ABSTRACT
Asymmetric cell divisions balance stem cell proliferation and differentiation to sustain tissue morphogenesis and homeostasis. During asymmetric divisions, fate determinants and niche contacts segregate unequally between daughters, but little is known on how this is achieved mechanistically. In Drosophila neuroblasts and murine mammary stem cells, the association of the spindle orientation protein LGN with the stem cell adaptor Inscuteable has been connected to asymmetry. Here we report the crystal structure of Drosophila LGN in complex with the asymmetric domain of Inscuteable, which reveals a tetrameric arrangement of intertwined molecules. We show that Insc:LGN tetramers constitute stable cores of Par3-Insc-LGN-GαiGDP complexes, which cannot be dissociated by NuMA. In mammary stem cells, the asymmetric domain of Insc bound to LGN:GαiGDP suffices to drive asymmetric fate, and reverts aberrant symmetric divisions induced by p53 loss. We suggest a novel role for the Insc-bound pool of LGN acting independently of microtubule motors to promote asymmetric fate specification.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Asymmetric Cell Division , Cytoskeletal Proteins/metabolism , Drosophila Proteins/metabolism , Drosophila/cytology , Drosophila/metabolism , Guanine Nucleotide Dissociation Inhibitors/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , Stem Cells/cytology , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/genetics , Animals , Cell Cycle Proteins , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , Drosophila/chemistry , Drosophila/genetics , Drosophila Proteins/chemistry , Drosophila Proteins/genetics , Guanine Nucleotide Dissociation Inhibitors/chemistry , Guanine Nucleotide Dissociation Inhibitors/genetics , Humans , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/genetics , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Models, Molecular , Protein Binding , Stem Cells/chemistry , Stem Cells/metabolismABSTRACT
Prestin is a unique ATP- and Ca(2+)-independent molecular motor with piezoelectric characteristics responsible for the electromotile properties of mammalian cochlear outer hair cells, i.e. the capacity of these cells to modify their length in response to electric stimuli. This 'electromotility' is at the basis of the exceptional sensitivity and frequency selectivity distinctive of mammals. Prestin belongs to the SLC26 (solute carrier 26) family of anion transporters and needs anions to function properly, particularly Cl(-). In the present study, using X-ray crystallography we reveal that the STAS (sulfate transporter and anti-sigma factor antagonist) domain of mammalian prestin, considered an 'incomplete' transporter, harbours an unanticipated anion-binding site. In parallel, we present the first crystal structure of a prestin STAS domain from a non-mammalian vertebrate prestin (chicken) that behaves as a 'full' transporter. Notably, in chicken STAS, the anion-binding site is lacking because of a local structural rearrangement, indicating that the presence of the STAS anion-binding site is exclusive to mammalian prestin.
Subject(s)
Anion Transport Proteins/metabolism , Amino Acid Sequence , Animals , Anion Transport Proteins/chemistry , Anion Transport Proteins/genetics , Anions , Binding Sites , Chickens , Crystallography, X-Ray , Humans , Models, Molecular , Molecular Sequence Data , Protein Conformation , Rats , Sequence Homology, Amino Acid , Sulfate TransportersABSTRACT
Prestin is the motor protein responsible for the somatic electromotility of cochlear outer hair cells and is essential for normal hearing sensitivity and frequency selectivity of mammals. Prestin is a member of mammalian solute-linked carrier 26 (SLC26) anion exchangers, a family of membrane proteins capable of transporting a wide variety of monovalent and divalent anions. SLC26 transporters play important roles in normal human physiology in different tissues, and many of them are involved in genetic diseases. SLC26 and related SulP transporters carry a hydrophobic membrane core and a C-terminal cytosolic portion that is essential in plasma membrane targeting and protein function. This C-terminal portion is mainly composed of a STAS (sulfate transporters and anti-sigma factor antagonist) domain, whose name is due to a remote but significant sequence similarity with bacterial ASA (anti-sigma factor antagonist) proteins. Here we present the crystal structure at 1.57 A resolution of the cytosolic portion of prestin, the first structure of a SulP transporter STAS domain, and its characterization in solution by heteronuclear multidimensional NMR spectroscopy. Prestin STAS significantly deviates from the related bacterial ASA proteins, especially in the N-terminal region, which-although previously considered merely as a generic linker between the domain and the last transmembrane helix-is indeed fully part of the domain. Hence, unexpectedly, our data reveal that the STAS domain starts immediately after the last transmembrane segment and lies beneath the lipid bilayer. A structure-function analysis suggests that this model can be a general template for most SLC26 and SulP anion transporters and supports the notion that STAS domains are involved in functionally important intramolecular and intermolecular interactions. Mapping of disease-associated or functionally harmful mutations on STAS structure indicates that they can be divided into two categories: those causing significant misfolding of the domain and those altering its interaction properties.