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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.


Viability of HUS box mutants.Missense mutations in the HUS box were tested for viability at room temperature by plasmid shuffle, spotted in half-log dilutions left to right.DOI:http://dx.doi.org/10.7554/eLife.12411.024
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fig6s3: Viability of HUS box mutants.Missense mutations in the HUS box were tested for viability at room temperature by plasmid shuffle, spotted in half-log dilutions left to right.DOI:http://dx.doi.org/10.7554/eLife.12411.024

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)

Viability of HUS box mutants.Missense mutations in the HUS box were tested for viability at room temperature by plasmid shuffle, spotted in half-log dilutions left to right.DOI:http://dx.doi.org/10.7554/eLife.12411.024
© Copyright Policy
Related In: Results  -  Collection

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

fig6s3: Viability of HUS box mutants.Missense mutations in the HUS box were tested for viability at room temperature by plasmid shuffle, spotted in half-log dilutions left to right.DOI:http://dx.doi.org/10.7554/eLife.12411.024
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.