The conserved formin homology 2 (FH2) domain nucleates actin filaments and remains bound to the barbed end of the growing filament. Here we report the crystal structure of the yeast Bni1p FH2 domain in complex with tetramethylrhodamine-actin. Each of the two structural units in the FH2 dimer binds two actins in an orientation similar to that in an actin filament, suggesting that this structure could function as a filament nucleus. Biochemical properties of heterodimeric FH2 mutants suggest that the wild-type protein equilibrates between two bound states at the barbed end: one permitting monomer binding and the other permitting monomer dissociation. Interconversion between these states allows processive barbed-end polymerization and depolymerization in the presence of bound FH2 domain. Kinetic and/or thermodynamic differences in the conformational and binding equilibria can explain the variable activity of different FH2 domains as well as the effects of the actin-binding protein profilin on FH2 function.

The autophagy factor ATG12~ATG5 conjugate exhibits E3 ligase-like activity by which the lipidation of members of the LC3 family is facilitated. The crystal structure of the human ATG12~ATG5 conjugate bound to the amino-terminal region of ATG16L1, the factor that recruits the conjugate to autophagosomal membranes, reveals an integrated architecture in which ATG12 docks onto ATG5 through conserved residues. ATG12 and ATG5 are oriented such that other conserved residues on each molecule, including the conjugation junction, form a continuous patch. Mutagenesis data support the importance of both the ATG12–ATG5 interface and the continuous patch for E3 activity. The ATG12~ATG5 conjugate interacts with the E2 enzyme ATG3 with high-affinity through another surface location that is exclusive to ATG12, suggesting a different role of the continuous patch in E3 activity. These findings provide a foundation for understanding the mechanism of LC3 lipidation.

Diaphanous-related formins (DRFs) regulate dynamics of unbranched actin filaments during cell contraction and cytokinesis. DRFs are autoinhibited through intramolecular binding of a Diaphanous autoinhibitory domain (DAD) to a conserved N-terminal regulatory element. Autoinhibition is relieved through binding of the GTPase RhoA to the N-terminal element. We report the crystal structure of the dimeric regulatory domain of the DRF, mDia1. Dimerization is mediated by an intertwined six-helix bundle, from which extend two Diaphanous inhibitory domains (DIDs) composed of five armadillo repeats. NMR and biochemical mapping indicate the RhoA and DAD binding sites on the DID partially overlap, explaining activation of mDia1 by the GTPase. RhoA binding also requires an additional structurally independent segment adjacent to the DID. This regulatory construction, involving a GTPase binding site spanning a flexibly tethered arm and the inhibitory module, is observed in many autoinhibited effectors of Ras superfamily GTPases, suggesting evolutionary pressure for this design.

An enigmatic step in de novo formation of the autophagosome membrane compartment is the expansion of the precursor membrane phagophore, which requires the acquisition of lipids to serve as building blocks. Autophagy-related 2 (ATG2), the rod-shaped protein that tethers phosphatidylinositol 3-phosphate (PI3P)-enriched phagophores to the endoplasmic reticulum (ER), is suggested to be essential for phagophore expansion, but the underlying mechanism remains unclear. Here, we demonstrate that human ATG2A is a lipid transfer protein. ATG2A can extract lipids from membrane vesicles and unload them to other vesicles. Lipid transfer by ATG2A is more efficient between tethered vesicles than between untethered vesicles. The PI3P effectors WIPI4 and WIPI1 associate ATG2A stably to PI3P-containing vesicles, thereby facilitating ATG2A-mediated tethering and lipid transfer between PI3P-containing vesicles and PI3P-free vesicles. Based on these results, we propose that ATG2-mediated transfer of lipids from the ER to the phagophore enables phagophore expansion.

Autophagy is an enigmatic cellular process in which double-membrane compartments, called "autophagosomes, form de novo adjacent to the endoplasmic reticulum (ER) and package cytoplasmic contents for delivery to lysosomes. Expansion of the precursor membrane phagophore requires autophagy-related 2 (ATG2), which localizes to the PI3P-enriched ER-phagophore junction. We combined single-particle electron microscopy, chemical cross-linking coupled with mass spectrometry, and biochemical analyses to characterize human ATG2A in complex with the PI3P effector WIPI4. ATG2A is a rod-shaped protein that can bridge neighboring vesicles through interactions at each of its tips. WIPI4 binds to one of the tips, enabling the ATG2A-WIPI4 complex to tether a PI3P-containing vesicle to another PI3P-free vesicle. These data suggest that the ATG2A-WIPI4 complex mediates ER-phagophore association and/or tethers vesicles to the ER-phagophore junction, establishing the required organization for phagophore expansion via the transfer of lipid membranes from the ER and/or the vesicles to the phagophore.