There is increasing evidence that ubiquitination of receptors provides an important endosomal sorting signal. Here we report that mammalian class E vacuolar protein-sorting (vps) proteins recognize ubiquitin. Both tumor susceptibility gene 101 (TSG101)/human VPS (hVPS)28 and hepatocyte growth factor receptor substrate (Hrs) cytosolic complexes bind ubiquitin-agarose. TSG101 and hVPS28 are localized to endosomes that contain internalized EGF receptor and label strongly for ubiquitinated proteins. Microinjection of anti-hVPS28 specifically retards EGF degradation and leads to endosomal accumulation of ubiquitin–protein conjugates. Likewise, depletion of TSG101 impairs EGF trafficking and causes dramatic relocalization of ubiquitin to endocytic compartments. Similar defects are found in cells overexpressing Hrs, further emphasizing the links between class E protein function, receptor trafficking, and endosomal ubiquitination.

The yeast vacuolar sorting protein Vps4p is an ATPase required for endosomal trafficking that couples membrane association to its ATPase cycle. To investigate the function of mammalian VPS4 in endosomal trafficking, we have transiently expressed wild-type or ATPase-defective human VPS4 (hVPS4) in cultured cells. Wild-type hVPS4 was cytosolic, whereas a substantial fraction of hVPS4 that was unable to either bind or hydrolyze ATP was localized to membranes, including those of specifically induced vacuoles. Vacuoles were exclusively endocytic in origin, and subsets of enlarged vacuoles stained with markers for each stage of the endocytic pathway. Sorting of receptors from the early endosome to the recycling compartment or to the trans-Golgi network was not significantly affected, and no mutant hVPS4 associated with these compartments. However, many hVPS4-induced vacuoles were substantially enriched in cholesterol relative to the endosomal compartments of untransfected cells, indicating that expression of mutant hVPS4 gives rise to a kinetic block in postendosomal cholesterol sorting. The phenotype described here is largely consistent with the defects in vacuolar sorting associated with class E vps mutants in yeast, and a role for mammalian VPS4 is discussed in this context.

Class E vacuolar protein sorting (vps) proteins are required for appropriate sorting of receptors within the yeast endocytic pathway, and most probably function in the biogenesis of multivesicular bodies. We have identified the mammalian orthologue of Vps28p as a 221-amino acid cytosolic protein that interacts with TSG101/ mammalian VPS23 to form part of a multiprotein complex. Co-immunoprecipitation and cross-linking experiments demonstrated that hVPS28 and TSG101 interact directly and that binding requires structural information within the conserved C-terminal portion of TSG101. TSG101 and hVPS28 are predominantly cytosolic. However, when endosomal vacuolization was induced by the expression of a dominant-negative mutant of another class E vps protein, human VPS4, a portion of both TSG101 and hVPS28 translocated to the surface of these vacuoles. We conclude that TSG101 and its interacting components are directly involved in endosomal sorting.

Recent evidence has proved that in addition to the well‐documented clathrin‐mediated endocytic route (vesicles of 100–150 nm), at least three distinct non‐clathrin‐coated endocytic pathways function at the surface of mammalian cells. Endocytosis via these pathways is initiated by caveolae (50–80 nm), macropinosomes (500–2000 nm) and micropinosomes (95–100 nm). The current state of knowledge about these non‐clathrin coated endocytic routes is presented and evidence that endocytic routes other than via clathrin‐coated vesicles are utilised by viruses is discussed. The recent advances in these areas have provided us with tools to investigate the entry of those viruses which appear to enter cells via endocytosis into non‐clathrin‐coated vesicles. Data indicate that these four endocytic pathways differ in the absence, presence and/or type of coat on the vesicles, the size of the vesicles, their sensitivity to a variety of inhibitors, and in the ligands endocytosed. A historical perspective of the discovery of these non‐clathrin‐coated endocytic pathways is provided and recent information is summarised and discussed. The entry of viruses via non‐clathrin‐coated pits is destined to be an exciting new area of viral‐cell entry, as has been indicated recently by the finding that entry of simian virus type 40 into cells occurs via caveolae. © 1997 by John Wiley & Sons, Ltd.

Hepatitis A virus (HAV) replication in BS-C-1 cells was studied in the presence of ten potential uncoating inhibitors. Strong inhibition of HAV replication was only observed in the presence of the phenothiazine compound chlorpromazine and the lysosomotropic agent chloroquine, but not by other lysosomotropic agents. Chlorpromazine and chloroquine were found to prevent virus uncoating. Chlorpromazine is known to inhibit endocytosis of non- clathrin-coated vesicles. Chloroquine is a weak base amine, and thought to inhibit virus replication by preventing endosomal acidification. These results therefore suggest that entry of HAV in BS-C-1 cells does not depend on the low pH encountered in the clathrin-coated endocytic entry pathway. A possible role of calcium ions in mediating viral uncoating is discussed, as calcium ions were found to destabilize HAV particles in vitro.

