Fig. 8: Physical constraints in recognition of a membrane-protein-linked Deg1 degron by Doa10 and working model for substrate recognition and polyubiquitination.
From: Substrate recognition mechanism of the endoplasmic reticulum-associated ubiquitin ligase Doa10
a Schematic diagram of the Deg1-Vma12-Ura3 model substrate. Left, original construct; right, minimal construct. Numbers indicate the amino acid positions. b Doa10-dependent yeast growth inhibition using various truncation variants of Deg1-Vma12-Ura3. c As in (b), but a Gly/Ser (GS) linker was inserted between the truncated Deg1 (Deg11–35) and Vma12Δ132. Two different GS linkers were tested: ‘10 GS’ = GGS GGS GGS G and ‘20 GS’ = GGS GGS GGS GGS GGS GGS GG. d Degradation of indicated Deg11–35-GS-Vma12Δ132 variants were measured by cycloheximide chase and immunoblotting (also see Supplementary Fig. 12b). Means and s.e.m. of three independent experiments. e Working model for the substrate-recognition mechanism of Doa10. Cytosolic substrates could be either soluble proteins with an N- or C- terminal degron (e.g., Deg1 and CL1) or TA membrane proteins that are extracted by the Msp1 and Spf1 ATPases (e.g., Pex15Δ30). Transmembrane substrates could be membrane proteins with a degron (either intrinsic or generated by damage or unfolding) in their cytosolic domains (e.g., Deg1-Vma12). Data in (b, c) are representative of two independent experiments. Data in (d) are mean ± s.e.m. from three independent experiments.
