Deletion of the Saccharomyces cerevisiae RACK1 homolog, ASC1, enhances autophagy and counteracts TDP-43 toxicity

Cytoplasmic aggregation of nuclear proteins such as TDP-43 (TAR DNA-binding protein 43) and FUS (fused in sarcoma) is associated with several neurodegenerative diseases. Studies in higher cells suggest that these aggregates of TDP-43 and FUS sequester polysomes by binding RACK1 (receptor for activated C kinase 1), a ribosomal protein, thereby inhibiting global translation and contributing to toxicity. But RACK1 is also a scaffold protein with many other roles including a role in autophagy. Using yeast we find that deletion of the RACK1 ortholog, ASC1, reduces TDP-43 toxicity, but not FUS toxicity. TDP-43 foci remain liquid like in the presence asc1{Delta} but they become smaller. This is consistent with the findings in cell culture. However, using double label tags we establish that ASC1 does not co-localize with TDP-43 foci, arguing against the sequestration hypothesis. Instead, ASC1 appears to influence toxicity through autophagy. We previously showed that expression of TDP-43 inhibits autophagy and TOROID (TORC1 Organized in Inhibited Domains) formation and that modifiers that rescue yeast from TDP-43 toxicity reverse these inhibitions. Here we show that FUS does not inhibit autophagy. This autophagy enhanced by asc1{Delta} is non-canonical, marked by reduced TOROID formation, and effectively counteracts the autophagy inhibition caused by TDP-43. Our findings suggest that ASC1 influences TDP-43 toxicity through autophagy regulation rather than polysome sequestration, highlighting autophagy as a key therapeutic target. SummaryTDP-43 and FUS aggregates are linked to neurodegenerative diseases. RACK1, a ribosomal protein, was previously thought to contribute to toxicity by co-localizing with these aggregates and sequestering polysomes. In yeast, deletion of ASC1--the RACK1 homolog--reduces TDP-43 toxicity but not FUS toxicity. TDP-43 foci remain liquid-like, but ASC1 does not co-localize with them, challenging the sequestration hypothesis. Instead, asc1{Delta} enhances autophagy, rescuing cells from the autophagy inhibition caused by TDP-43. Unlike TDP-43, FUS does not inhibit autophagy. These findings highlight autophagy, rather than polysome sequestration, as the key mechanism of TDP-43 toxicity and its mitigation via ASC1/RACK1 reduction.