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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.


GST pulldown analysis of Arf1 and Arl1 interaction with Sec7ΔC. Stable interaction of Arf1 and Arl1 with S. cerevisiae Sec7ΔC was assayed by pulldown of Sec7 with a great excess of GST-GTPase bound to GDP (denoted D) or GMPPNP (denoted T*). Following SDS-PAGE, protein was visualized by Coomassie staining. There was no apparent nucleotide-dependent interaction observed between Sec7ΔC and Arf1-GTP or Arl1-GTP in this experiment.DOI:http://dx.doi.org/10.7554/eLife.12411.016
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fig4s3: GST pulldown analysis of Arf1 and Arl1 interaction with Sec7ΔC. Stable interaction of Arf1 and Arl1 with S. cerevisiae Sec7ΔC was assayed by pulldown of Sec7 with a great excess of GST-GTPase bound to GDP (denoted D) or GMPPNP (denoted T*). Following SDS-PAGE, protein was visualized by Coomassie staining. There was no apparent nucleotide-dependent interaction observed between Sec7ΔC and Arf1-GTP or Arl1-GTP in this experiment.DOI:http://dx.doi.org/10.7554/eLife.12411.016

Mentions: Another potential role of the DCB/HUS domain which would not have been uncovered in our in vitro assay is its interaction with Arl1 (Christis and Munro, 2012). Despite extensive effort, we could not detect any stable interaction between S. cerevisiae Sec7ΔC and Arl1 in the presence or absence of membranes, nor did preincubation of Sec7ΔC with membrane-bound Arl1 show any effect on catalytic rate (Figure 4—figure supplements 3–5). As we previously reported an interaction between Arl1 and longer Sec7 constructs (McDonold and Fromme, 2014), this interaction appears to depend on one or more of the HDS domains in addition to the DCB/HUS domain in S. cerevisiae Sec7.


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

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

GST pulldown analysis of Arf1 and Arl1 interaction with Sec7ΔC. Stable interaction of Arf1 and Arl1 with S. cerevisiae Sec7ΔC was assayed by pulldown of Sec7 with a great excess of GST-GTPase bound to GDP (denoted D) or GMPPNP (denoted T*). Following SDS-PAGE, protein was visualized by Coomassie staining. There was no apparent nucleotide-dependent interaction observed between Sec7ΔC and Arf1-GTP or Arl1-GTP in this experiment.DOI:http://dx.doi.org/10.7554/eLife.12411.016
© Copyright Policy
Related In: Results  -  Collection

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

fig4s3: GST pulldown analysis of Arf1 and Arl1 interaction with Sec7ΔC. Stable interaction of Arf1 and Arl1 with S. cerevisiae Sec7ΔC was assayed by pulldown of Sec7 with a great excess of GST-GTPase bound to GDP (denoted D) or GMPPNP (denoted T*). Following SDS-PAGE, protein was visualized by Coomassie staining. There was no apparent nucleotide-dependent interaction observed between Sec7ΔC and Arf1-GTP or Arl1-GTP in this experiment.DOI:http://dx.doi.org/10.7554/eLife.12411.016
Mentions: Another potential role of the DCB/HUS domain which would not have been uncovered in our in vitro assay is its interaction with Arl1 (Christis and Munro, 2012). Despite extensive effort, we could not detect any stable interaction between S. cerevisiae Sec7ΔC and Arl1 in the presence or absence of membranes, nor did preincubation of Sec7ΔC with membrane-bound Arl1 show any effect on catalytic rate (Figure 4—figure supplements 3–5). As we previously reported an interaction between Arl1 and longer Sec7 constructs (McDonold and Fromme, 2014), this interaction appears to depend on one or more of the HDS domains in addition to the DCB/HUS domain in S. cerevisiae Sec7.

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.