Escherichia coli SecA Helicase Activity Is Not Required in Vivo for Efficient Protein Translocation or Autogenous Regulation

Schmidt, Marcel; Brosh, Robert M.; Oliver, Donald B.

doi:10.1074/jbc.m104584200

Cited by 20 publications

(14 citation statements)

References 65 publications

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“…Similar to CrhC, SecA possesses RNA helicase activity and is plasma membrane associated. Although the exact role that SecA driven RNA helicase activity performs in protein translocation is not known (Schmidt et al ., 2001; Sianidis et al ., 2001), it is possible that CrhC performs a function similar to that of SecA during cold acclimation in cyanobacteria. In fact, evidence has been presented indicating that the Sec‐dependent protein translocation process is cold sensitive (Pogliano and Beckwith, 1993).…”

Section: Discussionmentioning

confidence: 99%

Polar‐biased localization of the cold stress‐induced RNA helicase, CrhC, in the Cyanobacterium Anabaena sp. strain PCC 7120

El-Fahmawi

Owttrim²

2003

Molecular Microbiology

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SummaryShift of the filamentous cyanobacterium, Anabaena sp. strain PCC 7120, from 30 ∞ ∞ ∞ ∞ C to 20 ∞ ∞ ∞ ∞ C induces expression of a cold shock response gene encoding the RNA helicase CrhC. Subcellular localization using cellular fractionation and membrane purification indicated that CrhC is localized to the plasma membrane with no evidence of a soluble-cytoplasmic form. Treatment of spheroplasts with trypsin and membrane fractions with various denaturing agents identified CrhC as an integral membrane protein associated with the cytoplasmic face of the plasma membrane. Immunoelectron microscopy confirmed the plasma membrane association of CrhC. Interestingly, a higher specific labelling was observed at the cell poles on the septa between adjacent cells within cell filaments. On a per cell area basis, CrhC localization to the cell pole was 3.5-and > 1000-fold higher than to the lateral portion of the plasma membrane or cytoplasm respectively. In addition, CrhC also localizes to new cell poles forming within a dividing cell. Polar-biased localization of the CrhC RNA helicase implies a role in RNA metabolism that is plasma membrane associated and preferentially occurs at the cell poles during cyanobacterial response to cold stress.

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Section: Discussionmentioning

confidence: 99%

Polar‐biased localization of the cold stress‐induced RNA helicase, CrhC, in the Cyanobacterium Anabaena sp. strain PCC 7120

El-Fahmawi

Owttrim²

2003

Molecular Microbiology

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“…More complex regulatory models can be envisioned as well; for example, the buildup of translocation intermediates of other presecretory and membrane proteins could titrate away a factor that is needed to promote the translational pause release. A role for SecA RNA helicase activity in promoting secA autoregulation has been ruled out recently, since secA helicase-defective mutants showed normal secA regulation (46). This observation precludes models in which the translational pausing agent is an RNA secondary or tertiary structure that is unwound by SecA helicase activity in order to release the secM translational pause.…”

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confidence: 99%

Critical Regions of secM That Control Its Translation and Secretion and Promote Secretion-Specific secA Regulation

