The mammalian retromer complex consists of SNX1, SNX2, Vps26, Vps29, and Vps35, and retrieves lysosomal enzyme receptors from endosomes to the trans-Golgi network. The structure of human Vps26A at 2.1Å resolution reveals two curvedβ -sandwich domains connected by a polar core and a flexible linker. Vps26 has an unexpected structural relationship to arrestins. The Vps35-binding site on Vps26 maps to a mobile loop spanning residues 235-246, near the tip of the C-terminal domain. The loop is phylogenetically conserved and provides a mechanism for Vps26 integration into the complex that leaves the rest of the structure free for engagements with membranes and for conformational changes. Hydrophobic residues and a Gly in this loop are required for integration into the retromer complex and endosomal localization of human Vps26, and for the function of yeast Vps26 in carboxypeptidase Y sorting.The biosynthetic sorting of acid hydrolase precursors from the trans-Golgi network (TGN) to the endosomal-lysosomal system is central to the biogenesis of lysosomes in metazoans. In mammals, this sorting is directed by binding of mannose 6-phosphate groups on the hydrolases to two transmembrane receptors known as the cation-dependent and cation-independent mannose 6-phosphate receptors (CD-MPR and CI-MPR, respectively) 1 . At the TGN, the hydrolase-receptor complexes are packaged into carrier vesicles by several coat and adaptor proteins, including clathrin, AP-1 (adaptor protein 1) and GGA (Golgi-localized, gamma earcontaining, ADP ribosylation factor-binding) proteins 1,2 . These carrier vesicles bud from the TGN and fuse with endosomes. Within the endosome, the acid pH in the lumen triggers the release of the hydrolases from their receptors. The hydrolases are carried with the fluid phase to lysosomes, while the receptors return to the TGN to be reutilized in further rounds of sorting. Several proteins and complexes, including AP-1 3,4 , TIP47 (tail-interacting protein of 47 kDa) 5 and PACS-1 (phosphofurin acidic cluster sorting protein) 6 have been implicated in the transport of MPRs from endosomes to the TGN.A similar process has been described in yeast cells for the sorting of acid hydrolases to the vacuole, which is the fungal equivalent of the mammalian lysosome. Carboxypeptidase Y (CPY) is sorted by the Vps10 transmembrane receptor. Genetic screens in yeast have identified more than 60 Vps (vacuolar protein sorting) gene products 7 , which are involved in the transport of CPY to the vacuole. Five yeast Vps proteins, Vps5, Vps17, Vps26, Vps29 and Vps35, form a complex named "retromer" that is required for Vps10 sorting from endosomes to the late-5 To whom correspondence should be addressed: James H. Hurley, (301) 402-4703, fax (301) A homologous retromer complex consisting of five subunits termed SNX1, SNX2, Vps26, Vps29 and Vps35 has been described in humans 8 . Depletion of some of these subunits in vivo by RNA interference (RNAi) impairs retrieval of the CI-MPR and results in its missorting to lysosomes, where...

Niemann-Pick C1 protein (NPC1) is a late-endosomal membrane protein involved in trafficking of LDL-derived cholesterol, Niemann-Pick disease type C, and Ebola virus infection. NPC1 contains 13 transmembrane segments (TMs), five of which are thought to represent a "sterol-sensing domain" (SSD). Although present also in other key regulatory proteins of cholesterol biosynthesis, uptake, and signaling, the structure and mechanism of action of the SSD are unknown. Here we report a crystal structure of a large fragment of human NPC1 at 3.6 Å resolution, which reveals internal twofold pseudosymmetry along TM 2-13 and two structurally homologous domains that protrude 60 Å into the endosomal lumen. Strikingly, NPC1's SSD forms a cavity that is accessible from both the luminal bilayer leaflet and the endosomal lumen; computational modeling suggests that this cavity is large enough to accommodate one cholesterol molecule. We propose a model for NPC1 function in cholesterol sensing and transport.endosomal membrane | cholesterol traffic | sterol-sensing domain | crystal structure | allostery C holesterol is a critical component of cellular membranes, and it is either synthesized de novo or supplied from the diet. Although amphiphilic, cholesterol is only poorly soluble in water. Therefore, cholesterol associates with soluble proteins for transport between compartments (1), either as a single molecule or in the form of large lipoprotein particles (2). Cholesterol also functions as a covalently attached ligand in hedgehog-mediated