membranes (DRMs). The two cell lines had very similar DRM protein profiles. The yield of DRM protein was 2-fold higher in the parental than the mutant line, possibly reflecting cytoskeletal differences. The same amount of DRM lipid was isolated from both lines, and the lipid composition was similar except for up-regulation of sphingomyelin in the mutant that compensated for the lack of GSL. DRMs from the two lines had similar fluidity as measured by fluorescence polarization of diphenylhexatriene. Methyl-␤-cyclodextrin removed cholesterol from both cell lines with the same kinetics and to the same extent, and both a raft-associated glycosyl phosphatidylinositol-anchored protein and residual cholesterol showed the same distribution between DRMs and the detergent-soluble fraction after cholesterol removal in both cell lines. Finally, a glycosyl phosphatidylinositol-anchored protein was delivered to the cell surface at similar rates in the two lines, even after cholesterol depletion with methyl-␤-cyclodextrin. We conclude that GSL are not essential for the formation of rafts and do not play a major role in determining their properties.Recent studies suggest that plasma membrane lipids do not always mix homogeneously but that membranes may contain microdomains or rafts that are rich in sphingolipids and cholesterol (1-3). Rafts may be concentrated or stabilized in caveolae but also exist in cells that lack caveolae. Rafts have been proposed play important roles in signal transduction; for instance, recruitment of signaling proteins to rafts in T cells (4 -8) and basophils (9 -11) appears to be required for signaling. Rafts may also play a role in intracellular sorting. For instance, depletion of cholesterol, sphingolipids, or certain raftassociated proteins affects sorting of apical proteins in epithelial cells (12-17) and axonal proteins in neurons (18,19). Recent studies also suggest that rafts play a role in sorting in the endocytic pathway (20,21).Two models for the organization of lipids in rafts have been proposed. The first was developed by Simons and colleagues (1,22,23) as part of a model for sorting of apical and basolateral proteins in the trans-Golgi network of polarized epithelial cells. In this model, clusters of GSL 1 form spontaneously in the trans-Golgi net...

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