The YEp13-based yeast high copy number genomic library (ATCC 37323) (Nasmyth and Reed 1980) was used in the screen. Yeast strains arl1Δ and ypt6Δ were transformed with the library by using lithium acetate transformation, as described previously (Gietz and Woods 2002), and were plated onto either SC with glucose without leucine medium to determine number of total transformants (20,000 colonies for each strains) or SC with glucose without leucine containing 5 ng/ml rapamycin (Sigma-Aldrich, R0395). We previously determined using this concentration of rapamycin that the negative control, the atg1Δ strain, cannot grow at either 30 or 37°, while the wild-type (WT) parent can grow in the presence of rapamycin at both temperatures (Yang and Rosenwald 2016). Here, the rapamycin plates were incubated at 37°. Plasmids from the colonies showing resistance to rapamycin at 37° were isolated (Hoffman and Winston 1987) and transformed into Escherichia coli DH5α strain by electroporation, followed by purification from E. coli cells (QIAGEN QIAprep Spin Miniprep Kit). Plasmids were then retransformed into arl1Δ or ypt6Δ strains to confirm that the suppressing function was contained on the plasmid and not as a result of a chromosomal mutation in the original yeast transformant. The plasmids were sequenced with primers (MP10: CTTGGAGCCACTATCGAC, MP11: CCGCACCTGTGGCGCCG) adjacent to the unique BamHI site of YEp13, into which the genomic fragments were cloned. Sequencing was performed by Genewiz (Plainfield, NJ). The sequencing results were analyzed with the Basic Local Alignment Search Tool (BLAST, NCBI) (Altschul et al. 1990). The genomic regions contained in the plasmids were identified with the genome browser tool from the Saccharomyces genome database (SGD; yeastgenome.org). ORF functions as membrane traffic or autophagy regulators were selected and the high copy number plasmids containing a single ORF of interest were either obtained directly from this screen (some of the plasmids that passed the screen contained only a single gene) or from the yeast ORF collection (GE Dharmacon) (Gelperin et al. 2005). These plasmids were transformed into arl1Δ and ypt6Δ strains to test in more detail whether they suppressed the autophagy defect of arl1Δ and ypt6Δ strains at 37°. When the ORF collection plasmids were used, cells were grown in media containing galactose rather than glucose because the genes of interest are under the control of the GAL1 promoter.