. 2014 Oct 3;3:e03779. doi: 10.7554/eLife.03779

Table 1.

Known Ypk1 substrates and potential substrates predicted by MOTIPS listed under GO Slim terms^*

DOI: http://dx.doi.org/10.7554/eLife.03779.004

Gene	MOTIPS sites	Chemical Sensitivity/YeastMine Interaction(s)	SDL score	Ypk1 dosage rescue	In vitro substrate
Known Ypk1 Substrates
GPD1/YDL022W*	24^P	YeastMine	+++	N/A	+
FPK1/YNR047W	37, 200, 244, 436, 481	YeastMine	+	N/A	+
FPK1(D621A) [Kinase-dead mutant]	37, 200, 244, 436, 481	YeastMine	++	N/A	+
ORM1/YGR038W	52, 53	YeastMine	+++	N/A	+
ORM2/YLR350W	47, 48	YeastMine	++++	N/A	+
Cytoskeleton Organization
AVO1/YOL078W	552^P,597,1078	YeastMine	−	N/A	N/A
AVO2/YMR068W	273^P, 305, 407	YeastMine	−	N/A	N/A
BEM2/YER155C	83, 168, 1810, 1813	Myr, YeastMine	−	N/A	N/A
BNI1/YNL271C	119, 1138^P, 1533	AbA, Casp, YeastMine	−	N/A	N/A
CDH1/YGL003C	51, 195, 213^P	AbA, YeastMine	TOXIC	−	N/A
ENT1/YDL161W	160^P	YeastMine	−	N/A	N/A
GIC2/YDR309C	90, 312, 345^P	Myr, AbA	−	N/A	N/A
LSB3/YFR024C-A	262^P	YeastMine	−	N/A	N/A
PAL1/YDR348C	49^P, 391, 436	AbA, Casp	++++	N/A	−
SLA1/YBL007C	445, 447^P, 449^P, 477	YeastMine	−	N/A	N/A
TSC11/YER093C	19^P, 97, 188	YeastMine	N/A	N/A	N/A
YHR097C	58, 288^P, 294^P	Myr	+++	N/A	+/−
YSC84/YHR016C	274, 374^P	Myr, YeastMine	−	N/A	N/A
Biological Process Unknown
COM2/YER130C	251, 370, 380	Myr, YeastMine	−	N/A	N/A
ECM3/YOR092W*	312, 350	Myr, AbA, YeastMine	−	N/A	N/A
ICS2/YBR157C	14, 95, 136, 172^P	Myr	−	N/A	N/A
JIP4/YDR475C	348, 352, 592, 649	Myr, AbA	N/A	N/A	N/A
KKQ8/YKL168C	83, 113, 144, 146, 212, 293	YeastMine	−	N/A	N/A
RTS3/YGR161C*	30, 238^P	YeastMine	−	N/A	N/A
SEG1/YMR086W	56, 118^P, 217, 634, 752^P	Myr	+	N/A	N/A
YDR186C	334^P, 540^P, 542^P, 620, 715^P	YeastMine	−	N/A	N/A
YHR080C	401, 513, 667	YeastMine	−	N/A	N/A
YNR014W	54, 115, 156, 197	YeastMine	+++	N/A	+
YPK3/YBR028C	72, 73, 90^P	Myr, Casp	−	N/A	N/A
Transcription from RNA Polymerase II Promoter
EPL1/YFL024C	24, 28, 61	YeastMine	−	N/A	N/A
FKH1/YIL131C	404	Myr, YeastMine	TOXIC	−	−
GAL11/YOL051W	1003^P	Myr, YeastMine	−	N/A	N/A
HCM1/YCR065W*	80	AbA, YeastMine	−	N/A	N/A
HOT1/YMR172W*	387, 520, 586	Myr	−	N/A	N/A
RLM1/YPL089C*	20	Myr	TOXIC	−	N/A
SMP1/YBR182C*	20, 107	YeastMine	TOXIC	+	+
SSN2/YDR443C	608^P	Myr	−	N/A	N/A
YHP1/YDR451C*	180, 182	Myr	−	N/A	N/A
Mitotic Cell Cycle
BCK2/YER167W	12, 38, 373, 430	YeastMine	TOXIC	−	N/A
ESC2/YDR363W	114, 143, 145	YeastMine	−	N/A	N/A
PTK2/YJR059W	59^P, 82, 91, 171, 275	Myr, YeastMine	+	N/A	N/A
SET3/YKR029C	236, 405, 428	YeastMine	−	N/A	N/A
SWI4/YER111C	816^P	Myr, YeastMine	−	N/A	N/A
VHS2/YIL135C	316, 318, 325^P	Myr, Casp	−	N/A	N/A
ZDS1/YMR273C*	78, 370	AbA, YeastMine	−	N/A	N/A
ZDS2/YML109W	183, 267, 345	YeastMine	−	N/A	N/A
Protein Phosphorylation
HAL5/YJL165C	17^P, 217^P, 233	YeastMine	−	N/A	N/A
KIN1/YDR122W	652, 791^P, 879, 986^P	YeastMine	+	N/A	N/A
KIN2/YLR096W	665^P, 818, 1020	Myr	−	N/A	N/A
