We first transformed high copy plasmids containing COG4, SNX4, SLT2, TAX4, IVY1, ATG5, and PEP3, as well as ARL1 and empty vector (EV, YEp352) into the arl1Δ strain. In plasmids containing SLT2, TAX4, IVY1, ATG5, and PEP3, expression is controlled by the GAL1 promoter, thus, yeast with these plasmids were cultured in medium with galactose as the carbon source. The COG4- and SNX4-containing plasmids were isolated from the YEp13 library as fragments with a single gene and expression of these genes is under the control of their native promoters; thus, yeast containing these two plasmids were cultured using normal growth conditions with glucose as the carbon source. In order to test which of the seven candidates suppressed the autophagy defect at 37°, an assay that followed cleavage of a modified version of the protein Atg8, GFP-Atg8, was used to monitor the transport of Atg8 to the vacuole (Figure 1A). Once autophagy is triggered, Atg4 modifies Atg8 on its C-terminus. This modification enables Atg8 to be conjugated to PE on its C-terminus, which helps expand the membranes of the phagophore to become the autophagosome (Xie et al. 2008). When the autophagosome fuses with the vacuole, Atg8 is degraded. However, when the fusion protein GFP-Atg8 is transferred to the vacuole, Atg8 is degraded, but the GFP moiety is resistant to degradation and can therefore be detected as free GFP on western blots if autophagy proceeds normally. As the results show, at time 0, no free GFP was detected in any of the strains. When the cells were treated in starvation medium for 3 hr at 30°, free GFP could be detected in all strains, demonstrating normal autophagy. As the negative control, no free GFP could be detected in the cell with empty vector at 37°, meaning that Arl1 is required for autophagy at high temperature, as previously found (Yang and Rosenwald 2016). Cells overexpressing COG4, TAX4, SNX4, and ARL1 were able to process GFP-Atg8 normally at 37°, while overexpression of IVY1, ATG5, SLT2, or PEP3 did not suppress the autophagy defect of the arl1Δ strain at 37°.