title: A biochemical analysis of the constraints of tail-anchored protein biogenesis.
abstract: TA (tail-anchored) proteins utilize distinct biosynthetic pathways, including TRC40 (transmembrane domain recognition complex of 40 kDa)-mediated, chaperone-dependent and/or unassisted routes to the ER (endoplasmic reticulum) membrane. We have addressed the flexibility of cytosolic components participating in these pathways, and explored the thermodynamic constraints of their membrane insertion, by exploiting recombinant forms of Sec61β and Cytb5 (cytochrome b5) bearing covalent modifications within their TA region. In both cases, efficient membrane insertion relied on cytosolic factors capable of accommodating a surprising range of covalent modifications to the TA region. For Sec61β, we found that both SGTA (small glutamine-rich tetratricopeptide repeat-containing protein α) and TRC40 can bind this substrate with a singly PEGylated TA region. However, by introducing two PEG [poly(ethylene glycol)] moieties, TRC40 binding can be prevented, resulting in a block of subsequent membrane integration. Although TRC40 can bind Sec61β polypeptides singly PEGylated at different locations, membrane insertion is more sensitive to the precise location of PEG attachment. Modelling and experimentation indicate that this post-TRC40 effect results from an increased energetic cost of inserting different PEGylated TA regions into the lipid bilayer. We therefore propose that the membrane integration of TA proteins delivered via TRC40 is strongly dependent upon underlying thermodynamics, and speculate that their insertion is via a phospholipid-mediated process.
authors.affiliation: Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester M13 9PT, UK.
authors.forename: Pawel
authors.initials: P
authors.lastname: Leznicki
authors.forename: Jim
authors.initials: J
authors.lastname: Warwicker
authors.forename: Stephen
authors.initials: S
authors.lastname: High
mesh_terms: D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D015786:Cytochromes b5 / Q000096:biosynthesis; D003600:Cytosol / Q000378:metabolism; D029721:Drosophila Proteins / Q000096:biosynthesis; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis*; D018832:Molecular Chaperones; D011092:Polyethylene Glycols / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D011994:Recombinant Proteins / Q000378:metabolism; D000069816:SEC Translocation Channels
chemical_list: D002352:Carrier Proteins; D029721:Drosophila Proteins; D008565:Membrane Proteins; D018832:Molecular Chaperones; D011994:Recombinant Proteins; D000069816:SEC Translocation Channels; C114575:SGTA protein, human; C499386:Sec61beta protein, Drosophila; D011092:Polyethylene Glycols; D015786:Cytochromes b5