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Defining novel mechanisms that regulate phase separation of metabolic enzymes and stress granules


Compartmentalization of biochemical processes is a central principle in cell biology. Traditionally, understanding spatial organization of reactions has focused on their localization to membrane-bound organelles. However, recent work has highlighted the ability of proteins and RNAs to dynamically partition into large, membrane-less condensates. Formation of these assemblies have been observed in both eukaryotes and prokaryotes suggesting that this could be an ancient mechanism utilized for compartmentalizing and regulating specific processes. This thesis focuses on identifying novel mechanisms that regulate the assembly of two classes of membrane-less condensates: metabolic enzymes and stress granules (SGs). First, we further expanded the list of metabolic enzymes capable of forming a filament or foci structure to 60 proteins. This expansion allowed us to determine that metabolic enzymes, which acted at branch points or highly connected nodes in the metabolic network, had a higher propensity to assemble into intracellular structures than other enzymes. Our analysis of the de novo purine biosynthesis pathway not only revealed that assembly is based on the hierarchical position in a pathway, but also that a subset of enzymes in this pathway localizes to SGs. These results led us to reexamine the hits from our screen and conduct a secondary screen for SG localization. In total, we identify 17 metabolic enzymes that are associated with SGs providing a connection between metabolic activity and post-transcriptional gene regulation. We find that the product of the SG-localized enzyme Sam1, AdoMet, regulates composition and frequency of acute and chronic nutrient stress-induced SGs. Furthermore, AdoMet blocks fusion of SGs in proliferating cancer cell lines while suppressing SG formation in motor neurons derived from patients with amyotrophic lateral sclerosis. With the goal of understanding the role of RNA-RNA interactions in SG formation, we developed a reconstitution system using yeast cytoplasmic extracts and in vitro transcribed RNA. Our in vitro assembled SGs mimicked in vivo SGs and advanced the knowledge of how ATP levels regulate SG assembly, disassembly, morphology, and dynamics. Lastly, we find that building a canonical SG depends not only on the material state of a RNA, but also the composition of the RNA.

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