We have previously shown that Xenopus rabaptin-5 is cleaved in apoptotic extracts, with a concomitant reduction in the ability of these extracts to support endosomal membrane fusion (Cosulich, S. C., Horiuchi, H., Zerial, M., Clarke, P. R., and Woodman, P. G. (1997) EMBO J. 16, 6182-6191). In this report we demonstrate that caspasedependent cleavage is a conserved feature of rabaptin-5. Human rabaptin-5 is cleaved at two sites (HSLD 379 and DESD 438 ) in apoptotic HeLa extracts. Cleavage is effected by caspase-3, since it is prevented when caspase-3 activity is either inhibited by Ac-DEVD-CHO or removed by immunodepletion. Moreover, an identical pattern of cleavage is observed using recombinant caspase-3. The action of caspase-3 is highly selective; neither caspase-2 nor caspase-7 are able to cleave recombinant or cytosolic rabaptin-5. Caspase-dependent cleavage of rabaptin-5 generates two physically separated coiled coilforming domains, the C-terminal of which retains the ability to bind the Rab5 exchange factor rabex-5.Programmed cell death (apoptosis) plays a fundamental role in the development and homeostasis of multicellular organisms (1, 2). The primary feature of apoptosis is rapid engulfment and degradation of dying cells by their neighbors, so that an inflammatory response can be avoided. Since in many cases the engulfing cells are not specialized for phagocytic uptake (3), signals that expedite engulfment and degradation are likely to arise from the apoptotic cell. A critical event during apoptosis is therefore the expression of surface receptors that permit the specific recognition of a dying cell. One such receptor is probably phosphatidylserine, which is translocated from the inner leaflet to the outer leaflet of the plasma membrane during apoptosis (4). There is considerable evidence, however, that other surface moieties, including carbohydrate, form part of the recognition signal (5).In addition to changes at the surface, the changes in cellular function that occur in an apoptotic cell are characterized by a variety of striking morphological and biochemical alterations. These include fragmentation of the nucleus and activation of endonuclease(s) (6, 7), cell shrinkage and fragmentation, and plasma membrane blebbing (8). A further distinguishing feature of apoptotic cells is a loss of organized endomembrane structure; the nuclear envelope is frequently lost, and other recognizable membrane structures such as the Golgi complex are replaced by a disorganized array of vacuoles and vesicles (9, 10). The so-called execution phase of apoptosis is evolutionarily conserved (11), underlining its importance.It is now widely believed that many (although not all) apoptotic changes are linked to activation of a number of conserved cysteine proteases (caspases), which cleave specific substrates involved in key cellular processes (for review see Ref. 12). Caspases themselves are present as proenzymes that are readily cleaved, either autocatalytically (13, 14) or by upstream "activator" caspases (15, 16), during a...

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BackgroundAutism spectrum disorders (ASDs) are frequently occurring disorders diagnosed by deficits in three core functional areas: social skills, communication, and behaviours and/or interests. Mental retardation frequently accompanies the most severe forms of ASDs, while overall ASDs are more commonly diagnosed in males. Most ASDs have a genetic origin and one gene recently implicated in the etiology of autism is the Deleted-In-Autism-1 (DIA1) gene.Methodology/Principal FindingsUsing a bioinformatics-based approach, we have identified a human gene closely related to DIA1, we term DIA1R (DIA1-Related). While DIA1 is autosomal (chromosome 3, position 3q24), DIA1R localizes to the X chromosome at position Xp11.3 and is known to escape X-inactivation. The gene products are of similar size, with DIA1 encoding 430, and DIA1R 433, residues. At the amino acid level, DIA1 and DIA1R are 62% similar overall (28% identical), and both encode signal peptides for targeting to the secretory pathway. Both genes are ubiquitously expressed, including in fetal and adult brain tissue.Conclusions/SignificanceExamination of published literature revealed point mutations in DIA1R are associated with X-linked mental retardation (XLMR) and DIA1R deletion is associated with syndromes with ASD-like traits and/or XLMR. Together, these results support a model where the DIA1 and DIA1R gene products regulate molecular traffic through the cellular secretory pathway or affect the function of secreted factors, and functional deficits cause disorders with ASD-like symptoms and/or mental retardation.