FtsH degrades dihydrofolate reductase by recognizing a partially folded species

Abstract

AAA+ proteolytic machines play essential roles in maintaining and rebalancing the cellular proteome in response to stress, developmental cues, and environmental changes. Of the five AAA+ proteases in Escherichia coli, FtsH is unique in its attachment to the inner membrane and its function in degrading both membrane and cytosolic proteins. E. coli dihydrofolate reductase (DHFR) is a stable and biophysically well-characterized protein, which a previous study found resisted FtsH degradation despite the presence of an ssrA degron. By contrast, we find that FtsH degrades DHFR fused to a long peptide linker and ssrA tag. Surprisingly, we also find that FtsH degrades DHFR with shorter linkers and ssrA tag, and without any linker or tag. Thus, FtsH must be able to recognize a sequence element or elements within DHFR. We find that FtsH degradation of DHFR is noncanonical in the sense that it does not rely upon recognition of an unstructured polypeptide at or near the N-terminus or Cterminus of the substrate. Results using peptide-array experiments, mutant DHFR proteins, and fusion proteins suggest that FtsH recognizes an internal sequence in a species of DHFR that is partially unfolded. Overall, our findings provide insight into substrate recognition by FtsH and indicate that its degradation capacity is broader than previously reported.

Toxin-based screening of C-terminal tags inEscherichia colireveals the exceptional potency of ssrA-like degrons

“…These ClpXP-dependent degrons bore similarity to the ssrA tag, with a strongly conserved terminal Ala-Ala motif. Although E. coli possesses five ATPdependent proteases, four of which are capable of recognizing the ssrA tag [16,[23][24][25][26][27], our data suggest that the interaction between ClpXP and ssrA-like sequences is a singularly robust recognition modality among short C-terminal degrons. The preferential enrichment of ssrA-like tags likely reflects the importance of ribosome rescue in cellular fitness.…”

“…One of the most well-studied degrons is the ssrA tag, a ~10 amino acid sequence that is co-translationally appended to stalled polypeptides during tmRNA-mediated ribosomal rescue [22,23]. Proteins bearing the ssrA degron are robustly proteolyzed by ClpXP, and to a lesser extent by other cellular proteases [24][25][26][27]. The E. coli ssrA tag (AANDENYALAA) has been extensively characterized and has serves as a versatile tool for studying the biochemical and mechanochemical properties of ClpXP and other proteases [28][29][30][31][32].…”

The membrane-cytoplasmic linker defines activity of FtsH proteases in Pseudomonas aeruginosa clone C

“…This article contains supporting information ( 82 , 83 , 84 ). A PDF file with supporting information containing SI Figures S1-S11 and an Excel file with SI Tables S1-S4 are provided.…”

“…D – G , all reactions used C-terminal 6xHis-tagged proteins. E - G , all reactions contained 5 mM ATP and a creatine kinase (CK)-based ATP regeneration system ( 83 ). DNTR, diverse N-terminal region; MC, membrane-cytoplasmic; MC 7+ , membrane-cytoplasmic linker plus seven C-terminal amino acids into the AAA+ ATPase module boundary; MW, Molecular weight; M, Molecular weight marker; mAU, milli-Absorbance Unit; MC 7+ , membrane-cytoplasmic plus next seven C-terminal amino acids; Peri, periplasmic domain; TM1, transmembrane helix 1; TM2, transmembrane helix 2.…”

Mutations in the riboflavin biosynthesis pathway confer resistance to furazolidone and abolish the synergistic interaction between furazolidone and vancomycin inEscherichia coli

“…FtsH is an essential inner-membrane-anchored AAA + protease that degrades specific proteinaceous targets for removal of misfolded proteins or regulated proteolysis in response to stresses (15). At least 23 FtsH substrates have been reported, including membrane-anchored and cytoplasmic targets, such as SecY, PspC, KdtA, LpxC, RpoH, SoxS, FolA and Cfa to name a few (15)(16)(17). It is very likely that the observed phenotypes are due to one or more of these FtsH substrates, whose identity remains to be determined.…”