Table 1.

Genetic suppressors of the Ugp1 overexpression phenotype

Gene(s)	#	Description	Efficacy	Temp
PSK1	8	PAS kinase 1	S	35
PSK2	3	PAS kinase 2	S	30
PGM2	6	Phosphoglucomutase 2	S	30
ORM1	1	Required for resistance to agents that induce the unfolded protein response	S	35
MNN11	3	Subunit of Golgi mannosyltransferase complex	S	35
ATG2	1	Peripheral membrane protein required for vesicle formation during autophagy and for the CVT pathway	M	35
BET2, PRP4, HDA3	1	Subunit of Type II geranylgeranyltransferase required for ER/Golgi vesicular transport; Splicing factor; Trichostatin A-sensitive class II histone deacetylase subunit	M	35
ISD11	1	Mitochondrial Fe-S cluster (FSC) biosynthesis	M	35
LEU1	3	Isopropylmalate isomerase, catalyzes the second step in the leucine biosynthesis pathway	M	35
PRX1; KIP1	1	Mitochondrial peroxiredoxin; Required for mitotic spindle assembly and chromosome segregation	M	35
RDN58-2; ITS2-2; ITS1-2	5	5.8S ribosomal RNA, component of the 60S ribosomal subunit; Non-coding region between RDN58 and RDN25	M	35
STP22	2	Component of the ESCRT-I complex	M	35
STT4	1	Required for vacuole morphology, cell wall integrity, and actin cytoskeleton organization	M	35
TIM44	2	Peripheral mitochondrial membrane protein involved in mitochondrial protein import, tethers essential chaperone Ssc1 to the mitochondrial membrane	M	35
YRL125W	4	Putative protein of unknown function	M	35
OSH2; ERP3	1	Involved in sterol metabolism; Member of the p24 family involved in ER to Golgi transport	W	35
ECM25	1	Non-essential protein of unknown function; promoter contains a consensus binding sequence for factor Abf1p	W	35
HTB2; ECM15; NTH2	1	Histone H2B; Non-essential protein of unknown function that may have a roll in cell wall biogeneiss; Putative neutral trehalase required for thermotolerance	W	35

The column labeled # indicates the number of independent clones recovered. Efficacy is the strength of suppression of each clone as compared to the PAS kinase suppression. S = strong, M = moderate and W = weak. Temperature indicates the temperature at which the screen yielding that suppressor was performed (30°C or 35°C). The strain JRY277(psk1 psk2) freshly transformed with pJR3020b (UGP1) was retransformed with a pRS426 library using the standard lithium acetate transformation protocol⁵⁵ and screened for growth on SGal-trp-ura. Approximately 2 million transformants resulted in 401 initial candidate suppressors. These candidates were then patched to SD-trp containing dox (to inhibit UGP1 overexpression) and 5-FOA (to allow for the loss of the URA3-encoding pRS426 library plasmid) and then subsequently replica plated onto SGal-trp at the isolation temperature to test for plasmid-independent suppression. The high copy suppressor candidate (HCSC) library plasmids were then isolated and characterized by restriction endonuclease digestion with PvuII. The approximately 200 unique plasmids were then retransformed into JRY277 pJR3020B cells to confirm suppression on SGal-ura-trp (using empty pRS426 in place of the pRS426-library plasmid DNA as a control); only 71 were able to reproducibly rescue the Ugp1 gal^ts phenotype. An UGPase enzymatic assay⁵⁶ was used to eliminate regulation of Ugp1 activity as a possible mode of suppression. These candidate plasmids were then sequenced and the genes were identified using the Basic Local Alignment Search Tool for nucleotides (BLASTN) through the NIH National Center for Biotechnology Information website. The genes that are inferred to be suppressors by virtue of their rolls in cell integrity pathways are underlined.