• Figure 3.
    Download figureOpen in new tabFigure 3. The HDS4 domain of Sec7 is important for function but dispensable for TGN localization. 

    (A) Centromeric plasmids encoding GFP-Sec7 constructs expressed from the SEC7 promoter were introduced into a SEC7 plasmid shuffling strain (CFY409). Growth on 5-FOA measures the ability of the construct to complement the sec7∆ mutation. (B) The same plasmids were imaged in an otherwise wild-type strain expressing endogenously tagged Sec7-RFPMars (‘Sec7-RFP’) (CFY578).

    DOI: http://dx.doi.org/10.7554/eLife.12411.012

    Figure 5.
    Download figureOpen in new tabFigure 5. Conserved DCB/HUS surface regions mediate Sec7 function. 

    (A) Locations of all S. cerevisiae mutants tested are shown as space-filling spheres mapped on the T. terrestris structure, with backbone colored by conservation. Positions of mutations resulting in stronger temperature sensitive growth phenotypes are colored red (e.g., S. cerevisiae Q365/R368), positions with weaker phenotypes are colored orange (e.g., S. cerevisiae D297/K301/F305), and positions with no growth phenotype are colored blue; the position corresponding to the sec7-1 mutation (S402L) is colored magenta. (B) Using a plasmid-shuffling assay, CEN plasmids bearing GFP-tagged Sec7 or Sec7f with the indicated mutations expressed via their endogenous promoter were tested for their ability to rescue a genomic sec7 deletion in a sensitized arf1Δ/ARF2 strain (CFY863). Growth of serial 10-fold dilutions after 3 days at 37°C is shown, comparing the shuffled strains on 5-FOA to their parents growing in parallel on synthetic complete media (SC). Changes in the number or size of colonies indicates a growth defect.

    DOI: http://dx.doi.org/10.7554/eLife.12411.020

    Figure 7.
    Download figureOpen in new tabFigure 7. The DCB/HUS domain can inhibit GEF activity in trans

    (A) S. cerevisiae Sec7ΔC and isolated GEF constructs were assayed for rate of nucleotide exchange of myristoylated Arf1 in the presence of liposomes, with 16-fold excess DCB/HUS construct or sixfold additional liposomes added as indicated. (B) Wild-type S. cerevisiae Sec7ΔC was assayed for nucleotide exchange of myristoylated Arf1 in the presence of liposomes and a 12-fold excess of DCB/HUS constructs bearing the indicated mutations. The range of activity of interest is bounded by the WT and mock rates and is left unshaded. (C) A speculative model of DCB/HUS domain and GEF domain cooperation in Arf1 activation.

    DOI: http://dx.doi.org/10.7554/eLife.12411.026

  • Table 1.

    Data collection and refinement statistics

    DOI: http://dx.doi.org/10.7554/eLife.12411.007

    T. terrestris Sec7 DCB/HUS domain (residues 1-458)
    Wavelength (Å)0.987
    Resolution range (Å)50 - 2.6 (2.64–2.6)
    Space groupP 21 21 21
    Unit cella=62.472Å b=132.024Å c=247.664Å
    α=β=γ=90°
    Total reflections569136
    Unique reflections59606
    Multiplicity9.5 (4.7)
    Completeness (%)98.77 (88.46)
    Mean I/sigma(I)8.46 (1.48)
    Wilson B-factor65.26
    R-work0.2119 (0.3113)
    R-free0.2568 (0.3665)
    Number of atoms10944
    Macromolecules10887
    Water57
    Protein residues1383
    RMS(bonds)0.009
    RMS(angles)1.23
    Ramachandran favored (%)98
    Ramachandran outliers (%)0.075
    Clashscore7.63
    Average B-factor89.7
    Macromolecules89.8
    Solvent60.3