Abstract
The Golgi complex is the central membrane and protein-sorting station in eukaryotic cells. Activation of Arf (ADP-ribosylation factor) GTPases is essential for vesicle formation via recruitment of cargo adaptors and coat proteins necessary for Golgi trafficking. Arf activation is spatially and temporally regulated by distinct guanine nucleotide exchange factors (GEFs) at different Golgi compartments. The yeast Arf-GEF Sec7 is a conserved and essential activator of Arf1 at the trans-Golgi network. Sec7 contains a highly conserved regulatory region, the homology upstream of Sec7 (HUS) box, with an unknown mechanistic role. In this study we explore how the HUS box, which is N-terminal to the catalytic domain, acts together with C-terminal regulatory domains in the allosteric activation of Sec7. We report that mutation of the HUS box disrupts positive feedback and allosteric activation of Sec7 by the GTPase Ypt31, a yeast Rab11 homolog. Taken together, our results support a model in which the inter- and intramolecular interactions of the HUS box and the C terminus are necessary for the allosteric activation of Sec7.
Highlights
Transport of proteins and membranes within eukaryotic cells requires the biogenesis of membrane transport carriers such as vesicles and tubules
These results indicate that the homology upstream of Sec7 (HUS) box is important for Sec7 function and that the N653A and D655A single mutations represent alleles that are likely partially compromised for HUS box function
Regulatory domains surrounding the catalytic guanine nucleotide exchange factors (GEFs) domain are involved in recruitment of Sec7 to the membrane, and the C terminus is required for autoinhibition and allosteric activation [12, 14, 16]
Summary
We previously reported that the HUS box is important for Sec function, because the N653A mutation was lethal in a. The N653A mutation reduced the activity of the Sec7⌬CϩHDS1 construct to a level similar to that of the Sec7⌬C construct (Fig. 4, A and B) This suggested to us that the HUS box may be required for the HDS1 domain to exert its positive-feedback regulatory role in membrane association of Sec. HUS box had no effect on localization to punctate structures (Fig. 5B) This result is consistent with the in vitro membrane-binding assay results and indicates that the HDS2– 4 domains enable recruitment of Sec to the Golgi even when the HUS box is compromised by mutation. To test whether the HUS box was important for the allosteric activation of Sec by Ypt31/32, we performed GEF assays in the presence of the membrane-anchored Rab protein Ypt activated by binding to GTP. Required for the allosteric stimulation of Sec GEF activity by Ypt (Fig. 7)
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