Transmembrane helix hydrophobicity is an energetic barrier during the retrotranslocation of integral membrane ERAD substrates

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  • Christopher J. Guerriero
    Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260
  • Karl-Richard Reutter
    Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260
  • Andrew A. Augustine
    Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260
  • G. Michael Preston
    Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260
  • Kurt F. Weiberth
    Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260
  • Timothy D. Mackie
    Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260
  • Hillary C. Cleveland-Rubeor
    Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260
  • Neville P. Bethel
    Cardiovascular Research Institute, Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158
  • Keith M. Callenberg
    Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260
  • Kunio Nakatsukasa
    Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260
  • Michael Grabe
    Cardiovascular Research Institute, Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA 94158
  • Jeffrey L. Brodsky
    Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260
  • Anne Spang
    editor

Description

<jats:p>Integral membrane proteins fold inefficiently and are susceptible to turnover via the endoplasmic reticulum–associated degradation (ERAD) pathway. During ERAD, misfolded proteins are recognized by molecular chaperones, polyubiquitinated, and retrotranslocated to the cytoplasm for proteasomal degradation. Although many aspects of this pathway are defined, how transmembrane helices (TMHs) are removed from the membrane and into the cytoplasm before degradation is poorly understood. In this study, we asked whether the hydrophobic character of a TMH acts as an energetic barrier to retrotranslocation. To this end, we designed a dual-pass model ERAD substrate, Chimera A*, which contains the cytoplasmic misfolded domain from a characterized ERAD substrate, Sterile 6* (Ste6p*). We found that the degradation requirements for Chimera A* and Ste6p* are similar, but Chimera A* was retrotranslocated more efficiently than Ste6p* in an in vitro assay in which retrotranslocation can be quantified. We then constructed a series of Chimera A* variants containing synthetic TMHs with a range of ΔG values for membrane insertion. TMH hydrophobicity correlated inversely with retrotranslocation efficiency, and in all cases, retrotranslocation remained Cdc48p dependent. These findings provide insight into the energetic restrictions on the retrotranslocation reaction, as well as a new computational approach to predict retrotranslocation efficiency.</jats:p>

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