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SecY-mediated quality control prevents the translocation of non-gated porins.

ABSTRACT: OmpC and OmpF are among the most abundant outer membrane proteins in E. coli and serve as hydrophilic channels to mediate uptake of small molecules including antibiotics. Influx selectivity is controlled by the so-called constriction zone or eyelet of the channel. Mutations in the loop domain forming the eyelet can disrupt transport selectivity and thereby interfere with bacterial viability. In this study we show that a highly conserved motif of five negatively charged amino acids in the eyelet, which is critical to regulate pore selectivity, is also required for SecY-mediated transport of OmpC and OmpF into the periplasm. Variants with a deleted or mutated motif were expressed in the cytosol and translocation was initiated. However, after signal peptide cleavage, import into the periplasm was aborted and the mutated proteins were redirected to the cytosol. Strikingly, reducing the proof-reading capacity of SecY by introducing the PrlA4 substitutions restored transport of OmpC with a mutated channel domain into the periplasm. Our study identified a SecY-mediated quality control pathway to restrict transport of outer membrane porin proteins with a deregulated channel activity into the periplasm.

SUBMITTER: Jung S

PROVIDER: S-EPMC7530735 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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SecY-mediated quality control prevents the translocation of non-gated porins.

Jung Sebastian S Bader Verian V Natriashvili Ana A Koch Hans-Georg HG Winklhofer Konstanze F KF Tatzelt Jörg J

Scientific reports 20201001 1

OmpC and OmpF are among the most abundant outer membrane proteins in E. coli and serve as hydrophilic channels to mediate uptake of small molecules including antibiotics. Influx selectivity is controlled by the so-called constriction zone or eyelet of the channel. Mutations in the loop domain forming the eyelet can disrupt transport selectivity and thereby interfere with bacterial viability. In this study we show that a highly conserved motif of five negatively charged amino acids in the eyelet, ...[more]

PMID: 33004891

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Project description:BackgroundsInflammation underlies the mechanism of different kinds of heart disease. Cytoplasmic membrane localized N-terminal fragment of gasdermin-D (GSDMD-N) could induce inflammatory injury to cardiomyocyte. However, effects and dynamic changes of GSDMD during the process of lipopolysaccharide (LPS) related inflammatory stress induced cardiomyocyte injury are barely elucidated to date. In this study, LPS related cardiomyocyte injury was investigated based on potential interaction of GSDMD-N induced mitochondrial injury and mitophagy mediated mitochondria quality control.MethodsHL-1 cardiomyocytes were treated with LPS and Nigericin to induce inflammatory stress. The dual-fluorescence-labelled GSDMD expressed HL-1 cardiomyocytes were constructed to study the translocation of GSDMD. The mitochondrial membrane potential (MMP) was measured by JC-1 staining. Mitophagy and autophagic flux were recorded by transmission electron microscopy and fluorescent image.ResultsGSDMD-N showed a time-dependent pattern of translocation from mitochondria to cytoplasmic membrane under LPS and Nigericin induced inflammatory stress in HL-1 cardiomyocytes. GSDMD-N preferred to localize to mitochondria to permeablize its membrane and dissipate the MMP. This effect couldn't be reversed by cyclosporine-A (mPTP inhibitor), indicating GSDMD-N pores as alternative mechanism underlying MMP regulation, in addition to mitochondrial permeability transition pore (mPTP). Moreover, the combination between GSDMD-N and autophagy related Microtubule Associated Protein 1 Light Chain 3 Beta (LC3B) was verified by co-immunoprecipitation. Besides, mitophagy alleviating GSDMD-N induced mitochondrial injury was proved by pre-treatment of autophagy antagonist or agonist in GSDMD-knock out or GSDMD-overexpression cells. A time-dependent pattern of GSDMD translocation and mitochondrial GSDMD targeted mitophagy were verified.ConclusionHerein, our study confirmed a crosstalk between GSDMD-N induced mitochondrial injury and mitophagy mediated mitochondria quality control during LPS related inflammation induced cardiomyocyte injury, which potentially facilitating the development of therapeutic target to myocardial inflammatory disease. Our findings support pharmaceutical intervention on enhancing autophagy or inhibiting GSDMD as potential target for inflammatory heart disease treatment.

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SecY-mediated quality control prevents the translocation of non-gated porins.

Publications

SecY-mediated quality control prevents the translocation of non-gated porins.

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