Studying the biochemical basis for the action of ABERRANT LATERAL ROOT FORMATION4, a putative ubiquitylation tuner
Final Report Abstract
Ubiquitin (Ub)-regulated protein degradation is a universal and switchable process that regulates almost every aspect of plant development. A hierarchical enzyme cascade of Ub activation and Ub transfer (E1-E2-E3) enables specific modification (ubiquitylation) of target proteins. Modular Cullin1-RING E3-Ub ligases (SCF complexes) control a variety of biological processes and are subject to precise coordination at the levels of muti-protein complex formation, target protein recruitment and UB transfer. Despite profound effects of ubiquitylation on the homeostasis of cellular proteins, little is known about how catalysis and regulation of SCF complexes and Ub transfer to target proteins occur at the molecular level. Glomulin (GLMN) in Mammalia and ABERRANT LATERAL ROOT FORMATION4 (ALF4) in plants is a highly conserved protein that is generally encoded by a single gene. GLMN/ALF4 inhibits the activity of SCF complexes via competitive binding to their E2 docking site. The lethality of glmn/alf4 knockout mutants in both Mammalia and plants underscores the essential role of GLMN/ALF4 function. Mutants of the reference plant Arabidopsis thaliana with weak alf4 alleles are viable but show growth defects indicating hormone response disorders. We have shown that components of SCF complexes are deregulated in these alf4 mutants, thereby stabilizing native SCF target proteins that are ubiquitylated and degraded in response to phytohormones in the wild type. It is not yet known how ALF4 exerts its inhibitory effect on SCF E3-Ub ligases and the ubiquitylation of target proteins. The aim of this project is to implement biochemical and biophysical approaches for a mechanistic understanding of ALF4 activity. To this end, the ALF4 function for the ubiquitylation of SCF target proteins in vivo and in vitro as well as the ALF4-regulated proteome will be investigated. Furthermore, the ALF4 interaction network will be reconstructed to gain insights into the control of ALF4 activity. Ultimately, the investigations in this project will contribute to a better understanding of the assembly of SCF complexes and thus ubiquitin-mediated protein degradation.
