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A combination of yeast genetics and protein biochemistry define how the fused in sarcoma (FUS) protein might contribute to Lou Gehrig's disease., TDP-43 and FUS are RNA-binding proteins that form cytoplasmic inclusions in some forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Moreover, mutations in TDP-43 and FUS are linked to ALS and FTLD. However, it is unknown whether TDP-43 and FUS aggregate and cause toxicity by similar mechanisms. Here, we exploit a yeast model and purified FUS to elucidate mechanisms of FUS aggregation and toxicity. Like TDP-43, FUS must aggregate in the cytoplasm and bind RNA to confer toxicity in yeast. These cytoplasmic FUS aggregates partition to stress granule compartments just as they do in ALS patients. Importantly, in isolation, FUS spontaneously forms pore-like oligomers and filamentous structures reminiscent of FUS inclusions in ALS patients. FUS aggregation and toxicity requires a prion-like domain, but unlike TDP-43, additional determinants within a RGG domain are critical for FUS aggregation and toxicity. In further distinction to TDP-43, ALS-linked FUS mutations do not promote aggregation. Finally, genome-wide screens uncovered stress granule assembly and RNA metabolism genes that modify FUS toxicity but not TDP-43 toxicity. Our findings suggest that TDP-43 and FUS, though similar RNA-binding proteins, aggregate and confer disease phenotypes via distinct mechanisms. These differences will likely have important therapeutic implications., Author Summary Many human neurodegenerative diseases are associated with the abnormal accumulation of protein aggregates in the neurons of affected individuals. Amyotrophic lateral sclerosis (ALS), also known as Lou Gehrig's disease, is a fatal human neurodegenerative disease caused primarily by a loss of motor neurons. Recently, mutations in a gene called fused in sarcoma (FUS) were identified in some ALS patients. The basic mechanisms by which FUS contributes to ALS are unknown. We have addressed this question using protein biochemistry and the genetically tractable yeast Saccharomyces cerevisiae. We defined the regions of biochemically pure FUS protein that contribute to its aggregation and toxic properties. We then used genome-wide screens in yeast to identify genes and cellular pathways involved in the toxicity of FUS. Many of the FUS toxicity modifier genes that we identified in yeast have clear homologs in humans, suggesting that these might also be relevant for the human disease. Together, our studies provide novel insight into the basic mechanisms associated with FUS aggregation and toxicity. Moreover, our findings open new avenues that could be explored for therapeutic intervention.