title: Preparation and characterization of the extracellular domain of human Sid-1.
abstract: In C. elegans, the cell surface protein Sid-1 imports extracellular dsRNA into the cytosol of most non-neuronal cells, enabling systemic spread of RNA interference (RNAi) throughout the worm. Sid-1 homologs are found in many other animals, although for most a function for the protein has not yet been established. Sid-1 proteins are composed of an N-terminal extracellular domain (ECD) followed by 9-12 predicted transmembrane regions. We developed a baculovirus system to express and purify the ECD of the human Sid-1 protein SidT1. Recombinant SidT1 ECD is glycosylated and spontaneously assembles into a stable and discrete tetrameric structure. Electron microscopy (EM) and small angle x-ray scattering (SAXS) studies reveal that the SidT1 ECD tetramer is a compact, puck-shaped globular particle, which we hypothesize may control access of dsRNA to the transmembrane pore. These characterizations provide inroads towards understanding the mechanism of this unique RNA transport system from structural prospective.
authors.affiliation: Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, United States of America.
authors.forename: Ashley J
authors.initials: AJ
authors.lastname: Pratt
authors.forename: Robert P
authors.initials: RP
authors.lastname: Rambo
authors.forename: Pick-Wei
authors.initials: PW
authors.lastname: Lau
authors.forename: Ian J
authors.initials: IJ
authors.lastname: MacRae
mesh_terms: D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D005110:Extracellular Space / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D017510:Protein Folding; D055503:Protein Multimerization; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D018411:Spodoptera / Q000166:cytology
chemical_list: D026901:Membrane Transport Proteins; C571194:systemic RNA Interference-defective-1 transmembrane family member 1, human