Decision letter | The Sec7 N-terminal regulatory domains facilitate membrane-proximal activation of the Arf1 GTPase

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The Sec7 N-terminal regulatory domains facilitate membrane-proximal activation of the Arf1 GTPase

Decision letter

Affiliation details

Cornell University, United States
Chris G Burd, Reviewing editor, Yale University, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your work entitled "The Sec7 N-terminal regulatory domains facilitate membrane-proximal activation of the Arf1 GTPase" for consideration by eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors. The evaluation has been overseen by the Reviewing Editor and Randy Schekman as the Senior Editor.

The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.

Summary:

A structure of the T. terrestris DCB/HUS domain of Sec7 is described and the role of these domains in GEF activity toward Arf1 has been investigated. The structure work is well done and is clearly presented. in vitro GEF activity assays show that nucleotide exchange on Arf1 by the Sec7 GEF domain is potentiated by the adjacent DCB/HUS domain in the presence of liposomes, and that this requires the N-terminal helix of Arf1. The authors conclude that the DCB/HUS domain facilitates lipid insertion of the Arf1 amphipathic helix.

Essential revisions:

The reviewers agreed that the major conclusion of the paper, that the DCB/HUS domain facilitates lipid insertion of the Arf1 amphipathic helix, is not adequately supported by the data. While they consider the authors' interpretation of their data to be reasonable, it is not directly supported with experimental data; all that is shown is Trp fluorescence measurements as a proxy for nucleotide exchange. This is necessary to address, particularly since early work on Arf1 (see below) raises an alternative interpretation than the authors' whereby the DCB/HUS domain increase the specific activity of the GEF domain without directly promoting membrane binding by the N-terminal helix. Related, the reviewers are also concerned that the Trp fluorescence assay used to infer nucleotide exchange may be misleading when assayed in the presence of lipids due to the potential that these agents influence the local environment of the Trp residue that reports the occupancy state of the nucleotide binding site. In order to be further considered by eLife, the paper will need to provide direct evidence supporting the model that the DCB-HUS domain acts directly on the N-terminal helix of Arf to promote its insertion into the membrane. With regard to the effects of lipids on Arf Trp fluorescence, the reviewers noted that this latter concern might be adequately addressed by a more thorough description of the assay methods, including the strategy used to account for changes in fluorescence not due to nucleotide binding (i.e., in the text).

Background: DPMC micelles stimulate the intrinsic rate of hydrolysis on Arf1 (Khan and Gilman 1986). This underscores a concern with the authors' models. Does the DCB-HUS domain promote Arf1 membrane insertion or, alternatively, does DCB-HUS increase the specific activity of the GEF domain when presented when the N-domain of Arf1 is – through whatever mechanism – already pried away from its pocket in Arf1:GDP. Indeed, that specific conformational change (characterized by NMR) has been suggested to be a prerequisite for GTP binding:

"The [yeast Myr-Arf1:GDP] structure clearly places the myristoyl chain in a hydrophobic groove between a C-terminal helix and a loop connecting b strands 3 and 4. The positioning suggests a clash with this loop would occur as it exists in the GTP-bound form, a fact that may require membrane association to accommodate an expelled myristoyl chain before GDP exchange can occur." (Liu, Khan, Prestegard PMID: 19141284). A possibility consistent with the authors' membrane-first model is that the DCB-HUS domain might actually allow Arf1-GDP to stably associate with the membrane (in the absence or presence of the GEF domain). That might be a pretty straightforward floatation or pelleting experiment.

DOI: http://dx.doi.org/10.7554/eLife.12411.032