The Sec7 N-terminal regulatory domains facilitate membrane-proximal activation of the Arf1 GTPase
- Cornell University, United States
Figures
Figure 1 with 3 supplements
Crystal structure of T. terrestris Sec7 DCB/HUS domain (residues 1–458)
(A) Chains A (green) and D (white) are shown; chain A is colored light to dark N to C, and helices are numbered N to C. The entire asymmetric unit is shown in supplement 1. Electron density is shown …
Figure 1—figure supplement 1
Asymmetric unit of crystal structure.
The entire asymmetric unit is shown on the left, with each chain colored light to dark N to C. A=green, B=red, C=blue, D=gray. The asymmetric unit is shown in B-factor putty form on the right …
Figure 1—figure supplement 2
Electron density of crystal structure.
Weighted composite omit maps of helix 4 including residues D297, K301, and F305 are shown. Chain A, above, was used for structural analysis; chain D, below, was modeled on the basis of chain A.
Figure 1—figure supplement 3
Magnified view of the DCB/HUS interface.
Packing of helices 7 and 8 (DCB) and 10 and 11 (HUS) is shown, including participating side chains. Conserved residues are colored in green.
Figure 2 with 3 supplements
Sec7 dimerizes primarily via the HDS 4 domain.
(A) CORAL (Petoukhov et al., 2012) was used to fit the T. terrestris Sec7 DCB/HUS domain structure (residues 1–458) to SAXS data collected on the same construct, accounting for the presence of …
Figure 2—figure supplement 1
SAXS analysis of S. cerevisiae Sec7ΔC.
BUNCH (Petoukhov and Svergun, 2005) and CRYSOL (Svergun et al., 1995) were used to model T. terrestris Sec7 DCB/HUS domain (residues 1–458), S. cerevisiae Gea2 GEF domain (residues 570–714), and …
Figure 2—figure supplement 2
MALS analysis of S. cerevisiae Gea2.
Solution molecular weights of the indicated S. cerevisiae Gea2 constructs were determined by MALS.
Figure 2—figure supplement 3
MALS traces of S. cerevisiae and T. terrestris Sec7 constructs.
Normalized gel filtration peak profiles of all Sec7 constructs are shown with MALS molecular weight measurements superimposed. The right-hand molecular weight axis for each panel is scaled to each …
Figure 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 …
Figure 4 with 6 supplements
Stimulation of GEF activity by the DCB/HUS domain depends on the presence of lipids.
(A) Triplicate nucleotide exchange curves of S. cerevisiae Sec7ΔC (green traces) or isolated GEF domain constructs (yellow traces) acting on myristoylated Arf1 substrate in the presence of liposomes …
Figure 4—figure supplement 1
Measurement of Sec7 GEF kinetics by liposome flotation.
2 μM myristoylated Arf1-GDP was incubated with liposomes, excess GMPPNP, and. 67 μM Sec7ΔC or GEF domain for the indicated amount of time and put on ice to stop the reaction. Lipid-bound protein …
Figure 4—figure supplement 2
GEF activity of T. terrestris Sec7.
T. terrestris Sec7ΔC and isolated GEF constructs were assayed for rate of nucleotide exchange of myristoylated Arf1 in the presence of liposomes (left) and ΔN17Arf1 in the absence of liposomes …
Figure 4—figure supplement 3
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, …
Figure 4—figure supplement 4
Liposome pelleting analysis of Arf1 and Arl1 interaction with Sec7ΔC.
Stable interaction of membrane-bound Arf1 and Arl1 with S. cerevisiae Sec7ΔC (as a potential effector) was assayed in the context of membranes by liposome pelleting as described previously (Paczkowsk…
Figure 4—figure supplement 5
Effect of Arl1 preincubation on Sec7 GEF activity.
S. cerevisiae Sec7ΔC was assayed for rate of nucleotide exchange of Arf1 in the presence of liposomes and Arl1-GMPPNP as described previously (McDonold and Fromme, 2014).
Figure 4—figure supplement 6
Purity of constructs used for kinetic assays.
2.5 μg of each construct used for biochemical assays were separated by SDS-PAGE and stained by Coomassie to assess purity.
Figure 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 …
Figure 6 with 4 supplements
Helices 4 and 6 of the DCB/HUS domain mediate GEF stimulation.
(A) sec7Δ/arf1Δ strains bearing the indicated GFP-Sec7 alleles on a centromeric plasmid expressed from the SEC7 promoter were imaged at permissive and restrictive temperatures. (B) Surface residue …
Figure 6—figure supplement 1
Atomic basis of the sec7-1 phenotype.
The temperature sensitive allele sec7-1 represents an S402L mutation, corresponding to T. terrestris Sec7 residue S156. This serine stabilizes a loop near the interface between the DCB and HUS …
Figure 6—figure supplement 2
Sec7ΔC mutant activity in the absence of membranes.
Purifiable mutants in the S. cerevisiae Sec7ΔC construct were assayed for rate of nucleotide exchange of ΔN17Arf1 in the absence of membranes.
Figure 6—figure supplement 3
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.
Figure 6—figure supplement 4
Expression of HUS box mutants.
Expression of the indicated GFP-Sec7f alleles was assayed by anti-GFP immunoblot.
Figure 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 …
Tables
Table 1
Data collection and refinement statistics
| T. terrestris Sec7 DCB/HUS domain (residues 1-458) | |
|---|---|
| Wavelength (Å) | 0.987 |
| Resolution range (Å) | 50 - 2.6 (2.64–2.6) |
| Space group | P 21 21 21 |
| Unit cell | a=62.472Å b=132.024Å c=247.664Å α=β=γ=90° |
| Total reflections | 569136 |
| Unique reflections | 59606 |
| Multiplicity | 9.5 (4.7) |
| Completeness (%) | 98.77 (88.46) |
| Mean I/sigma(I) | 8.46 (1.48) |
| Wilson B-factor | 65.26 |
| R-work | 0.2119 (0.3113) |
| R-free | 0.2568 (0.3665) |
| Number of atoms | 10944 |
| Macromolecules | 10887 |
| Water | 57 |
| Protein residues | 1383 |
| RMS(bonds) | 0.009 |
| RMS(angles) | 1.23 |
| Ramachandran favored (%) | 98 |
| Ramachandran outliers (%) | 0.075 |
| Clashscore | 7.63 |
| Average B-factor | 89.7 |
| Macromolecules | 89.8 |
| Solvent | 60.3 |
Additional files
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Supplementary file 1
T. terrestris intron assignment
C. thermophilum, M. thermophila, and T. terrestris Sec7 genomic sequences, each containing a single annotated intron in the Sec7ΔC region, are aligned with annotated introns shown in lowercase. Conservation suggests that the T. terrestris intron should instead match that of the other two species; the intron assignment assumed for this work is highlighted in gray, and this correction to the construct was required for its expression (not shown).
- https://doi.org/10.7554/eLife.12411.027
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Supplementary file 2
Plasmids and strain tables
- https://doi.org/10.7554/eLife.12411.028
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Supplementary file 3
Estimation of exocytic Arf1 flux.
Values for Arf1 trafficking calculations are provided.
- https://doi.org/10.7554/eLife.12411.029
