Translocation of proteins across biological membranes is essential for life. Proteins that clog the endoplasmic reticulum (ER) translocon prevent the movement of other proteins into the ER. Eukaryotes have multiple translocon quality control (TQC) mechanisms to detect and destroy proteins that persistently engage the translocon. TQC mechanisms have been defined using a limited panel of substrates that aberrantly occupy the channel. The extent of substrate overlap among TQC pathways is unknown. In this study, we found that two TQC enzymes, the ER-associated degradation (ERAD) ubiquitin ligase Hrd1 and zinc metalloprotease Ste24, promote degradation of characterized translocon-associated substrates of the other enzyme in Saccharomyces cerevisiae. While both enzymes contribute to substrate turnover, our results suggest a prominent role for Hrd1 in TQC. Yeast lacking both Hrd1 and Ste24 exhibit a profound growth defect, consistent with overlapping function. Remarkably, two mutations that mildly perturb post-translational translocation and reduce the extent of aberrant translocon engagement by a model substrate diminish cellular dependence on TQC enzymes. Our data reveal previously unappreciated mechanistic complexity in TQC substrate detection and suggest that a robust translocon surveillance infrastructure maintains functional and efficient translocation machinery.
A prominent source of mutation in cancer is single-stranded DNA cytosine deamination by cellular APOBEC3 enzymes, which results in C-to-T and C-to-G mutations in TCA and TCT motifs. Although multiple enzymes have been implicated, reports conflict and it is unclear which enzyme(s) are responsible. Here we develop a selectable system to quantify genome mutation and compare the mutagenic activities of three leading candidates - APOBEC3A, APOBEC3B, and APOBEC3H. The human cell line, HAP1, was engineered to express the thymidine kinase (TK) gene of HSV-1, which confers sensitivity to ganciclovir. Clonal expression of APOBEC3A and APOBEC3B, but not catalytic mutant controls or APOBEC3H, triggered elevated DNA damage responses and increased frequencies of TK mutation. Mutant TK DNA sequences revealed nearly indistinguishable cytosine mutation patterns. Whole genome sequences from TK mutant clones confirmed these results and enabled broader bioinformatic analyses. Most importantly, comparisons of “pure” APOBEC3A- and APOBEC3B-inflicted mutation signatures from this system and the actual APOBEC3 signature from breast cancer indicated that most tumors manifest a composite signature. These studies help resolve a long-standing etiologic debate in the cancer field and indicate that future diagnostic and therapeutic efforts should focus on both APOBEC3A and APOBEC3B.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.