RESUMEN
Plant growth and architecture is regulated by the polar distribution of the hormone auxin. Polarity and flexibility of this process is provided by constant cycling of auxin transporter vesicles along actin filaments, coordinated by a positive auxin-actin feedback loop. Both polar auxin transport and vesicle cycling are inhibited by synthetic auxin transport inhibitors, such as 1-N-naphthylphthalamic acid (NPA), counteracting the effect of auxin; however, underlying targets and mechanisms are unclear. Using NMR, we map the NPA binding surface on the Arabidopsis thaliana ABCB chaperone TWISTED DWARF1 (TWD1). We identify ACTIN7 as a relevant, although likely indirect, TWD1 interactor, and show TWD1-dependent regulation of actin filament organization and dynamics and that TWD1 is required for NPA-mediated actin cytoskeleton remodeling. The TWD1-ACTIN7 axis controls plasma membrane presence of efflux transporters, and as a consequence act7 and twd1 share developmental and physiological phenotypes indicative of defects in auxin transport. These can be phenocopied by NPA treatment or by chemical actin (de)stabilization. We provide evidence that TWD1 determines downstream locations of auxin efflux transporters by adjusting actin filament debundling and dynamizing processes and mediating NPA action on the latter. This function appears to be evolutionary conserved since TWD1 expression in budding yeast alters actin polarization and cell polarity and provides NPA sensitivity.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácidos Indolacéticos/metabolismo , Proteínas de Unión a Tacrolimus/metabolismo , Citoesqueleto de Actina/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico/genética , Transporte Biológico/fisiología , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Proteínas de Unión a Tacrolimus/genéticaRESUMEN
Recently we have shown that the peptidyl-prolyl cis/trans isomerase parvulin 17 (Par17) interacts with tubulin in a GTP-dependent manner, thereby promoting the formation of microtubules. Microtubule assembly is regulated by Ca(2+)-loaded calmodulin (Ca(2+)/CaM) both in the intact cell and under in vitro conditions via direct interaction with microtubule-associated proteins. Here we provide the first evidence that Ca(2+)/CaM interacts also with Par17 in a physiologically relevant way, thus preventing Par17-promoted microtubule assembly. In contrast, parvulin 14 (Par14), which lacks only the first 25 N-terminal residues of the Par17 sequence, does not interact with Ca(2+)/CaM, indicating that this interaction is exclusive for Par17. Pulldown experiments and chemical shift perturbation analysis with (15)N-labeled Par17 furthermore confirmed that calmodulin (CaM) interacts in a Ca(2+)-dependent manner with the Par17 N terminus. The reverse experiment with (15)N-labeled Ca(2+)/CaM demonstrated that the N-terminal Par17 segment binds to both CaM lobes simultaneously, indicating that Ca(2+)/CaM undergoes a conformational change to form a binding channel between its two lobes, apparently similar to the structure of the CaM-smMLCK(796-815) complex. In vitro tubulin polymerization assays furthermore showed that Ca(2+)/CaM completely suppresses Par17-promoted microtubule assembly. The results imply that Ca(2+)/CaM binding to the N-terminal segment of Par17 causes steric hindrance of the Par17 active site, thus interfering with the Par17/tubulin interaction. This Ca(2+)/CaM-mediated control of Par17-assisted microtubule assembly may provide a mechanism that couples Ca(2+) signaling with microtubule function.
Asunto(s)
Calcio/metabolismo , Calmodulina/metabolismo , Isomerasa de Peptidilprolil/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Secuencias de Aminoácidos , Calmodulina/genética , Dominio Catalítico , Humanos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Peptidilprolil Isomerasa de Interacción con NIMA , Isomerasa de Peptidilprolil/química , Isomerasa de Peptidilprolil/genética , Polimerizacion , Unión ProteicaRESUMEN
A 25-residue elongation at the N-terminus endows parvulin 17 (Par17) with altered functional properties compared to parvulin 14 (Par14), such as an enhanced influence on microtubule assembly. Therefore the three-dimensional structure of this N-terminal elongation is of particular interest. Here, we report the nearly complete (1)H, (13)C and (15)N chemical shift assignments of Par17. Subsequent chemical shift index analysis indicated that Par17 features a parvulin-type PPIase domain at the C-terminus, analogous to Par14, and an unstructured N-terminus encompassing the first 60 residues. Hence the N-terminus of Par17 apparently adopts a functionally-relevant structure only in presence of the respective interaction partner(s).
Asunto(s)
Resonancia Magnética Nuclear Biomolecular , Isomerasa de Peptidilprolil/química , Protones , Secuencia de Aminoácidos , Isótopos de Carbono , Humanos , Peptidilprolil Isomerasa de Interacción con NIMA , Isótopos de NitrógenoRESUMEN
The Atfkbp42 gene is associated with reduced and disoriented growth of Arabidopsis thaliana. Resonance assignments are reported for the FKBP-type PPIase domain of AtFKBP42. Signal intensities reveal an additional structure element that is atypical for such FKBP domains.
