Heteromeric amino acid transporters (HATs) comprise a group of membrane proteins that belong to the solute carrier (SLC) superfamily. They are formed by two different protein components: a light chain subunit from an SLC7 family member and a heavy chain subunit from the SLC3 family. The light chain constitutes the transport subunit whereas the heavy chain mediates trafficking to the plasma membrane and maturation of the functional complex. Mutation, malfunction, and dysregulation of HATs are associated with a wide range of pathologies or represent the direct cause of inherited and acquired disorders. Here we report the cryogenic electron microscopy structure of the neutral and basic amino acid transport complex (b[0,+]AT1-rBAT) which reveals a heterotetrameric protein assembly composed of two heavy and light chain subunits, respectively. The previously uncharacterized interaction between two HAT units is mediated via dimerization of the heavy chain subunits and does not include participation of the light chain subunits. The b(0,+)AT1 transporter adopts a LeuT fold and is captured in an inward-facing conformation. We identify an amino-acid-binding pocket that is formed by transmembrane helices 1, 6, and 10 and conserved among SLC7 transporters.

Plant growth and architecture is regulated by the polar distribution of the hormone auxin. Polarity and flexibility of this process is provided by constant cycling of auxin transporter vesicles along actin filaments, coordinated by a positive auxin-actin feedback loop. Both polar auxin transport and vesicle cycling are inhibited by synthetic auxin transport inhibitors, such as 1-N-naphthylphthalamic acid (NPA), counteracting the effect of auxin; however, underlying targets and mechanisms are unclear. Using NMR, we map the NPA binding surface on the Arabidopsis thaliana ABCB chaperone TWISTED DWARF1 (TWD1). We identify ACTIN7 as a relevant, although likely indirect, TWD1 interactor, and show TWD1-dependent regulation of actin filament organization and dynamics and that TWD1 is required for NPA-mediated actin cytoskeleton remodeling. The TWD1-ACTIN7 axis controls plasma membrane presence of efflux transporters, and as a consequence act7 and twd1 share developmental and physiological phenotypes indicative of defects in auxin transport. These can be phenocopied by NPA treatment or by chemical actin (de)stabilization. We provide evidence that TWD1 determines downstream locations of auxin efflux transporters by adjusting actin filament debundling and dynamizing processes and mediating NPA action on the latter. This function appears to be evolutionary conserved since TWD1 expression in budding yeast alters actin polarization and cell polarity and provides NPA sensitivity.

ATG101 is an essential component of the ULK complex responsible for initiating cellular autophagy in mammalian cells; its 3-dimensional structure and molecular function, however, are currently unclear. Here we present the X-ray structure of human ATG101. The protein displays an open HORMA domain fold. Both structural properties and biophysical evidence indicate that ATG101 is locked in this conformation, in contrast to the prototypical HORMA domain protein MAD2. Moreover, we discuss a potential mode of dimerization with ATG13 as a fundamental aspect of ATG101 function.

BACKGROUND:Both disrupted-in-schizophrenia 1 (DISC1) and dysbindin have been identified as schizophrenia candidate genes in independent genetic linkage studies. The proteins have been assigned distinct subcellular locations and functions. We investigated whether both proteins converge into a common pathway specific for schizophrenia or mental diseases.METHODS:DISC1 and dysbindin were expressed as recombinant proteins with or without a fluorescent protein-tag in human or mouse neuroblastoma cells and as recombinant proteins in E. coli. Postmortem brains of patients with mental diseases from the Stanley Research Medical Institute's Consortium Collection were used to demonstrate molecular interactions in biochemically purified protein fractions.RESULTS:First, upon overexpression in neuroblastoma cells, DISC1 formed aggresomes that recruited homologous soluble C-terminal DISC1 fragment or heterologous dysbindin. Domains involved in binding could be mapped to DISC1 (316-597) and dysbindin (82-173), indicating a specific interaction. In addition, recruitment was demonstrated when externally added, purified DISC1 aggresomes penetrated recipient cells after coincubation. Second, a direct interaction between soluble DISC1 protein and dysbindin was demonstrated in a cell free system using E. coli-expressed proteins. Third, co-aggregation of DISC1 and dysbindin was demonstrated in postmortem brains for a subgroup of cases with chronic mental disease but not healthy control subjects.CONCLUSIONS:A direct interaction of soluble and insoluble DISC1 protein with dysbindin protein demonstrates convergence of so far considered independent mental disease genes by direct molecular interaction. Our findings highlight protein aggregation and recruitment as a biological mechanism in mental disease.

Genetic studies have established a role of disrupted-in-schizophrenia-1 (DISC1) in chronic mental diseases (CMD). Limited experimental data are available on the domain structure of the DISC1 protein although multiple interaction partners are known including a self-association domain within the middle part of DISC1 (residues 403-504). The DISC1 C-terminal domain is deleted in the original Scottish pedigree where DISC1 harbors two coiled-coil domains and disease-associated polymorphisms at 607 and 704, as well as the important nuclear distribution element-like 1 (NDEL1) binding site at residues 802-839. Here, we performed mutagenesis studies of the C-terminal domain of the DISC1 protein (residues 640-854) and analyzed the expressed constructs by biochemical and biophysical methods. We identified novel DISC1 self-association motifs and the necessity of their concerted action for orderly assembly: the region 765-854 comprising a coiled-coil domain is a dimerization domain and the region 668-747 an oligomerization domain; dimerization was found to be a prerequisite for orderly assembly of oligomers. Consistent with this, disease-associated polymorphism C704 displayed a slightly higher oligomerization propensity. The heterogeneity of DISC1 multimers in vitro was confirmed with a monoclonal antibody binding exclusively to HMW multimers. We also identified C-terminal DISC1 fragments in human brains, suggesting that C-terminal fragments could carry out DISC1-dependent functions. When the DISC1 C-terminal domain was transiently expressed in cells, it assembled into a range of soluble and insoluble multimers with distinct fractions selectively binding NDEL1, indicating functionality. Our results suggest that assembly of the C-terminal domain is controlled by distinct domains including the disease-associated polymorphism 704 and is functional in vivo.