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. 2022 Aug 30;40(9):111282.
doi: 10.1016/j.celrep.2022.111282.

Structural basis for activation of Arf1 at the Golgi complex

Arnold J Muccini  1 , Margaret A Gustafson  1 , J Christopher Fromme  2
Affiliations

Structural basis for activation of Arf1 at the Golgi complex

Arnold J Muccini et al. Cell Rep. .

Abstract

The Golgi complex is the central sorting station of the eukaryotic secretory pathway. Traffic through the Golgi requires activation of Arf guanosine triphosphatases that orchestrate cargo sorting and vesicle formation by recruiting an array of effector proteins. Arf activation and Golgi membrane association is controlled by large guanine nucleotide exchange factors (GEFs) possessing multiple conserved regulatory domains. Here we present cryoelectron microscopy (cryoEM) structures of full-length Gea2, the yeast paralog of the human Arf-GEF GBF1, that reveal the organization of these regulatory domains and explain how Gea2 binds to the Golgi membrane surface. We find that the GEF domain adopts two different conformations compatible with different stages of the Arf activation reaction. The structure of a Gea2-Arf1 activation intermediate suggests that the movement of the GEF domain primes Arf1 for membrane insertion upon guanosine triphosphate binding. We propose that conformational switching of Gea2 during the nucleotide exchange reaction promotes membrane insertion of Arf1.

Keywords: ARF1; CP: Cell biology; GTPase; Golgi; cryoEM; membrane; membrane insertion.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

Figure 1.
Figure 1.. Structure of Gea2 determined by cryoEM
(A) Schematic of Gea2 primary structure indicating conserved domains. DCB, dimerization and cyclophilin binding; HUS, homology upstream of Sec7; GEF, guanine nucleotide exchange factor (also known as “Sec7 domain”); HDS, homology downstream of Sec7. (B) CryoEM density of the Gea2 dimer in its closed/open conformation. One monomer adopts an open conformation of the GEF domain and the other monomer adopts a closed conformation. The GEF-HDS1 linker is colored magenta. (C) Atomic model of the Gea2 dimer, shown in cartoon depiction. (D) Close-up view of the closed monomer. (E) Close-up view of the open monomer. See also Figures S1-S5 and S7.
Figure 2.
Figure 2.. Gea2 dimerizes via the DCB-HUS domains
(A) Gea2 dimer, with a dashed box indicating the region depicted in (B) and (C). (B) View of the dimerization interface peeled apart and colored by calculated charge potential. (C) View of the dimerization interface peeled apart and colored by hydrophobicity. (D) Close-up view highlighting a homotypic hydrophobic interaction at the dimer interface. (E) Close-up view highlighting electrostatic interactions at the dimer interface. (F) Close-up view highlighting hydrophobic interactions at the dimer interface.
Figure 3.
Figure 3.. A conserved amphipathic α helix mediates Gea2 membrane binding
(A) Gea2 depicted on a modeled membrane surface. (B) Close-up view of the amphipathic helix predicted by both secondary and tertiary structure prediction methods but absent from the experimentally determined cryoEM density. The structural model determined by cryoEM is superimposed onto the AlphaFold prediction (Jumper et al., 2021). The AlphaFold prediction is colored by conservation, with dark red representing the most conserved residues and cyan representing the least conserved residues. (C) Sequence alignment highlighting conservation of the helix; colors highlight conserved residues based on their biochemical properties. (D) Helical wheel indicating the amphipathic nature of the helix. Red box indicates Tyr residue mutated for functional experiments. (E) GEA2 complementation test (plasmid shuffling). (F) Localization analysis of Gea2 and amphipathic helix mutants. Scale bar, 2 μm. (G) In vitro membrane-binding assay (liposome pelleting) using purified proteins and synthetic liposomes. S, supernatant; P, pellet. ***p < 0.001. (H) In vitro GEF activity assay using purified Gea2 proteins (200 nM), purified myristoylated-Arf1 substrate (1 μM), and synthetic liposomes. nd, not detectable. (I) In vitro GEF activity assay using purified Gea2 proteins (25 nM) and the ΔN17-Arf1 substrate (500 nM) without liposomes. ns, not significant. For data quantitation in (G), (H), and (I), data are presented as mean (bars) and individual data values (closed circles). Error bars are 95% confidence intervals.
Figure 4.
Figure 4.. CryoEM structure of a Gea2-Arf1 activation intermediate complex
(A) Schematic of the Gea2-Arf1 activation intermediate complex used for cryoEM. (B) CryoEM density of the Gea2-Arf1 complex, colored and labeled as in Figure 1, with Arf1 colored purple. (C) Atomic model of the Gea2-Arf1 complex. (D) Views of the Gea2 GEF domain and GEF-HDS1 linker for each of the three conformations adopted by Gea2 in the Gea2 only (closed and open) and Arf1-bound conformations. See also Figures S5-S7.
Figure 5.
Figure 5.. Steric constraints appear to enforce Gea2 conformational change
(A) Structure of the closed/closed Gea2 dimer shown for context. (B) Structure of the Gea2-Arf1 complex shown for context. (C) Close-up view of the modeled Gea2 closed-Arf1-GDP complex. (D) Close-up view of the modeled Gea2 closed-Arf1-NF (nucleotide-free) complex. (E) Close-up view of the Gea2-Arf1-NF cryoEM structure. (F) Magnified view of (C). (G) Magnified view of (D). Note the steric clash between Arf1 and the GEF-HDS1 linker. (H) Magnified view of (E). (I) Comparison of the modeled closed/closed Gea2-Arf1-GDP complex with the modeled open/open Gea2-Arf1-GDP complex. Note how in the closed conformation, the GEF domain appears more readily able to encounter freely diffusing Arf1-GDP, compared with the open conformation.
Figure 6.
Figure 6.. Model for activation of Arf1 by Gea2 on the Golgi membrane surface
(A) Gea2 in the closed/closed conformation shown for context. (B) In step 1, at least one of the Gea2 monomers adopts the closed conformation while bound to the membrane surface (the cryoEM structure of one side of the closed/closed conformation is shown on a modeled membrane). (C) In step 2, Arf1-GDP binds to the GEF domain (the modeled closed-Arf1-GDP complex is shown). (D) In step 3,GDP dissociates from Arf1 (Arf1-NF = nucleotide-free), and the resulting conformation change in Arf1 causes the GEF domain to switch from the closed state to an open state in order to avoid steric clash with Arf1 (the Gea2-Arf1 cryoEM structure is shown). (E) In step 4, GTP binding causes another conformation change in Arf1, resulting in folding of its amphipathic helix (colored red) at the membrane surface and dissociation from Gea2 (the NMR structure of Arf1-GTP and cryoEM structure of the closed/closed conformation of Gea2 are shown). The structures of Arf1-GDP and Arf1-GTP were derived from RCSB entries PDB: 1R8S (Renault et al., 2003) and 2KSQ (Liu et al., 2010). See also Video S1.
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