Fig 13. The Cyc8 prion domain (PrD) contributes to inclusion formation in the absence of sumoylation.

(A) Domain schematic of Cyc8 with the polyglutamine tract and glutamine-rich PrD (residues 441–677) are shown in green, and the sumoylation sites identified in this study are shown in blue. (B) SIM images of cells expressing GFP-tagged, wild-type Cyc8 or sumoylation-deficient Cyc8^4KtoR with or without the PrD. Cells were exposed to hyperosmotic stress (1.2M sorbitol) for the indicated times. All constructs were integrated at the gene’s endogenous locus and expressed from the endogenous promoter. (C) Analysis of data from (B). The presence of inclusions was analyzed by counting the cells with at least one inclusion. At least 200 cells in at least 5 fields were analyzed for each time point. Data in the graph represent the average of 3 independent experiments. Error bars represent the standard deviation. (D) 6His-FLAG-SMT3 cells expressing wild-type Cyc8-3HSV or Cyc8^ΔPrD-3HSV were examined for Cyc8 sumoylation at 0 or 15 minutes of hyperosmotic stress (1.2M sorbitol). Cell lysates (input) and purified sumoylated proteins (SUMO pulldown) were subject to western analyses using anti-HSV antibodies to detect Cyc8. Relative levels of the Cyc8^ΔPrD protein compared with the full-length Cyc8 protein are indicated below the input and SUMO pulldown lanes.