title: Molecular determinants of the sensory and motor neuron-derived factor insertion into plasma membrane.
abstract: The sensory and motor neuron-derived factor (SMDF) is a type III neuregulin that regulates development and proliferation of Schwann cells. Although SMDF has been shown to be a type II protein, the molecular determinants of membrane biogenesis, insertion, and topology remain elusive. Here we used heterologous expression of a yellow fluorescent protein-SMDF fusion protein along with a stepwise deletion strategy to show that the apolar/uncharged segment (Ile(76)-Val(100)) acts as an internal, uncleaved membrane insertion signal that defines the topology of the protein. Unexpectedly, removal of the transmembrane segment (TM) did not eliminate completely membrane association of C-terminal fragments. TM-deleted fusion proteins, bearing the amino acid segment (Ser(283)-Glu(296)) located downstream to the epidermal growth factor-like motif, strongly interacted with plasma membrane fractions. However, synthetic peptides patterned after this segment did not insert into artificial lipid vesicles, suggesting that membrane interaction of the SMDF C terminus may be the result of a post-translational modification. Subcellular localization studies demonstrated that the 40-kDa form, but not the 83-kDa form, of SMDF was segregated into lipid rafts. Deletion of the N-terminal TM did not affect the interaction of the protein with these lipid microdomains. In contrast, association with membrane rafts was abolished completely by truncation of the protein C terminus. Collectively, these findings are consistent with a topological model for SMDF in which the protein associates with the plasma membrane through both the TM and the C-terminal end domains resembling the topology of other type III neuregulins. The TM defines its characteristic type II membrane topology, whereas the C terminus is a newly recognized anchoring motif that determines its compartmentalization into lipid rafts. The differential localization of the 40- and 83-kDa forms of the neuregulin into rafts and non-raft domains implies a central role in the protein biological activity.
authors.affiliation: Centro de Biologia Molecular y Celular and Instituto de Neurociencias-Consejo Superior de Investigaciones Cientificas, Universidad Miguel Hernández, Alicante 03202, Spain.
authors.forename: Hugo
authors.initials: H
authors.lastname: Cabedo
authors.forename: Carolina
authors.initials: C
authors.lastname: Luna
authors.forename: Asia M
authors.initials: AM
authors.lastname: Fernandez
authors.forename: Juana
authors.initials: J
authors.lastname: Gallar
authors.forename: Antonio
authors.initials: A
authors.lastname: Ferrer-Montiel
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D019556:COS Cells; D002152:Calorimetry, Differential Scanning; D002462:Cell Membrane / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008954:Models, Biological; D008969:Molecular Sequence Data; D020880:Neuregulins / Q000737:chemistry / Q000378:metabolism; D017830:Octoxynol / Q000494:pharmacology; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013696:Temperature; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D018076:DNA, Complementary; D008164:Luminescent Proteins; D020880:Neuregulins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C054562:yellow fluorescent protein, Bacteria; D014443:Tyrosine; D017830:Octoxynol