KSP1/YHR082C	594, 827^P, 884^P	Myr, AbA, YeastMine	TOXIC	−	N/A
NPR1/YNL183C	125^P, 255^P, 257^P, 317^P	YeastMine	++++	N/A	−
SKY1/YMR216C	383^P	Myr, YeastMine	−	N/A	N/A
YAK1/YJL141C	128^P, 206, 240	Myr, Casp, YeastMine	−	N/A	N/A
YPL150W	371^P, 452, 890	YeastMine	−	N/A	N/A
Lipid Metabolic Process
ADR1/YDR216W	180, 230^P, 756	Myr, AbA	−	N/A	N/A
CDC1/YDR182W*	9	N/A	−	N/A	+
CKI1/YLR133W	14^P, 25^P, 30^P	Myr, AbA, YeastMine	−	N/A	N/A
GPT2/YKR067W	27, 652	Myr	+++	N/A	+
LAC1/YKL008C*	23, 24	Myr, YeastMine	+++	N/A	+
LAG1/YHL003C	24^P	Myr, YeastMine	+++	N/A	+
LCB3/YJL134W	16^P	Myr, YeastMine	−	N/A	+
Cellular Ion Homeostasis and Transport
AVT3/YKL146W	55, 59^P, 172, 174	Myr, AbA, YeastMine	−	N/A	N/A
CCH1/YGR217W^†	146, 148, 347	YeastMine	−	N/A	+
FPS1/YLL043W	147, 181, 185, 570^P	Myr, YeastMine	+++++	N/A	+
MNR2/YKL064W	165, 620, 621, 826	AbA	−	N/A	N/A
NHA1/YLR138W*	544, 830	Myr, YeastMine	−	N/A	N/A
PPZ1/YML016C	122, 203, 250^P	Myr, YeastMine	TOXIC	−	N/A
Translation
DED1/YOR204W	84, 576^P	YeastMine	−	N/A	N/A
HCR1/YLR192C*	223	Myr, AbA, YeastMine	−	N/A	N/A
HEF3/YNL014W*	898	Myr, AbA, YeastMine	−	N/A	N/A
RPL3/YOR063W	24^P, 337	Myr, AbA, YeastMine	−	N/A	N/A
SUI2/YJR007W*	58	Myr	−	N/A	N/A
TEF1/YPR080W*	72^P	Myr	−	N/A	N/A
Cell Wall Organization or Biogenesis
BPH1/YCR032W	1334, 1336, 1963	Casp	−	N/A	N/A
CSR2/YPR030W	61, 103, 525, 987	Myr	−	N/A	N/A
ROM2/YLR371W	76^P, 193^p, 396	YeastMine	−	N/A	N/A
SSD1/YDR293C	164^P, 482^P, 503	Myr, AbA, YeastMine	TOXIC	−	N/A
Golgi Vesicle Transport
BRE5/YNR051C	398^P	Myr, YeastMine	+++	N/A	−
EXO84/YBR102C	76, 313, 494, 554	YeastMine	−	N/A	N/A
MUK1/YPL070W	173, 184^P, 185^P	Myr	+++	N/A	+/−
RGP1/YDR137W	220, 364^P, 450, 452	YeastMine	−	N/A	N/A
Signaling
IRA2/YOL081W	882, 884, 1578, 1745, 3069	YeastMine	N/A	N/A	N/A
GIS3/YLR094C*	249, 333	Myr, AbA	−	N/A	N/A
MDS3/YGL197W	757, 824, 842, 851, 1204	Myr, AbA, YeastMine	+++	N/A	−
SYT1/YPR095C	277^P, 410, 728	YeastMine	N/A	N/A	N/A
DNA Replication
CDC13/YDL220C	314, 333^P	YeastMine	TOXIC	−	N/A
CTI6/YPL181W	155, 216^P	Myr, YeastMine	−	N/A	N/A
RIM4/YHL024W	93, 429, 525, 607	YeastMine	−	N/A	N/A
Endocytosis
ALY2/YJL084C	166^P, 201, 225, 803	Myr	−	N/A	N/A
ROD1/YOR018W	563, 617, 807, 823	Myr	+++	N/A	+/−
ROG3/YFR022W	425, 584, 718	YeastMine	−	N/A	N/A
Other
FRT1/YOR324C	167, 201, 203, 228^P, 385	Myr, YeastMine	−	N/A	N/A
HER1/YOR227W	28^P, 102^p, 157^P	Myr, AbA	TOXIC	−	+
YSP2/YDR326C	326, 518, 1237	Myr, YeastMine	+++	N/A	+
RNA Catabolic Process
JSN1/YJR091C	174, 275^P, 600	YeastMine	−	N/A	N/A
PUF2/YPR042C	55, 143, 246, 902	Myr	N/A	N/A	N/A
Cytokinesis
CYK3/YDL117W	159, 207^P, 746	AbA, YeastMine	+++	N/A	−
Chromosome Segregation
DSN1/YIR010W	240, 250^P	YeastMine	−	N/A	N/A
Peroxisome Organization
PEX31/YGR004W	432^P	YeastMine	++	N/A	+
Pseudohyphal Growth
PAM1/YDR251W	471, 553^P, 625	Myr, AbA, Casp, YeastMine	−	N/A	N/A
Response to Starvation
ATG21/YPL100W	191, 237^P	Myr	+++	N/A	+