signal transduction (3). Not surprisingly, many proteins involved in cholesterol biosynthesis, transport, or signaling pathways are polytopic, integral membrane proteins (4). Structures of the critical transmembrane region of these polytopic membrane proteins have thus far not been determined, except for an NADPH-dependent reductase in the cholesterol biosynthetic pathway (5), the structure of which yielded insights into the mechanism of intramembrane catalysis.A subgroup of the polytopic integral membrane proteins of cholesterol-related pathways shares a highly conserved region comprised of five transmembrane segments (TMs) that are thought to represent a key regulatory element in response to cholesterol in the bilayer (6). This transmembrane region has been termed the "sterol-sensing domain" (SSD) (7,8). Because crystal structures of these SSDs have not been determined, it has remained unclear precisely how an SSD detects cholesterol in the bilayer and conveys this information to the rest of the protein to influence its activity, stability, or trafficking.Niemann-Pick C1 protein (NPC1) is an SSD-containing, ubiquitous, cholesterol-trafficking protein in the cholesterol uptake pathway (9). Cholesterol is transported throughout the body as cholesterol esters that are packaged into lipoprotein particles including "low-density lipoprotein" (LDL) (2, 10). LDL is endocytosed and transported to late endosomes and lysosomes, where the ∼25-nm particle is subject to lipolysis by lysosomal acid lipase (11,12)...

Pre-mRNA splicing requires the function of a number of RNAdependent ATPases͞helicases, yet no three-dimensional structure of any spliceosomal ATPases͞helicases is known. The highly conserved DECD-box protein UAP56͞Sub2 is an essential splicing factor that is also important for mRNA export. The expected ATPase͞helicase activity appears to be essential for the UAP56͞ Sub2 functions. Here, we show that purified human UAP56 is an active RNA-dependent ATPase, and we also report the crystal structures of UAP56 alone and in complex with ADP, as well as a DECD to DEAD mutant. The structures reveal a unique spatial arrangement of the two conserved helicase domains, and ADPbinding induces significant conformational changes of key residues in the ATP-binding pocket. Our structural analyses suggest a specific protein-RNA displacement model of UAP56͞Sub2. The detailed structural information provides important mechanistic insights into the splicing function of UAP56͞Sub2. The structures also will be useful for the analysis of other spliceosomal DExD-box ATPases͞helicases.helicase ͉ RNA processing ͉ export ͉ crystallography T he spliceosome is a complex molecular machine responsible for the removal of noncoding introns and the joining of exons. Pre-mRNA splicing also plays important roles in subsequent cellular processes such as mRNA export, translation, and mRNA degradation by altering the composition of ribonucleoprotein complexes assembled on spliced mRNAs (1, 2). A number of ATP-using enzymes containing the characteristic DExD sequence motif are required for the assembly, remodeling, and disassembly of the spliceosome (3, 4).Human UAP56 (56-kDa U2AF-associated protein) and its yeast homolog Sub2 are essential DECD-box splicing factors (5-8). UAP56 is required for the association of U2 small nuclear ribonucleoprotein with pre-mRNA (5), and Sub2 has been implicated in both ATP-independent and -dependent steps of prespliceosome assembly (6, 7). Interestingly, deletion of Mud2, the yeast homolog of U2AF65, can bypass the requirement of Sub2 (6). It has been proposed that splicing may occur through Sub2-Mud2 dependent and Sub2-Mud2 independent pathways. A possible function of Sub2 is to displace Mud2 and͞or SF1, a branchpoint binding protein, from pre-mRNA before the binding of U2 small nuclear ribonucleoprotein. The functions of Sub2 require intact ATPase͞helicase motifs, suggesting that ATP hydrolysis is essential for its activities (8).UAP56͞Sub2 also plays important roles in the export of mRNA from the nucleus to the cytoplasm (9-12). Reduction of cellular UAP56 levels by RNA interference in Drosophila or Caenorhabditis elegans resulted in the retention of significant fractions of mRNAs in the nucleus (13-15). UAP56͞Sub2 couples transcription to mRNA export in the context of a transcription-export (TREX) complex, which is recruited to activated genes and travels the entire length of the gene with RNA polymerase II during transcription (16).Here, we report the crystal structures of UAP56 alone and in complex with ADP as well...