EH domains of AtEH1/Pan1 differ in their Ca2+-binding capacities. a Domain organization of AtEH1/Pan1 and AtEH2/Pan1. Both proteins contain two Eps15 homology domains (EH), a coiled-coil domain (CC), and an acidic (A)-motif. A schematic representation of a multiple sequence alignment (MSA) - shows strong conservation of the EH domains (blue lines) across the plant kingdom. Percentages indicate the relative number of identical amino acids. b–g Cartoon representation of the X-ray structure of EH1.1 and NMR/all-atom molecular dynamics structure of EH1.2. Ions are shown as orange (Ca2+) or grey (Na+) spheres. Insets show the ion coordination in each EF-hand loop. Ca2+ coordinating residues and water molecules (W) are indicated in (c, d) and (f, g).

Savvas N. Savvides, Kostas Tripsianes, Roman Pleskot, and Daniel Van Damme Research Groups

Significance

Clathrin-mediated endocytosis (CME) is the gatekeeper of the plasma membrane. In contrast to animals and yeasts, CME in plants depends on the TPLATE complex (TPC), an evolutionary ancient adaptor complex. The mechanistic contribution of the individual TPC subunits to plant CME remains however elusive. In this study, we used a multidisciplinary approach to elucidate the structural and functional roles of the evolutionary conserved N-terminal Eps15 homology (EH) domains of the TPC subunit AtEH1/Pan1. By integrating high-resolution structural information obtained by X-ray crystallography and NMR spectroscopy with all-atom molecular dynamics simulations, we provide structural insight into the function of both EH domains. Both domains bind phosphatidic acid with a different strength, and only the second domain binds phosphatidylinositol 4,5-bisphosphate. Unbiased peptidome profiling by mass-spectrometry revealed that the first EH domain preferentially interacts with the double N-terminal NPF motif of a previously unidentified TPC interactor, the integral membrane protein Secretory Carrier Membrane Protein 5 (SCAMP5). Furthermore, we show that AtEH/Pan1 proteins control the internalization of SCAMP5 via this double NPF peptide interaction motif. Collectively, our structural and functional studies reveal distinct but complementary roles of the EH domains of AtEH/Pan1 in plant CME and connect the internalization of SCAMP5 to the TPLATE complex.

Yperman, K., Papageorgiou, A. C., Merceron, R., De Munck, S., Bloch, Y., Eeckhout,D., Jiang, Q., Tack, P., Grigoryan, R., Evangelidis, T., Van Leene, J., Vincze, L., , Vandenabeele, P., Vanhaecke, F., Potocký, M., De Jaeger, G., Savvides, S. N. , Tripsianes , K., Pleskot, R.  & Van Damme D.: Distinct EH domains of the endocytic TPLATE complex confer lipid and protein binding, Nature Comm. (2021) **, **  https://doi.org/10.1038/s41467-021-23314-6

The essential fungal‐specific translation elongation factor 3 (eEF3) has been implicated in tRNA binding and release. Combined in vitro and in vivo analyses show that its critical is in release of E‐site‐tRNA from the ribosome during late steps of translocation.

Daniel N. Wilson Research Group

Significance

In addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the ribosomal A and E sites, its exact mechanism of action is unclear. Here, we show that eEF3 acts at the mRNA–tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl‐tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slowdown in translation elongation due to accumulation of ribosomes with an occupied A site. Cryo‐EM analysis of native eEF3‐ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non‐rotated ribosomal states, as well as by opening the L1 stalk to release the E‐site tRNA. Additionally, our analysis provides structural insights into novel translation elongation states, enabling presentation of a revised yeast translation elongation cycle.

Ranjan, N; Pochopien, A ; hih-Chien Wu, C; Beckert, B; Blanchet, S; Green, R; Rodnina, M; Wilson, DN: Yeast translation elongation factor eEF3 promotes late stages of tRNA translocation during RF3-mediated recycling of RF1 EMBO J (2021) 40: e106449; https://doi.org/10.15252/embj.2020106449