Dataset Information

The AAA+ FtsH Protease Degrades an ssrA-Tagged Model Protein in the Inner Membrane of Escherichia coli.

ABSTRACT: In eubacteria, the tmRNA system frees ribosomes that stall during protein synthesis and adds an ssrA tag to the incompletely translated polypeptide to target it for degradation. The AAA+ ClpXP protease degrades most ssrA-tagged proteins in the Escherichia coli cytoplasm and was recently shown to degrade an ssrA-tagged protein in the inner membrane. However, we find that tmRNA-mediated tagging of E. coli ProW1-182, a different inner-membrane protein, results in degradation by the membrane-tethered AAA+ FtsH protease. ClpXP played no role in the degradation of ProW1-182 in vivo. These studies suggest that a complex distribution of proteolytic labor maintains protein quality control in the inner membrane.

SUBMITTER: Hari SB

PROVIDER: S-EPMC5218843 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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The AAA+ FtsH Protease Degrades an ssrA-Tagged Model Protein in the Inner Membrane of Escherichia coli.

Hari Sanjay B SB Sauer Robert T RT

Biochemistry 20160930 40

In eubacteria, the tmRNA system frees ribosomes that stall during protein synthesis and adds an ssrA tag to the incompletely translated polypeptide to target it for degradation. The AAA+ ClpXP protease degrades most ssrA-tagged proteins in the Escherichia coli cytoplasm and was recently shown to degrade an ssrA-tagged protein in the inner membrane. However, we find that tmRNA-mediated tagging of E. coli ProW<sub>1-182</sub>, a different inner-membrane protein, results in degradation by the membr ...[more]

PMID: 27677373

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Project description:In bacteria, chromosomal DNA resides in the cytoplasm, and most transcription factors are also found in the cytoplasm. However, some transcription factors, called membrane-bound transcription factors (MTFs), reside in the cytoplasmic membrane. Here, we report the identification of a new MTF in the Gram-positive pathogen Staphylococcus aureus and its regulation by the protease FtsH. The MTF, named MbtS (membrane-bound transcription factor of Staphylococcus aureus), is encoded by SAUSA300_2640 and predicted to have an N-terminal DNA binding domain and three transmembrane helices. The MbtS protein was degraded by membrane vesicles containing FtsH or by the purified FtsH. MbtS bound to an inverted repeat sequence in its promoter region, and the DNA binding was essential for its transcription. Transcriptional comparison between the ftsH deletion mutant and the ftsH mbtS double mutant showed that MbtS could alter the transcription of over 200 genes. Although the MbtS protein was not detected in wild-type (WT) cells grown in a liquid medium, the protein was detected in some isolated colonies on an agar plate. In a murine model of a skin infection, the disruption of mbtS increased the lesion size. Based on these results, we concluded that MbtS is a new S. aureus MTF whose activity is proteolytically regulated by FtsH.IMPORTANCEStaphylococcus aureus is an important pathogenic bacterium causing various diseases in humans. In the bacterium, transcription is typically regulated by the transcription factors located in the cytoplasm. In this study, we report an atypical transcription factor identified in S. aureus Unlike most other transcription factors, the newly identified transcription factor is located in the cytoplasmic membrane, and its activity is proteolytically controlled by the membrane-bound AAA+ protease FtsH. The newly identified MTF, named MbtS, has the potential to regulate the transcription of over 200 genes. This study provides a molecular mechanism by which a protease affects bacterial transcription and illustrates the diversity of the bacterial transcriptional regulation.

Project description:Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ?19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ?25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with biological membrane surfaces that we are only beginning to decipher.

Dataset Information

The AAA+ FtsH Protease Degrades an ssrA-Tagged Model Protein in the Inner Membrane of Escherichia coli.

Publications

The AAA+ FtsH Protease Degrades an ssrA-Tagged Model Protein in the Inner Membrane of Escherichia coli.

Similar Datasets

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