Escherichia coli membrane microdomain SPFH protein HflC interacts with YajC and contributes to aminoglycoside and oxidative stress tolerance

Eukaryotic cells segregate many membrane-dependent functions into membrane microdomains also known as lipid rafts. These domains are enriched in polyisoprenoid lipids and in scaffolding proteins belonging to the Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) protein superfamily, which are also found in prokaryotes. Whereas Gram-positive bacteria were also shown to possess functional membrane microdomains (FMMs) structurally and functionally similar to eukaryotic lipid rafts, little is still known about Gram-negative bacteria FMMs. Escherichia coli K12 possesses 4 SPFH proteins, YqiK, QmcA, HflK, and HflC, previously shown to localize in discrete polar or lateral inner-membrane locations, raising the possibility that E. coli SPFH proteins could contribute to the assembly of inner-membrane FMMs regulating cellular processes. Here we studied the determinants of the native, chromosomal QmcA and HflC cell localization using a domain swap analysis and fluorescent and super-resolution microscopy. We showed that full QmcA and HflC protein is required for achieving their native inner-membrane localization and that impairing the synthesis of cardiolipin and isoprenoid lipids known to associate with FMMs alters QmcA and HflC localization pattern. Finally, using Biolog phenotypic arrays, we showed that a mutant lacking all SPFH genes displayed increased sensitivity to aminoglycosides and oxidative stress. This phenotype is exclusively due to the absence of HflKC and a cross-linking and mass spectrometry analysis showed that YajC, a SecDF translocon accessory protein, interacts with HflC and also contributes to E. coli stress tolerance. Our study therefore provides insights into the function and interactions associated with SPFH proteins in E. coli FMMs. IMPORTANCE Eukaryotic cells segregate many cellular processes in cholesterol-rich functional membrane micro-domain also called lipid rafts, which contain proteins of the Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) superfamily. Whereas SPFH proteins are also present in bacteria, they were mostly studied in Gram-positive bacteria and less is known on the function of SPFH proteins in Gram-negative bacteria. Here, we showed that the cell localization of the E. coli SPFH proteins QmcA and HflKC is altered in absence of cardiolipin and isoprenoid lipid synthesis, suggesting that these lipids could contribute to E. coli membrane microdomain assembly. Using a broad phenotypic analysis and cross-linking coupled with spectrometry approaches, we identified that YajC, a SecDF-YajC translocon accessory protein, interacts with HflC and that both proteins contribute to E. coli tolerance to aminoglycosides and oxidative stress. Our study, therefore, provides new insights into the cellular processes associated with SPFH proteins in E. coli functional membrane microdomains.