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

Richardson BC, Halaby SL, Gustafson MA, Fromme JC - Elife (2016)

Bottom Line: We demonstrate that the established role of the N-terminal region in dimerization is not conserved; instead, a C-terminal autoinhibitory domain is responsible for dimerization of Sec7.We find that the DCB/HUS domain amplifies the ability of Sec7 to activate Arf1 on the membrane surface by facilitating membrane insertion of the Arf1 amphipathic helix.This enhancing function of the Sec7 N-terminal domains is consistent with the high rate of Arf1-dependent trafficking to the plasma membrane necessary for maximal cell growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, United States.

ABSTRACT
The Golgi complex is the central sorting compartment of eukaryotic cells. Arf guanine nucleotide exchange factors (Arf-GEFs) regulate virtually all traffic through the Golgi by activating Arf GTPase trafficking pathways. The Golgi Arf-GEFs contain multiple autoregulatory domains, but the precise mechanisms underlying their function remain largely undefined. We report a crystal structure revealing that the N-terminal DCB and HUS regulatory domains of the Arf-GEF Sec7 form a single structural unit. We demonstrate that the established role of the N-terminal region in dimerization is not conserved; instead, a C-terminal autoinhibitory domain is responsible for dimerization of Sec7. We find that the DCB/HUS domain amplifies the ability of Sec7 to activate Arf1 on the membrane surface by facilitating membrane insertion of the Arf1 amphipathic helix. This enhancing function of the Sec7 N-terminal domains is consistent with the high rate of Arf1-dependent trafficking to the plasma membrane necessary for maximal cell growth.

No MeSH data available.


Expression of HUS box mutants.Expression of the indicated GFP-Sec7f alleles was assayed by anti-GFP immunoblot.DOI:http://dx.doi.org/10.7554/eLife.12411.025
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4764562&req=5

fig6s4: Expression of HUS box mutants.Expression of the indicated GFP-Sec7f alleles was assayed by anti-GFP immunoblot.DOI:http://dx.doi.org/10.7554/eLife.12411.025

Mentions: A notable absence from both the structure presented here and the available structures of the GEF domain is the highly conserved ‘HUS-box’ located between helix 15 and the helix 16 truncated from our crystallized construct (Mouratou et al., 2005). A previous study reported that the HUS-box was required for a yeast 2-hybrid interaction between the DCB and HUS domains (Ramaen et al., 2007), but our structural data do not support such a role. To assess whether the HUS-box plays a role in GEF stimulation by the DCB/HUS domain, we generated single alanine mutants in the HUS-box (Figure 6D—figure supplements 3 and 4). One mutation, N653A, was both inviable in the sensitized strain and compatible with purification. The N653A Sec7ΔC construct showed no difference in GEF activity compared to the wild-type allele in the presence of membranes, indicating that the HUS-box plays no role in the observed exchange rate enhancement by the DCB/HUS domain.


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

Richardson BC, Halaby SL, Gustafson MA, Fromme JC - Elife (2016)

Expression of HUS box mutants.Expression of the indicated GFP-Sec7f alleles was assayed by anti-GFP immunoblot.DOI:http://dx.doi.org/10.7554/eLife.12411.025
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4764562&req=5

fig6s4: Expression of HUS box mutants.Expression of the indicated GFP-Sec7f alleles was assayed by anti-GFP immunoblot.DOI:http://dx.doi.org/10.7554/eLife.12411.025
Mentions: A notable absence from both the structure presented here and the available structures of the GEF domain is the highly conserved ‘HUS-box’ located between helix 15 and the helix 16 truncated from our crystallized construct (Mouratou et al., 2005). A previous study reported that the HUS-box was required for a yeast 2-hybrid interaction between the DCB and HUS domains (Ramaen et al., 2007), but our structural data do not support such a role. To assess whether the HUS-box plays a role in GEF stimulation by the DCB/HUS domain, we generated single alanine mutants in the HUS-box (Figure 6D—figure supplements 3 and 4). One mutation, N653A, was both inviable in the sensitized strain and compatible with purification. The N653A Sec7ΔC construct showed no difference in GEF activity compared to the wild-type allele in the presence of membranes, indicating that the HUS-box plays no role in the observed exchange rate enhancement by the DCB/HUS domain.

Bottom Line: We demonstrate that the established role of the N-terminal region in dimerization is not conserved; instead, a C-terminal autoinhibitory domain is responsible for dimerization of Sec7.We find that the DCB/HUS domain amplifies the ability of Sec7 to activate Arf1 on the membrane surface by facilitating membrane insertion of the Arf1 amphipathic helix.This enhancing function of the Sec7 N-terminal domains is consistent with the high rate of Arf1-dependent trafficking to the plasma membrane necessary for maximal cell growth.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, United States.

ABSTRACT
The Golgi complex is the central sorting compartment of eukaryotic cells. Arf guanine nucleotide exchange factors (Arf-GEFs) regulate virtually all traffic through the Golgi by activating Arf GTPase trafficking pathways. The Golgi Arf-GEFs contain multiple autoregulatory domains, but the precise mechanisms underlying their function remain largely undefined. We report a crystal structure revealing that the N-terminal DCB and HUS regulatory domains of the Arf-GEF Sec7 form a single structural unit. We demonstrate that the established role of the N-terminal region in dimerization is not conserved; instead, a C-terminal autoinhibitory domain is responsible for dimerization of Sec7. We find that the DCB/HUS domain amplifies the ability of Sec7 to activate Arf1 on the membrane surface by facilitating membrane insertion of the Arf1 amphipathic helix. This enhancing function of the Sec7 N-terminal domains is consistent with the high rate of Arf1-dependent trafficking to the plasma membrane necessary for maximal cell growth.

No MeSH data available.