Genes that are not bioinformatically predicted Ypk1 substrates, but contain Ypk1 motifs and were included in this study are marked with an asterisk. SDL assay results are listed for each bioinformatically predicted Ypk1 substrate. The scoring system reports growth phenotypes of the ypk1-as ypk2Δ strain transformed with the indicated P_GAL1-SUBSTRATE plasmid upon overexpression on galactose with varying levels of 3-MB-PP1-imposed Ypk1-as inhibition. A growth phenotype is defined as at least 1 serial dilution spot less growth than YCpLG-GFP control at the given 3-MB-PP1 concentration. A strong growth phenotype is defined as no growth at the given 3-MB-PP1 concentration. (+++++) indicates a growth phenotype with no 3-MB-PP1. (++++) is a strong growth phenotype on 1 μM 3-MB-PP1. (+++) indicates a growth phenotype on 1 μM 3-MB-PP1. (++) is defined as no phenotype on 1 μM 3-MB-PP1, but a strong growth phenotype on 2 μM 3-MB-PP1. (+) indicates no phenotype on 1 μM 3-MB-PP1, but a detectable growth phenotype on 2 μM 3-MB-PP1. (−) indicates no growth phenotype at any concentration of 3-MB-PP1 tested. TOXIC indicates overexpression of the putative substrate on galactose-containing medium was deleterious to growth even in the wild-type control strain (BY4741). These toxic genes were then tested for Ypk1 dosage rescue (for details, see ‘Materials and methods’); here, (+) indicates that Ypk1 overexpression could at least partially rescue the overexpression toxicity of the indicated gene and (−) indicates that Ypk1 overexpression could not rescue the overexpression toxicity. Lastly, the results of testing the indicated purified predicted Ypk1 target as a substrtate in the in vitro protein kinase assay with purified Ypk1-as; here, (+) indicates that Ypk1-as- dependent (3-MB-PP1 inhibitable) incorporation was detectable for the substrate at a level comparable to incorporation into the positive control [the known Ypk1 substrate, GST-Orm1(1–85)]; (+/−) indicates that readily detectable Ypk1-as-dependent incorporation was found, but at a level lower than that seen for an equivalent amount of GST-Orm1(1–85) protein. (N/A) indicates that the indicated gene product was not tested in the indicated assay.

^†

The SDL assay was performed with a plasmid constitutively overexpressing CCH1 under the TDH3 promoter [pBCT-CCH1H, (Iida et al., 2007)], as our efforts to generate a P_GAL1-CCH1 vector failed.