Light-triggered and phosphorylation-dependent 14-3-3 association with NON-PHOTOTROPIC HYPOCOTYL 3 is required for hypocotyl phototropism

Abstract

NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) is a key component of the auxin-dependent plant phototropic growth response. We report that NPH3 directly binds polyacidic phospholipids, required for plasma membrane association in darkness. We further demonstrate that blue light induces an immediate phosphorylation of a C-terminal 14-3-3 binding motif in NPH3. Subsequent association of 14-3-3 proteins is causal for the light-induced release of NPH3 from the membrane and accompanied by NPH3 dephosphorylation. In the cytosol, NPH3 dynamically transitions into membraneless condensate-like structures. The dephosphorylated state of the 14-3-3 binding site and NPH3 membrane recruitment are recoverable in darkness. NPH3 variants that constitutively localize either to the membrane or to condensates are non-functional, revealing a fundamental role of the 14-3-3 mediated dynamic change in NPH3 localization for auxin-dependent phototropism. This regulatory mechanism might be of general nature, given that several members of the NPH3-like family interact with 14-3-3 via a C-terminal motif.

BRXL4‐LAZY1 interaction at the plasma membrane controls Arabidopsis branch angle and gravitropism

“…Proteins may associate with membranes via basic and hydrophobic (BH) domains (Barbosa et al., 2016 ). A software program (Brzeska et al., 2010 ; https://hpcwebapps.cit.nih.gov/bhsearch/ ) that successfully predicted membrane‐associating BH domains in Arabidopsis proteins (Reuter et al., 2021 ) predicted two BH domains in BRXL4, one in each of the BRX domains (Figure 2a ). The same analysis of the LAZY1 sequence predicted a BH domain in region I (Figure 2b ) that included the residues previously found to be necessary for localizing LAZY1 to the plasma membrane (Yoshihara et al., 2013 ).…”

Phototropin phosphorylation of ROOT PHOTOTROPISM 2 and its role in mediating phototropism, leaf positioning, and chloroplast accumulation movement in Arabidopsis

“…Mutation of the RxSΦS phosphorylation site in NPH3 reduced its functionality for phototropism in etiolated seedlings (Reuter et al., 2021, Sullivan et al., 2021). Phototropism was also partially reduced in transgenic seedlings expressing the S591A mutant of RPT2 under the same conditions (Figure 6b) indicating that phot phosphorylation of S591 and concomitant 14‐3‐3 binding within the C‐terminus contributes to full phototropic responsiveness.…”

“…In the case of NPH3, one consequence of RxSΦS phosphorylation is to elicit early cellular events such as dephosphorylation (Reuter et al., 2021; Sullivan et al., 2021) at residues within the N‐terminal region of the protein (Kimura et al., 2020) as well as changes in subcellular relocalization (Reuter et al., 2021; Sullivan et al., 2021). Dephosphorylation of NPH3 can be readily observed by immunoblotting because of its increased electrophoretic mobility (Haga et al., 2015; Sullivan et al., 2019).…”

Condensate formation of the human RNA-binding protein SMAUG1 is controlled by its intrinsically disordered regions and interactions with 14-3-3 proteins

“…While several motif-mediated interactions were described that help drive biological condensation [57][58][59], to our knowledge this is the first instance of 14-3-3 motifs shown to directly regulate LLPS. Previous studies have established connection between the localization and function the LLPS driver, nonphototropic hypocotyl 3 (NPH3) and its 14-3-3 binding capacity [60]. 14-3-3 binding was also shown to regulate whether tristetraprolin (TTP) resides in stress granules [61], thus 14-3-3 serves as a regulator of an LLPS client or co-scaffold, as stress granule assembly does not depend on the presence of TTP.…”