SINGLE-POINT MUTATION OF UBL4A ABOLISHES ITS INTERACTION WITH THE ARP2/3 COMPLEX Yao, Qi Cellular biology Arp2/3 cell biology Immunoblotting Protein Interaction Ubl4A Ubl4A is a member of the ubiquitin-like protein family with multiple functions, such as pro-survival and anti-tumor. Our group previously found that Ubl4A directly interacts with actin-related protein Arp2/3 complex to promote Arp2/3-dependent actin “Y shape” branching formation. However, the binding region of Ubl4A for Arp2/3 still remains unknown. To address this question, we generated several Ubl4A mutant and truncated constructs, and cloned them into a GST vector in which GST was fused in-frame with Ubl4A at the N-terminus. We used Glutathione-beads to purify GST fusion proteins and performed pull-down assay with purified Arp2/3 complex. We show that C-terminus of Ubl4A is the region where the Arp2/3 complex interacts with. A single point mutation (D122A) in Ubl4A C-terminal “DYD” motif can abolish Ubl4A ability to bind Arp2/3. These results indicate that C-terminus, especially D122, is critical for Ubl4A association with Arp2/3. Xiang, Jialing 2018 Thesis application/pdf islandora:1001315 http://hdl.handle.net/10560/islandora:1001315 Illinois Institute of Technology BIOL / Biology en In Copyright http://rightsstatements.org/page/InC/1.0/ Restricted Access