Sphingolipid and cholesterol-rich Triton X-100-insoluble membrane fragments (detergent-resistant membranes, DRMs) containing lipids in a state similar to the liquid-ordered phase can be isolated from mammalian cells, and probably exist as discrete domains or rafts in intact membranes. We postulated that proteins with a high affinity for such an ordered lipid environment might be targeted to rafts. Saturated acyl chains should prefer an extended conformation that would fit well in rafts. In contrast, prenyl groups, which are as hydrophobic as acyl chains but have a branched and bulky structure, should be excluded from rafts. Here, we showed that at least half of the proteins in Increasing evidence suggests that cholesterol and sphingolipid-rich lipid microdomains or rafts exist in eukaryotic cell membranes and have important functions there (1-3). These rafts are likely to be important in the structure and function of caveolae, plasma membrane invaginations that are implicated in signal transduction (4, 5), endocytosis (6), transcytosis across endothelial cells (7,8), and cholesterol trafficking (9 -11). However, rafts are not restricted to caveolae (2, 3, 12) and recent evidence suggests that they act in signal transduction in cells that lack distinct caveolae, such as T lymphocytes (13-16) and basophils (17)(18)(19). Rafts have also been implicated in protein and lipid sorting in the secretory and endocytic pathways (1, 20 -22).Cholesterol and sphingolipid-rich detergent-resistant membranes (DRMs) 1 can be isolated from mammalian cells (23). DRM lipids are in a state similar to the liquid-ordered (l o ) phase (3, 24 -26). The l o phase, which requires cholesterol to form, is favored by lipids like sphingolipids, whose long saturated acyl chains give them a high degree of order and a high acyl-chain melting temperature (3). Acyl chain order explains the detergent-insolubility of DRMs (3). We hypothesize that DRMs are an in vitro correlate of rafts in intact membranes. It is important to note that detergent insolubility can underestimate the association of proteins and lipids with the l o phase; some proteins and lipids that are in rafts can be solubilized (25). Nevertheless, DRM association provides a powerful tool for identifying molecules that are likely to have a high affinity for rafts. DRMs isolated from cells contain a number of proteins (27-29) which are undoubtedly crucial for the function of the domains in vivo. For this reason, it is important to determine how proteins associate with DRMs. Three DRM targeting signals have been defined. First, glycosylphosphatidylinositol (GPI)-anchored proteins are targeted to DRMs through acyl chain interactions (23)(24)(25)30). An N-terminal Met-Gly-Cys motif that is present in some Src family kinases and heterotrimeric G protein ␣ subunits, in which Gly is myristoylated and Cys is palmitoylated, can also serve as a DRM targeting signal (31,32). Third, dual palmitoylated Cys residues are required for raft association of the T cell adaptor protein LAT (15) and the neu...

The multisubunit protein, dynactin, is a critical component of the cytoplasmic dynein motor machinery. Dynactin contains two distinct structural domains: a projecting sidearm that interacts with dynein and an actin-like minifilament backbone that is thought to bind cargo. Here, we use biochemical, ultrastructural, and molecular cloning techniques to obtain a comprehensive picture of dynactin composition and structure. Treatment of purified dynactin with recombinant dynamitin yields two assemblies: the actin-related protein, Arp1, minifilament and the p150Glued sidearm. Both contain dynamitin. Treatment of dynactin with the chaotropic salt, potassium iodide, completely depolymerizes the Arp1 minifilament to reveal multiple protein complexes that contain the remaining dynactin subunits. The shoulder/sidearm complex contains p150Glued, dynamitin, and p24 subunits and is ultrastructurally similar to dynactin's flexible projecting sidearm. The dynactin shoulder complex, which contains dynamitin and p24, is an elongated, flexible assembly that may link the shoulder/sidearm complex to the Arp1 minifilament. Pointed-end complex contains p62, p27, and p25 subunits, plus a novel actin-related protein, Arp11. p62, p27, and p25 contain predicted cargo-binding motifs, while the Arp11 sequence suggests a pointed-end capping activity. These isolated dynactin subdomains will be useful tools for further analysis of dynactin assembly and function.

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We previously isolated detergent-resistant membrane complexes (DRMs) that were not solubilized after extraction of Madin-Darby canine kidney cells with Triton X-100 on ice. The complexes were rich in glycosphingolipids, cholesterol, and glycosylphosphatidylinositol (GPI)-anchored proteins. In this study, we examined the protein composition of DRMs and further characterized the detergent solubility of these structures. Eight to ten cell-surface proteins, including proteins from both apical and basolateral membranes, were recovered in DRMs. Most DRM proteins, however, were not exposed to the surface of whole cells, and we did not detect the complex of cell-surface proteins described by Sargiacomo et al. in a similar study [Sargiacomo, M., et al. (1993) J. Cell Biol. 122, 789-807]. Almost all proteins in DRMs were solubilized by Triton X-100 at temperatures above 30 degrees C or by octyl glucoside on ice. In contrast, a GPI-anchored protein, placental alkaline phosphatase, was mostly solubilized by Triton X-100 after extraction at 10 degrees C. This protein was insoluble in ice-cold Triton X-100 when first delivered to the plasma membrane and remained so for at least 6 h after synthesis. A fraction of the lipids in DRMs remained insoluble after extraction with Triton X-100 at 37 degrees C. DRM lipids were not solubilized by octyl glucoside, suggesting that this detergent selectively extracts proteins from DRMs.

Dynamitin is a commonly used inhibitor of cytoplasmic dynein-based motility in living cells. Dynamitin does not inhibit dynein directly but instead acts by causing disassembly of dynactin, a multiprotein complex required for dynein-based movement. In dynactin, dynamitin is closely associated with the subunits p150Glued and p24, which together form the shoulder and projecting arm structures of the dynactin molecule. In this study, we explore the way in which exogenous dynamitin effects dynactin disruption. We find that pure, recombinant dynamitin is an elongated protein with a strong propensity for self-assembly. Titration experiments reveal that free dynamitin binds dynactin before it causes release of subunits. When dynamitin is added to dynactin at an equimolar ratio of exogenous dynamitin subunits to endogenous dynamitin subunits (1؋ ‫؍‬ 4 mol of exogenous dynamitin per mole of dynactin), exogenous dynamitin exchanges with endogenous dynamitin, and partial release of p150Glued is observed. When added in vast excess (>25؋; 100 mol of exogenous dynamitin per mole of dynactin), recombinant dynamitin causes complete release of both p150Glued subunits, two dynamitins and one p24, but not other dynactin subunits. Our data suggest that dynamitin mediates disruption of dynactin by binding to endogenous dynamitin subunits. This binding destabilizes the shoulder structure that links the p150 Glued arm to the Arp1 filament and leads to subunit release.