Conformational State of the SecYEG-Bound SecA Probed by Single Tryptophan Fluorescence Spectroscopy

Natale, Paolo; Blaauwen, Tanneke den; Does, Chris van der; Driessen, Arnold J. M.

doi:10.1021/bi047488r

Cited by 17 publications

(17 citation statements)

References 54 publications

(151 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The ATP-binding pocket of SecA is formed between the two NBFs, but nucleotide binding to the isolated soluble form of SecA does not induce a large conformational change, and the catalytic arginine finger remains far from the ␥-phosphate of ATP (10), explaining the low intrinsic ATPase activity. In contrast, ATP-dependent conformational change is detectable for SecA in association with the SecYEG channel (33).…”

Section: Discussionmentioning

confidence: 84%

The Long α-Helix of SecA Is Important for the ATPase Coupling of Translocation

Mori

Ito

2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

SecA contains two ATPase folds (NBF1 and NBF2) and other interaction/regulatory domains, all of which are connected by a long helical scaffold domain (HSD) running along the molecule. Here we identified a functionally important and spatially adjacent pair of SecA residues, Arg-642 on HSD and Glu-400 on NBF1. A charge-reversing substitution at either position as well as disulfide tethering of these positions inactivated the translocation activity. Interestingly, however, the translocation-inactive SecA variants fully retained the ability to up-regulate the ATPase in response to a preprotein and the SecYEG translocon. The translocation defect was suppressible by second site alterations at the hinge-forming boundary of NBF2 and HSD. Based on these results, we propose that the motor function of SecA is realized by ligand-activated ATPase engine and its HSD-mediated conversion into the mechanical work of preprotein translocation.

show abstract

Section: Discussionmentioning

confidence: 84%

The Long α-Helix of SecA Is Important for the ATPase Coupling of Translocation

Mori

Ito

2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…SecA binds SecYEG with an even higher affinity than its association with the lipids (21). This interaction of SecA to SecYEG is likely to be important for sub-strate export, since it changes the conformation of SecA in an ATP-dependent fashion (37). Whether SecA2 interacts with the cell membrane or the translocon in a similar manner remains to be investigated.…”

Section: Discussionmentioning

confidence: 99%

Differential Localization of the Streptococcal Accessory Sec Components and Implications for Substrate Export

Yen

Cameron

Bensing

et al. 2012

Journal of Bacteriology

View full text Add to dashboard Cite

The accessory Sec system of Streptococcus gordonii is comprised of SecY2, SecA2, and five proteins (Asp1 through -5) that are required for the export of a serine-rich glycoprotein, GspB. We have previously shown that a number of the Asps interact with GspB, SecA2, or each other. To further define the roles of these Asps in export, we examined their subcellular localization in S. gordonii and in Escherichia coli expressing the streptococcal accessory Sec system. In particular, we assessed how the locations of these accessory Sec proteins were altered by the presence of other components. Using fluorescence microscopy, we found in E. coli that SecA2 localized within multiple foci at the cell membrane, regardless of whether other accessory Sec proteins were expressed. Asp2 alone localized to the cell poles but formed a similar punctate pattern at the membrane when SecA2 was present. Asp1 and Asp3 localized diffusely in the cytosol when expressed alone or with SecA2. However, these proteins redistributed to the membrane in a punctate arrangement when all of the accessory Sec components were present. Cell fractionation studies with S. gordonii further corroborated these microscopy results. Collectively, these findings indicate that Asp1 to -3 are not integral membrane proteins that form structural parts of the translocation channel. Instead, SecA2 serves as a docking site for Asp2, which in turn attracts a complex of Asp1 and Asp3 to the membrane. These protein interactions may be important for the trafficking of GspB to the cell membrane and its subsequent translocation.

show abstract

“…Tryptophan residues have been successfully used as fluorescent probes to investigate conformational changes in membrane proteins [6,7,12,14,[22][23][24][25][26][27][28][29][30]. Our recent success in functional hSGLT1 expression and purification from P. pastoris and identification of different conformational states of hSGLT1 in solution [7] raised the question whether these conformational changes can also be observed in proteoliposomes in a reconstituted form, an experimental condition closer to the in vivo situation of membrane proteins.…”

Section: Discussionmentioning

confidence: 99%