Purines serve as the building blocks for DNA and RNA, confer cellular energy and signaling. Purines are generated by de novo synthesis pathway and salvage pathway. Under purine-depleted or other cellular stresses, enzymes in the purine de novo synthesis pathway form a dynamic and reversible condensate called purinosome, but the underlying mechanism of purinosome formation is unknown. In this thesis, we found that ASB11-based Cul5 E3 ligase promotes ubiquitination of PAICS, a purine de novo synthesis enzyme. This ubiquitination does not lead to PAICS degradation, but drives purinosome assembly. We provide evidence that purinosome assembly involves a liquid-liquid phase separation (LLPS) process and identify several ubiquitin binding proteins that may bind ubiquitinated PAICS through a multivalent mode to drive LLPS and purinosome assembly. Importantly, ASB11 is upregulated under the stresses that promote purinosome assembly, thus increasing the formation of ASB11/PAICS complex. Finally, we demonstrated that melanoma cells express a high level of ASB11 to confer a constitutive purinosome formation, which support their viability. In summary, our study identifies ASB11-mediated PAICS ubiquitination as a driving mechanism for purinosome assembly, the regulation of this mechanism under stressed conditions, and the importance of this regulation in cell viability.