TY  - GEN
T1  - Isolated THATCH domain of End4 is unable to bind F-actin independently in the fission yeast Schizosaccharomyces pombe
AU  - Ren, Yuan
AU  - Berro, Julien
DO  - 10.17912/micropub.biology.000508
UR  - http://beta.micropublication.org/journals/biology/micropub-biology-000508/
AB  - End4 is an endocytic protein that mediates the connection between the lipid membrane and the actin cytoskeleton in fission yeast (Gottfried, Ehrlich, and Ashery 2010, 4; Iwaki et al. 2004, 4; Lacy et al. 2018, 4; Engqvist-Goldstein et al. 2001; Skruzny et al. 2012). The N-terminus of End4 binds to PIP2 on the membrane together with Ent1p, and the C-terminus of End4 shares sequence similarities to the actin binding domain of talin, and is therefore named THATCH (talin-HIP1/R/Sla2p actin-tethering C-terminal homology) (Legendre-Guillemin 2004, 4; Baggett, D’Aquino, and Wendland 2003, 4; McCann and Craig 1997; Brett et al. 2006). The crystal structures of THATCH domains from HIP1R and talin are five-helix-bundles (Brett et al. 2006; Gingras et al. 2008). The THATCH domain of Sla2 is critical for linking F-actin to the endocytic coat during CME in budding yeast (Baggett, D’Aquino, and Wendland 2003, 2; Brett et al. 2006; Abella et al. 2021). The affinity of purified THATCH domains for actin filaments has been measured in vitro by multiple groups (Brett et al. 2006; Gingras et al. 2008; Senetar, Foster, and McCann 2004). Although structurally similar, the THATCH domain from HIP1R has the lowest affinity to actin among all tested THATCH domains, in some cases not significantly higher than that of the negative control with purified GST (Senetar, Foster, and McCann 2004). This discrepancy in the actin-binding ability of the THATCH domain from in vitro and in vivo data could be explained by cryptic actin-binding sites that are activated in vivo, by a) post-translational modifications, b) the binding of other molecules, or c) force transmitted through the rest of the End4 molecule. All three binding site activation mechanisms have been found on talin, the molecule responsible for connecting the membrane to F-actin during cell adhesion through its 13 rod domains including the THATCH domain (Goult, Yan, and Schwartz 2018; Goult, Brown, and Schwartz 2021). A clear way to distinguish mechanism c) from a) and b) is through in vivo protein cleaving. By isolating the THATCH domain of End4 from the rest of End4 in the cytoplasm of the fission yeast, the native environment for possible post-translational modifications or binding partners of THATCH are preserved, but force transmission to the THATCH domain is prevented. Consequently, the cellular localization of the isolated THATCH domain is indicative of its direct affinity to F-actin in vivo.
PY  - 2022
JO  - microPublication Biology
ER  -