Quality control machinery in the endoplasmic reticulum (ER) helps ensure that only properly folded and assembled proteins accumulate in the ER or continue along the secretory pathway. Aberrant proteins are retrotranslocated to the cytosol and degraded by the proteasome, a process called ER-associated degradation. Doa10, a transmembrane protein of the ER/nuclear envelope, is one of the primary ubiquitin ligases (E3s) participating in ER-associated degradation in Saccharomyces cerevisiae. Here we report the membrane organization of the 1319-residue Doa10 polypeptide. The topology was determined by fusing a dual-topology reporter after 16 different Doa10 fragments. Our results indicate that Doa10 contains 14 transmembrane helices (TMs). Based on protease digestion of yeast microsomes, both the N-terminal RING-CH domain and the C terminus face the cytosol. Notably, the experimentally derived topology was not predicted correctly by any of the generally available TM prediction algorithms. Bioinformatic analysis and in silico mutagenesis guided the topological studies through problematic regions. The conserved TD domain in Doa10 includes three TMs. These TMs might function in cofactor binding or substrate recognition, or they might be part of a retrotranslocation channel. The Derlins were previously proposed to provide such channels, but we show that the two yeast Derlins are not required for degradation of Doa10 membrane substrates, as was found before for the Sec61 translocon. Finally, we provide evidence that the likely human Doa10 ortholog, TEB4 (MARCH-VI), adopts a topology similar to that of Doa10.
|Evidence ID||Analyze ID||Interactor||Interactor Systematic Name||Interactor||Interactor Systematic Name||Type||Assay||Annotation||Action||Modification||Phenotype||Source||Reference||Note|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Gene Ontology Term||Gene Ontology Term ID||Qualifier||Aspect||Method||Evidence||Source||Assigned On||Annotation Extension||Reference|
|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
|Evidence ID||Analyze ID||Regulator||Regulator Systematic Name||Target||Target Systematic Name||Experiment||Assay||Construct||Conditions||Strain Background||Reference|