title: The role of cytosolic proteins in the insertion of tail-anchored proteins into phospholipid bilayers.
abstract: Tail-anchored (TA) proteins are membrane proteins that contain an N-terminal domain exposed to the cytosol and a single transmembrane segment near the C-terminus followed by few or no polar residues. TA proteins with a mildly hydrophobic transmembrane domain, such as cytochrome b5 (b5), are able to insert post-translationally into pure lipid vesicles without assistance from membrane proteins. Here, we investigated whether any cytosolic proteins are needed to maintain b5 in a competent state for transmembrane integration. Using b5 constructs translated in vitro or produced in bacteria, we demonstrate that cytosolic proteins are neither necessary nor facilitatory for the unassisted translocation of b5. Furthermore, we demonstrate that no cytosolic protein is involved in the translocation of a C-terminal domain of 85 residues appended to the transmembrane domain of b5. Nevertheless, b5 does bind cytosolic proteins, and in their presence but not in their absence, its insertion into liposomes is inhibited by the thiol oxidant diamide and the alkylating agent N-ethylmaleimide. The effect of diamide is also observed in living cells. Thus, the specific in vivo targeting of b5 might be achieved by interaction with redox-sensitive targeting factors that hinder its nonspecific insertion into any permissive bilayer.
authors.affiliation: CNR Institute for Neuroscience and Department of Pharmacology, Università degli Studi di Milano, Italy.
authors.forename: Sara F
authors.initials: SF
authors.lastname: Colombo
authors.forename: Renato
authors.initials: R
authors.lastname: Longhi
authors.forename: Nica
authors.initials: N
authors.lastname: Borgese
mesh_terms: D000477:Alkylating Agents / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D015786:Cytochromes b5 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D003958:Diamide / Q000494:pharmacology; D005033:Ethylmaleimide / Q000494:pharmacology; D057927:Hydrophobic and Hydrophilic Interactions; D008051:Lipid Bilayers / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D018832:Molecular Chaperones / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D010743:Phospholipids / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism; D013439:Sulfhydryl Reagents / Q000494:pharmacology; D013552:Swine; D013997:Time Factors
chemical_list: D000477:Alkylating Agents; D008051:Lipid Bilayers; D018832:Molecular Chaperones; D016877:Oxidants; D010743:Phospholipids; D011994:Recombinant Proteins; D013439:Sulfhydryl Reagents; D003958:Diamide; D015786:Cytochromes b5; D005033:Ethylmaleimide