Ubiquitin-Conjugating Enzymes − Elucidating the Regulatory Mechanisms of Ubiquitin Chain Assembly

• Applicant: Professor Dr. Marco Trujillo Linke
• Subject Area: Plant Biochemistry and Biophysics
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 462017488

Ubiquitination is a central process intricately involved in the regulation of most cellular processes. The E2 ubiquitin-conjugating enzymes (UBCs) are key mediators of ubiquitin chain assembly by coordinating a multitude of interactions that include ubiquitin itself, the E1 ubiquitin-activating enzyme and an E3 ubiquitin ligase. Moreover, they are largely responsible for the type of chain built, endowing the modified protein with different fates, including changes in activity, localization, as well as degradation. Ubiquitin attachment is responsive to cellular cues, and orchestrated by an intertwined cross-talk with kinase-mediated signaling pathways. However, in spite of the E2’s central importance in ubiquitination, there is scarce information on mechanisms regulating their activity. UBC35 and its close homologue UBC36, are the main sources of Lys63-linked ubiquitin chains in Arabidopsis, which is the second most abundant type of ubiquitin chain. Lys63-linked ubiquitin chains play key roles in the plasma membrane to vacuole transport of integral membrane proteins, by acting as a signal that coordinates various steps of vesicle traffic. By doing so, it contributes to the regulation of protein levels at the plasma membrane, and to the general maintenance of cellular homeostasis. We showed that the pairing between UBC35 and the E3 ligase PUB22 is enhanced in response to the activation of the immune response. UBC35, and the closely related UBC36, are potentially phosphorylated in various residues located on surfaces known to participate in the interaction with the E1 and the E3. Further, UBC35-interacting UEV1a-UEV1d are phosphorylated in N-terminal residues conserved in UBC35/36. Thus, the multitude of interactions and modification converging on the UBC35/36 duo, are indicative of a multi-tiered regulation and reflect their pivotal function as a central signaling hub through the generation of Lys63-linked chains. In this project, we aim to elucidate the mechanisms regulating Lys63-linked chain assembly by (i) confirming and identifying phosphorylation sites on UBC35/36, (ii) dissecting the impact of phosphorylation on ubiquitination activity, as well as on the interaction with known reaction partners, including ubiquitin-activating enzyme (E1), UEVs and E3s. In addition, (iii) we aim to identify new interacting proteins with a focus on kinases. (iv) Finally, we will functionally characterize the relationship of UEVs and UBC35/36 and analyse their subcellular localization. The expected outcome is to understand how cellular signaling impinges of the assembly of Lys63-linked chains by UBC35/36, enabling cells to react to challenges such as infection, as well as intrinsic cues during physiological processes.

• DFG Programme: Research Grants