The ssrA-Tag Facilitated Degradation of an Integral Membrane Protein

Chai, Qian; Wang, Zhaoshuai; Webb, Stacy R.; Dutch, Rebecca Ellis; Wei, Yinan

doi:10.1021/acs.biochem.6b00038

Cited by 16 publications

(22 citation statements)

References 32 publications

(57 reference statements)

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“…We were the first to report that ClpXP could facilitate the degradation of an ssrA-tagged integral membrane protein AcrB. 14 We confirmed that the deletion of the clpX or clpP gene in E. coli strain hindered the degradation. AcrB-ssrA was membrane integrated before degradation, as confirmed by the reduction of efflux activity after the induced expression of ClpX in a clpX knockout strain.…”

supporting

confidence: 56%

“…10−12 Before our discovery that ClpXP mediates the degradation of AcrB-ssrA, FtsH was the only protease that has been reported to completely degrade membrane integrated proteins. 13,14 FtsH is a protease anchored to the cytoplasmic side of the inner membrane. This protease has been shown to degrade several integral membrane proteins with a metastable structure.…”

mentioning

confidence: 99%

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Accessibility from the Cytoplasm Is Critical for ssrA Tag-Mediated Degradation of Integral Membrane Proteins by ClpXP Protease

et al. 2018

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The AAA+ protease ClpXP has long been established as the cellular rescue system that degrades ssrA-tagged proteins resulting from stalled ribosomes. Until recently, in all of these studies soluble proteins were used as model substrates, since the ClpXP complex and the related adapter SspB are all cytosolic proteins. In a previous study, we found that the introduction of an ssrA tag can facilitate complete degradation of a large and stable trimeric integral membrane protein AcrB, which is the first reported example of a membrane protein substrate. To investigate the mechanism of degradation of a membrane protein by a soluble protein complex, we experimented with the truncation of the C-terminal tail of AcrB. We found that the C-terminal tail is important for degradation, as systematic truncation of the tail diminished degradation. Thus, we hypothesize that membrane proteins need a cytosolic tail/domain for ClpXP-SspB to latch on to initiate degradation. To test this hypothesis, we introduced the ssrA tag at the C-terminal of several membrane proteins, including AqpZ, YiiP, YajR, as well as their truncation fragments, and examined their degradation. We found that the ssrA-facilitated degradation of membrane proteins by ClpXP-SspB depends on the presence of a CT tail or domain, which is critical for accessibility of the tag by ClpXP-SspB. When the ssrA tag is not well-exposed to the cytosol, FtsH can access and degrade the tagged protein, given that the substrate protein is metastable.

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confidence: 56%

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confidence: 99%

Accessibility from the Cytoplasm Is Critical for ssrA Tag-Mediated Degradation of Integral Membrane Proteins by ClpXP Protease

et al. 2018

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“…12 Challenging this assumption, however, a recent study showed that ClpXP degrades ssrA-tagged AcrB, an E. coli inner-membrane protein. 13 Here, we investigate a different ssrA-tagged integral membrane protein and find that its degradation requires FtsH but not ClpXP or ClpAP in vivo . Thus, which AAA+ protease degrades which ssrA-tagged inner-membrane protein depends on factors that are currently poorly defined.…”

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confidence: 97%

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

Hari

Sauer

2016

Biochemistry

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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 E. 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 degradation of ProW1-182 in vivo. These studies suggest that a complex distribution of proteolytic labor maintains protein quality control in the inner membrane.

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“…The cell culture was returned to the shaker, and aliquots of samples were withdrawn at the indicated time and analyzed. In a previous study, we found that the intensity of the radioactively labeled AcrB did not decrease over 24 hours [20]. Here we extended the experiment to 7 days (Figure 2A and 2B).…”

Section: Resultsmentioning

confidence: 55%

Study of the degradation of a multidrug transporter using a non-radioactive pulse chase method

et al. 2016

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Proteins are constantly synthesized and degraded in living cells during their growth and division, often in response to metabolic and environmental conditions. The synthesis and breakdown of proteins under different conditions reveal information about their mechanism of function. The metabolic incorporation of non-natural amino acid azidohomoalanine (AHA) and subsequent labeling via click chemistry emerged as a non-radioactive strategy useful in the determination of protein kinetics and turnover. We used the method to monitor the degradation of two proteins involved in the multidrug efflux in E. coli, the inner membrane transporter AcrB and its functional partner membrane fusion protein AcrA. Together they form a functional complex with an outer membrane channel TolC to actively transport various small molecule compounds out of E. coli cells. We found that both AcrA and AcrB lasted for approximately six days in live E. coli cells, and the stability of AcrB depended on the presence of AcrA but not on active efflux. These results lead to new insight into the multidrug resistance in Gram-negative bacteria conferred by efflux.

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The ssrA-Tag Facilitated Degradation of an Integral Membrane Protein

Cited by 16 publications

References 32 publications

Accessibility from the Cytoplasm Is Critical for ssrA Tag-Mediated Degradation of Integral Membrane Proteins by ClpXP Protease

Accessibility from the Cytoplasm Is Critical for ssrA Tag-Mediated Degradation of Integral Membrane Proteins by ClpXP Protease

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

Study of the degradation of a multidrug transporter using a non-radioactive pulse chase method

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