Sarker

Oliver

2002

J Bacteriol

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SecA is an essential ATP-driven motor protein that binds to presecretory or membrane proteins and the translocon and promotes the translocation or membrane integration of these proteins. secA is subject to a protein secretion-specific form of regulation, whereby its translation is elevated during secretion-limiting conditions. A novel mechanism that promotes this regulation involves translational pausing within the gene upstream of secA, secM. The secM translational pause prevents formation of an RNA helix that normally blocks secA translational initiation. The duration of this pause is controlled by the rate of secretion of nascent SecM, which in turn depends on its signal peptide and a functional translocon. We characterized the atypical secM signal peptide and found that mutations within the amino-terminal region specifically affect the secM translational pause and secA regulation, while mutations in the hydrophobic core region affect SecM secretion as well as translational pausing and secA regulation. In addition, mutational analysis of the 3 end of secM allowed us to identify a conserved region that is required to promote the translational pause that appears to be operative at the peptide level. Together, our results provide direct support for the secM translational pause model of secA regulation, and they pinpoint key sequences within secM that promote this important regulatory system.In bacteria nascent or fully synthesized presecretory or membrane proteins are selectively targeted to the translocon by interactions with SecB and SecA or the signal recognition particle and its receptor (3,24,40,47,50). These pathways converge at the translocon, which consists of the integral membrane proteins SecYEG and SecDFyajC and the peripheral membrane protein SecA ATPase. SecYE forms the preprotein channel and SecA receptor (10,20,27,30), while SecG and SecDFyajC greatly enhance the rate of protein translocation by regulating SecA membrane cycling (11,28,34). SecA is central to protein translocation since it binds to the signal peptides or transmembrane segments of presecretory and membrane proteins, interacts with the SecB chaperone to promote release of the bound preprotein, and acts as a motor protein to drive protein translocation at the translocon (for a review, see reference 26). Considerable evidence suggests that SecA undergoes ATP-driven cycles of insertion and retraction at SecYE, thereby promoting the stepwise translocation of proteins across the plasma membrane (12, 13, 52).The selectivity of the translocon for its protein cargo is remarkable, since erroneous translocation of cytoplasmic proteins is essentially undetectable. Current evidence suggests that the translocon possesses a proofreading activity that is responsible for aborting the translocation of preproteins that lack a functional signal peptide (for a review, see reference 7). prl alleles of secA, secY, secE, and secG have been isolated that allow translocation of preproteins with a defective signal peptide (4,15,17,23,48). A recent study sugge...

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“…These observations suggest that Motif III allows optimal DEAD motor response to individual substrates. Remarkably, SecA Motif III is required for both RNA helicase (Park et al , 1997; Schmidt et al , 2001) and protein translocase (Fig 1E) activities. This extraordinary example of enzymatic adaptation shows that the two seemingly unrelated chemistries may be catalysed by similar DEAD motor mechanisms.…”

Section: Discussionmentioning

confidence: 99%

“…The function of this tripeptide is poorly understood (Caruthers & McKay, 2002), but it has been implicated in coupling RNA binding to helicase activity (Pause & Sonenberg, 1992; Linder et al , 2000). Motif III of SecA, however, is known to be required for its RNA helicase activity (Schmidt et al , 2001). We now examine the possible additional role of helicase Motif III (Fig 1A–C) in protein translocation.…”

Section: Introductionmentioning

confidence: 99%

Helicase Motif III in SecA is essential for coupling preprotein binding to translocation ATPase

et al. 2004

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The SecA ATPase is a protein translocase motor and a superfamily 2 (SF2) RNA helicase. The ATPase catalytic core ('DEAD motor') contains the seven conserved SF2 motifs. Here, we demonstrate that Motif III is essential for SecA-mediated protein translocation and viability. SecA Motif III mutants can bind ligands (nucleotide, the SecYEG translocase 'channel', signal and mature preprotein domains), can catalyse basal and SecYEG-stimulated ATP hydrolysis and can be activated for catalysis. However, Motif III mutation specifically blocks the preprotein-stimulated 'translocation ATPase' at a step of the reaction pathway that lies downstream of ligand binding. A functional Motif III is required for optimal ligand-driven conformational changes and kinetic parameters that underlie optimal preprotein-modulated nucleotide cycling at the SecA DEAD motor. We propose that helicase Motif III couples preprotein binding to the SecA translocation ATPase and that catalytic activation of SF2 enzymes through Motif-III-mediated action is essential for both polypeptide and nucleic-acid substrates.

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Escherichia coli SecA Helicase Activity Is Not Required in Vivo for Efficient Protein Translocation or Autogenous Regulation

Cited by 20 publications

References 65 publications

Polar‐biased localization of the cold stress‐induced RNA helicase, CrhC, in the Cyanobacterium Anabaena sp. strain PCC 7120

Polar‐biased localization of the cold stress‐induced RNA helicase, CrhC, in the Cyanobacterium Anabaena sp. strain PCC 7120

Critical Regions of secM That Control Its Translation and Secretion and Promote Secretion-Specific secA Regulation

Helicase Motif III in SecA is essential for coupling preprotein binding to translocation ATPase

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