Abstract 1332 Suppressor mutations in yeast Vps13 suggest a cooperative mechanism in lipid transport

Yeast cells lacking the mitochondrial-ER contact site complex ERMES cannot grow in the absence of glucose due to mitochondrial defects. However, spontaneous suppressors arise that allow growth on non-fermentable carbon sources. These suppressors are caused by point mutations in the yeast VPS13 gene that, presumably, enhance lipid transfer to the mitochondrion and compensate for the loss of ERMES. We have found that the same mutation that bypasses the need for ERMES can also partially compensate for defects in Vps13 function at the endosome and at the prospore membrane, These results suggest that the mutations make the mutant Vps13 more efficient at lipid transfer. More than a dozen different "suppressor" alleles of VPS13 have been isolated. These mutations are spread out over a 1500aa amino acid region of the protein comprising most of the RBG repeats that make up the hydrophobic channel. Mapping these mutations onto the Alphafold structure reveals that they can occur in at least seven different RBG repeats (of 12 total) and that they are enriched at positions in the hinge region of the repeat. A structural alignment of different RBG domains revealed that several suppressors are located at the same position in different repeats. Moreover, we were able to engineer new suppressor mutations using these alignments. The observation that mutations in a (possibly) flexible hinge region of the RBG repeat gives rise to alleles with enhanced lipid transfer activity suggests that the mechanism of transfer involves a conformational change in the RBG repeat. That single mutations in different RBG repeats produce the same phenotype further suggests that this conformational change involves cooperative movement of the repeats. A model incorporating these observations will be discussed. This work was supported by NIH grants GM072540 and GM145606