The dispensability and requirement of SecA N-terminal aminoacyl residues for complementation, membrane binding, lipid-specific domains and channel activities

Floyd, Jeanetta Holley; You, Zhipeng; Hsieh, Ying-Hsin; Ma, Yamin; Yang, Hsuichin; Tai, Phang C.

doi:10.1016/j.bbrc.2014.09.080

Cited by 15 publications

(27 citation statements)

References 40 publications

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“…Since SecA 1-825/826 failed to be overexpressed in our hands, we purified SecA 22-828 and SecA 22-829 and used them as representatives of the complementation non-active and active minimal length SecA proteins, respectively. The intrinsic-, membrane- and translocation-ATPase levels of the two complementation-active and non-active truncated SecAs were similar, though greatly reduced comparing to wild-type (data not shown), indicating that high ATPase activity is not obligatory for cell viability, as shown previously [30]. …”

Section: Resultssupporting

confidence: 79%

“…Based on our previous work on the deletion of N-termini, we further define the minimal length of N-/C-termini of active SecA for complementation [30]. Wild-type SecA (901 residues) was deleted from its N- and/or C- termini and tested for complementing ts strain BL21.19 at 42°C (Table 1).…”

Section: Resultsmentioning

confidence: 99%

“…We previously identified two specific N- terminal domains that are crucial for initial SecA/membrane interactions [30]. The first small helix, the linker and part of the second helix (Δ2-22) were found to be dispensable for SecA activity in complementing the growth of the SecA ts mutant.…”

Section: Introductionmentioning

confidence: 99%

“…However, deletions of N-terminal aminoacyl residues #23-25 resulted in severe progressive retardation of growth. Moreover, a decrease of SecA activity caused by N-terminal deletions correlated to the loss of SecA membrane binding, formation of lipid-specific domains and channel activity [30]. Here we further investigated the roles of the C-terminal residues of SecA with or without N-terminal deletions.…”

Section: Introductionmentioning

confidence: 99%

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Characterization of the minimal length of functional SecA in Escherichia coli

You

Yang

et al. 2015

Biochemical and Biophysical Research Communications

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Previous studies showed that certain regions of E. coli SecA can be deleted from its N- and/or C-termini to complement a SecA amber ts mutant. In this study, we determined and characterized the dispensability of both ends of SecA molecules. With N-terminal intact or 9-aa deleted, 826aa (SecA1-826 and SecA10-826 respectively) is the minimum for complementation activity, while with N-terminus deleted by 2-21aa, SecA22-829 is the minimum. Further deletion at the C-terminus of SecA1-826/SecA10-826/SecA22-829 abolished the complementation activity in the cells. A hydrophobic amino acid is required for the 826th residue in the minimal-length SecAs. Chemical crosslinking and gel filtration result showed that both purified SecA22-828 and SecA22-829 could form a dimer. Moreover, the in vitro ATPase and protein translocation activities of SecA22-828 and SecA22-829 were similar, though lower than wild-type SecA. The active mutants had more truncated SecA in soluble than membrane-bound form, but was more stably embedded in membranes. In contrast, the inactive mutants tended to have truncated SecA more membrane-bound than soluble form, and were more loosely bound and easily chased out. Thus, the loss of complementation appears to be related to their altered subcellular localization and stability in the membranes. This study defines the substantial regions of N- and C-termini of SecA that may be deleted without losing complementation activity.

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Section: Resultssupporting

confidence: 79%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Characterization of the minimal length of functional SecA in Escherichia coli

You

Yang

et al. 2015

Biochemical and Biophysical Research Communications

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“…That is, there is no consensus about the oligomeric state during translocation (Allen et al 2016). The 20 N-terminal residues of SecA are important for binding to SecYEG and are also required for SecA-lipid interactions (Floyd et al 2014; Bauer et al 2014; Gouridis et al 2013; Hendrick und Wickner 1991). Binding also depends on the nucleotide in SecA’s binding pocket: AMP-PNP, the non-hydrolysable analogue of ATP, augments SecA’s affinity to SecY by three orders of magnitude as compared to ADP (Deville et al 2011).…”

Section: Introductionmentioning

confidence: 99%

Driving Forces of Translocation Through Bacterial Translocon SecYEG

et al. 2018

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This review focusses on the energetics of protein translocation via the Sec translocation machinery. First we complement structural data about SecYEG’s conformational rearrangements by insight obtained from functional assays. These include measurements of SecYEG permeability that allow assessment of channel gating by ligand binding and membrane voltage. Second we will discuss the power stroke and Brownian ratcheting models of substrate translocation and the role that the two models assign to the putative driving forces: (i) ATP (SecA) and GTP (ribosome) hydrolysis, (ii) interaction with accessory proteins, (iii) membrane partitioning and folding, (iv) proton motive force (PMF), and (v) entropic contributions. Our analysis underlines how important energized membranes are for unravelling the translocation mechanism in future experiments.

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Using Chemical Probes to Assess the Feasibility of Targeting SecA for Developing Antimicrobial Agents against Gram‐Negative Bacteria

et al. 2016

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With the wide-spread emergence of drug resistance, there is an urgent need to search for new antimicrobials, especially those against Gram-negative bacteria. Along this line, the identification of viable targets is a critical first step. SecA is a protein translocase and is commonly believed to be an excellent target for the development of broad-spectrum antimicrobials. In recent years, we have developed three structural classes of SecA inhibitors, which have proven to be very effective against Gram-positive bacteria. However, we have not achieved the same level of success against Gram-negative bacteria, despite the potent inhibition of SecA in enzyme assays by the same inhibitors. In this study, we use representative inhibitors as chemical probes to gain understanding as to why these inhibitors were not effective against Gram-negative bacteria. The results validate our initial postulation that the major difference in effectiveness against Gram-positive and Gram-negative bacteria is in the additional permeability barrier posed by the outer membrane in Gram-negative bacteria. We have also found that expression of efflux pumps, which are responsible for multi-drug resistance, have no effect on the effectiveness of these SecA inhibitors. Identification of an inhibitor-resistant mutant and complementation tests of the plasmids containing secA in a secAts mutant showed that a single secA-azi-9 mutation increased the resistance, providing genetic evidence that SecA indeed is the target of these inhibitors in bacteria. Such results strongly suggest SecA as an excellent target for developing effective antimicrobials against Gram-negative bacteria with the intrinsic ability to overcome MDR. A key future research direction should be the optimization of membrane permeability.

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The dispensability and requirement of SecA N-terminal aminoacyl residues for complementation, membrane binding, lipid-specific domains and channel activities

Cited by 15 publications

References 40 publications

Characterization of the minimal length of functional SecA in Escherichia coli

Characterization of the minimal length of functional SecA in Escherichia coli

Driving Forces of Translocation Through Bacterial Translocon SecYEG

Using Chemical Probes to Assess the Feasibility of Targeting SecA for Developing Antimicrobial Agents against Gram‐Negative Bacteria

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