Juvenile mucopolysaccharidosis plus disease caused by a missense mutation in VPS33A.

A rare and fatal disease resembling mucopolysaccharidosis in infants, is caused by impaired intracellular endocytic trafficking due to deficiency of core components of the intracellular membrane-tethering protein complexes, HOPS, and CORVET. Whole exome sequencing identified a novel VPS33A mutation in a patient suffering from a variant form of mucopolysaccharidosis. Electron and confocal microscopy, immunoblotting, and glycosphingolipid trafficking experiments were undertaken to investigate the effects of the mutant VPS33A in patient-derived skin fibroblasts. We describe an attenuated juvenile form of VPS33A-related syndrome-mucopolysaccharidosis plus in a man who is homozygous for a hitherto unknown missense mutation (NM_022916.4: c.599 G>C; NP_075067.2:p. Arg200Pro) in a conserved region of the VPS33A gene. Urinary glycosaminoglycan (GAG) analysis revealed increased heparan, dermatan sulphates, and hyaluronic acid. We showed decreased abundance of VPS33A in patient derived fibroblasts and provided evidence that the p.Arg200Pro mutation leads to destablization of the protein and proteasomal degradation. As in the infantile form of mucopolysaccharidosis plus, the endocytic compartment in the fibroblasts also expanded-a phenomenon accompanied by increased endolysosomal acidification and impaired intracellular glycosphingolipid trafficking. Experimental treatment of the patient's cultured fibroblasts with the proteasome inhibitor, bortezomib, or exposure to an inhibitor of glucosylceramide synthesis, eliglustat, improved glycosphingolipid trafficking. To our knowledge this is the first report of an attenuated juvenile form of VPS33A insufficiency characterized by appreciable residual endosomal-lysosomal trafficking and a milder mucopolysaccharidosis plus than the disease in infants. Our findings expand the proof of concept of redeploying clinically approved drugs for therapeutic exploitation in patients with juvenile as well as infantile forms of mucopolysaccharidosis plus disease.

FCHO controls AP2's initiating role in endocytosis through a PtdIns(4,5)P2-dependent switch.

Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane-localized Fer/Cip4 homology domain-only proteins (FCHO). Here, live-cell enhanced total internal reflection fluorescence-structured illumination microscopy shows that FCHO marks sites of clathrin-coated pit (CCP) initiation, which mature into uniform-sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Epidermal growth factor potential receptor substrate number 15 (Eps15) ring. We dissect the network of interactions between the FCHO interdomain linker and AP2, which concentrates, orients, tethers, and partially destabilizes closed AP2 at the plasma membrane. AP2's subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with phosphatidylinositol 4,5-bisphosphate, clathrin, cargo, and CME accessory factors. FCHO can now relocate toward a CCP's outer edge to engage and activate further AP2s to drive CCP growth/maturation.

Architecture of the AP2/clathrin coat on the membranes of clathrin-coated vesicles.

Clathrin-mediated endocytosis (CME) is crucial for modulating the protein composition of a cell's plasma membrane. Clathrin forms a cage-like, polyhedral outer scaffold around a vesicle, to which cargo-selecting clathrin adaptors are attached. Adaptor protein complex (AP2) is the key adaptor in CME. Crystallography has shown AP2 to adopt a range of conformations. Here, we used cryo-electron microscopy, tomography, and subtomogram averaging to determine structures, interactions, and arrangements of clathrin and AP2 at the key steps of coat assembly, from AP2 in solution to membrane-assembled clathrin-coated vesicles (CCVs). AP2 binds cargo and PtdIns(4,5)P 2 (phosphatidylinositol 4,5-bisphosphate)-containing membranes via multiple interfaces, undergoing conformational rearrangement from its cytosolic state. The binding mode of AP2 ß2 appendage into the clathrin lattice in CCVs and buds implies how the adaptor structurally modulates coat curvature and coat disassembly.

On the structure of the stator of the mitochondrial ATP synthase.

