A biochemical analysis of the constraints of tail-anchored protein biogenesis

Pawel Leznicki , Jim Warwicker , Stephen High (1)

1 Faculty of Life Sciences [Manchester]

Tail-anchored (TA) proteins utilise distinct biosynthetic pathways, including TRC40-mediated, chaperone-dependent and/or unassisted routes to the endoplasmic reticulum (ER) 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 Sec61beta and Cytb5 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 Sec61beta we find that both SGTA and TRC40 can bind this substrate with a singly PEGylated TA-region. However, by introducing two PEG moieties, TRC40 binding can be prevented resulting in a block of subsequent membrane integration. Whilst TRC40 can bind Sec61beta 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.

Mots clés: Life Sciences

Pawel Leznicki, Jim Warwicker, Stephen High. A biochemical analysis of the constraints of tail-anchored protein biogenesis. Biochemical Journal, 2011, 436 (3), pp.719-727. ⟨10.1042/BJ20101737⟩. ⟨hal-00596276⟩