As sequencing of the human genome nears completion, the genes that cause many human diseases are being identified and functionally described. This has revealed that many human diseases are due to defects of intracellular trafficking. This ‘Toolbox’ catalogs and briefly describes these diseases.

As more details emerge on the mechanisms that mediate and control intracellular transport, the molecular basis for variety of human diseases has been revealed. In turn, disease pathology and physiology shed light on the intricate controls that regulate intracellular transport to assure proper cellular and tissue function and

Clathrin-coated vesicles (CCV) mediate protein sorting and vesicular trafficking from the plasma membrane and the trans-Golgi network. Before delivery of the vesicle contents to the target organelles, the coat components, clathrin and adaptor protein complexes (APs), must be released. Previous work has established that hsc70/the uncoating ATPase mediates clathrin release in vitro without the release of APs. AP release has not been reconstituted in vitro, and nothing is known about the requirements for this reaction. We report a novel quantitative assay for the ATP-and cytosol-dependent release of APs from CCV. As expected, hsc70 is not sufficient for AP release; however, immunodepletion and reconstitution experiments establish that it is necessary. Interestingly, complete clathrin release is not a prerequisite for AP release, suggesting that hsc70 plays a dual role in recycling the constituents of the clathrin coat. This assay provides a functional basis for identification of the additional cytosolic factor(s) required for AP release. INTRODUCTIONMost, if not all, intracellular transport vesicles are encased in a proteinaceous coat, one class of which is clathrin-coated vesicles (CCV). 1 CCV mediate the transport of lysosomal hydrolases from the transGolgi network, as well as the efficient internalization of extracellular solutes such as nutrients, hormones, growth factors, and immunoglobulins at the plasma membrane. Their coat contains two components: clathrin (heavy and light chains) and adaptor protein complexes (AP). There are two distinct classes of CCVassociated APs. The AP1 complex is found on transGolgi network-derived CCV, and AP2 is found on plasma membrane-derived CCV (Robinson, 1987;Ahle et al., 1988). More recently, a new AP complex AP3 has been identified that is partially associated with clathrin-coated structures in vivo (Simpson et al., 1997;Dell' Angelica et al., 1998). Both clathrin-associated AP complexes are heterotetramers consisting of two different subunits of ϳ100 kDa (␥ and ␤1 in AP1, ␣ and ␤2 in AP2), one medium chain subunit (1 or 2) of ϳ50 kDa, and a small chain subunit (1 or 2) of ϳ17 kDa (reviewed by Robinson, 1994;Kirchhausen et al., 1997;Schmid, 1997).A fully assembled clathrin coat, viewed by electron microscopy, appears as a polygonal lattice of clathrin on the outer surface of the vesicle, with APs located at each vertex (Vigers et al., 1986a(Vigers et al., , 1986bHeuser and Keen, 1988). The APs form the innermost shell of the coat and associate with the plasma membrane via saturable, high-affinity, and protease-sensitive receptors (Mahaffey et al., 1990). In low-ionic strength, low-pH buffers, clathrin can spontaneously self-assemble into a closed polygonal lattice (Kartenbeck, 1978;Woodward and Roth, 1978;Crowther and Pearse, 1981). APs coassemble with clathrin and promote coat assembly under physiological conditions that do not otherwise support clathrin self-assembly (Keen et al., 1979;Pearse and Robinson, 1984;Ahle and Ungewickell, 1989). Given the ability of APs to stimu...

Abstract. Glycosyl-phosphatidylinositol (GPI)-anchored proteins are sorted to the apical surface of many epithelial cell types. To better understand the mechanism for apical segregation of these proteins, we analyzed the lateral mobility and molecular associations of a model GPI-anchored protein, herpes simplex virus gD1 fused to human decay accelerating factor (gD1-DAF) (Lisanti, M. P., I. W. Caras, M. A. Davitz, and E. . J. Cell Biol. 109:2145-2156 shortly after arrival and after longterm residence at the surface of confluent, polarized MDCK cells. FRAP measurements of lateral diffusion showed that the mobile fraction of newly arrived gD1-DAF molecules was much less than the mobile fraction of long-term resident molecules (40 vs. 80-90%). Fluorescence resonance energy transfer measurements showed that the newly arrived molecules were clustered, while resident molecules were not. Newly delivered gD1-DAF molecules were clustered but not immobilized in mutant, Concanavalin A-resistant MDCK cells that failed to sort gD1-DAE Our results indicate that GPI-anchored proteins in MDCK cells are clustered before delivery to the surface. However, clustering alone does not target molecules for apical delivery. The immobilization observed when gD1-DAF is correctly sorted suggests that the clusters must associate some component of the cell's cytoplasm.

Abstract. Glycosylphosphatidylinositol-anchored pro-teins, GPI-proteins, are selectively delivered to the apical surfaces of many types of morphologically polarized epithelial cells. It has been proposed that the unit for targeting GPI-proteins to the apical surface is a membrane lipid domain. This sorting domain or molecular cluster has been equated to detergent (Triton X-100)-insoluble membrane fractions that are enriched in enriched in GPI-proteins, glycosphingolipids, and cholesterol. To determine the role of cholesterol in the formation of sorting domains and to examine its importance in the intracellular traffic and membrane polarity of GPI-proteins, we studied the behavior of a model GPIprotein, gD1-DAF, in MDCK cells cultured for 3 or 14 d without their principal source of cholesterol, serum LDL. LDL deprivation affects the intracellular traffic of gD1-DAF. Surface expression of gD1-DAF is reduced in LDL-deprived cells; this reduction is most marked after 3 d of LDL deprivation. We also find a great reduction in the fraction of gD1-DAF that is detergent-insoluble in these cells and a change in its membrane milieu defined by susceptibility to cleavage with PI-specific phospholipase C. Despite these changes, the surface polarity of gD1-DAF is no different in LDLdeprived cells than in control cells.