The structure of most of the peripheral stalk, or stator, of the F-ATPase from bovine mitochondria, determined at 2.8 A resolution, contains residues 79-183, 3-123 and 5-70 of subunits b, d and F6, respectively. It consists of a continuous curved alpha-helix about 160 A long in the single b-subunit, augmented by the predominantly alpha-helical d- and F6-subunits. The structure occupies most of the peripheral stalk in a low-resolution structure of the F-ATPase. The long helix in subunit b extends from near to the top of the F1 domain to the surface of the membrane domain, and it probably continues unbroken across the membrane. Its uppermost region interacts with the oligomycin sensitivity conferral protein, bound to the N-terminal region of one alpha-subunit in the F1 domain. Various features suggest that the peripheral stalk is probably rigid rather than resembling a flexible rope. It remains unclear whether the transient storage of energy required by the rotary mechanism takes place in the central stalk or in the peripheral stalk or in both domains.

The expression, purification, crystallization and preliminary X-ray analysis of a subcomplex of the peripheral stalk of ATP synthase from bovine mitochondria.

A subcomplex of the peripheral stalk or stator domain of the ATP synthase from bovine mitochondria has been expressed to high levels in a soluble form in Escherichia coli. The subcomplex consists of residues 79-184 of subunit b, residues 1-124 of subunit d and the entire F6 subunit (76 residues). It has been purified and crystallized by vapour diffusion. The morphology and diffraction properties of the crystals of the subcomplex were improved by the presence of thioxane or 4-methylpyridine in the crystallization liquor. With a synchrotron-radiation source, these crystals diffracted to 2.8 A resolution. They belong to the monoclinic space group P2(1).

The peripheral stalk of the mitochondrial ATP synthase.

The peripheral stalk of F-ATPases is an essential component of these enzymes. It extends from the membrane distal point of the F1 catalytic domain along the surface of the F1 domain with subunit a in the membrane domain. Then, it reaches down some 45 A to the membrane surface, and traverses the membrane, where it is associated with the a-subunit. Its role is to act as a stator to hold the catalytic alpha3beta3 subcomplex and the a-subunit static relative to the rotary element of the enzyme, which consists of the c-ring in the membrane and the attached central stalk. The central stalk extends up about 45 A from the membrane surface and then penetrates into the alpha3beta3 subcomplex along its central axis. The mitochondrial peripheral stalk is an assembly of single copies of the oligomycin sensitivity conferral protein (the OSCP) and subunits b, d and F6. In the F-ATPase in Escherichia coli, its composition is simpler, and it consists of a single copy of the delta-subunit with two copies of subunit b. In some bacteria and in chloroplasts, the two copies of subunit b are replaced by single copies of the related proteins b and b' (known as subunits I and II in chloroplasts). As summarized in this review, considerable progress has been made towards establishing the structure and biophysical properties of the peripheral stalk in both the mitochondrial and bacterial enzymes. However, key issues are unresolved, and so our understanding of the role of the peripheral stalk and the mechanism of synthesis of ATP are incomplete.

ATP synthase from Saccharomyces cerevisiae: location of subunit h in the peripheral stalk region.

Subunit h is a component of the peripheral stalk region of ATP synthase from Saccharomyces cerevisiae. It is weakly homologous to subunit F6 in the bovine enzyme, and F6 can replace the function of subunit h in a yeast strain from which the gene for subunit h has been deleted. The removal of subunit h (or F6) uncouples ATP synthesis from the proton motive force. A biotinylation signal has been introduced following the C terminus of subunit h. It becomes biotinylated in vivo, and allows avidin to be bound quantitatively to the purified enzyme complex in vitro. By electron microscopy of the ATP synthase-avidin complex in negative stain and by subsequent image analysis, the C terminus of subunit h has been located in a region of the peripheral stalk that is close to the Fo membrane domain of ATP synthase. Models of the peripheral stalk are proposed that are consistent with this location and with reconstitution experiments conducted with isolated peripheral stalk subunits.