(A) The three-part screening strategy to identify Ypk1 substrates is shown schematically as a flow chart. Numbers indicate the number of hits/considered genes at each step in the screen. (B) The …
(A) A diagram of the membrane topology of Lac1 and Lag1 derived from (Kageyama-Yahara and Riezman, 2006); Lac1 and Lag1 are experimentally determined to have eight transmembrane domains. The N …
(A) 3xHA-Lac1 (yAM165–A) 3xHA-Lac1(S23A S24A) (yAM166–A), 3xFLAG-Lag1 (yAM159–A) and 3xFLAG-Lag1(S23A S24A) (yAM163–A) strains were grown to mid-exponential phase in YPD. Cells were then harvested …
(A) 3xHA-Lac1 (yAM165–A) cells were grown to early exponential phase in YPD. Cultures were then treated with sublethal doses of myriocin (0.625 μM) or methanol (vehicle) or aureobasidin A (1.8 μM) …
(A) Wildtype (BY4741) or cna1Δ cna2Δ (JTY5574) strains were transformed with a centromeric plasmid encoding 3xHA-Lac1 expressed under control of its endogenous promoter (pAX136). Cultures were grown …
(A) Cultures of LAC1 LAG1 (yAM205–A), Lac1SSAA Lag1SSAA (yAM207B) and Lac1SSEE Lag1SSEE (yAM210) strains were grown to mid-exponential phase and then harvested. Sphingolipids were extracted and …
(A) LAC1 LAG1 (yAM205–A) or Lac1SSAA Lag1SSAA (yAM207–B) were transformed with PYPK1-Ypk1D242A (shown as Ypk1*) (pFR273) or empty vector pRS316 (EV). Transformants were grown to exponential phase in …
(A) Diagram of yeast de novo sphingolipid biosynthesis shown is derived from (Dickson, 2008). Enzymes are in ovals. Metabolites are in boxes. Increased color intensity indicates level of metabolite …
Known Ypk1 substrates and potential substrates predicted by MOTIPS listed under GO Slim terms*
Gene | MOTIPS sites | Chemical Sensitivity/YeastMine Interaction(s) | SDL score | Ypk1 dosage rescue | In vitro substrate |
---|---|---|---|---|---|
Known Ypk1 Substrates | |||||
GPD1/YDL022W* | 24P | 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 | 552P,597,1078 | YeastMine | − | N/A | N/A |
AVO2/YMR068W | 273P, 305, 407 | YeastMine | − | N/A | N/A |
BEM2/YER155C | 83, 168, 1810, 1813 | Myr, YeastMine | − | N/A | N/A |
BNI1/YNL271C | 119, 1138P, 1533 | AbA, Casp, YeastMine | − | N/A | N/A |
CDH1/YGL003C | 51, 195, 213P | AbA, YeastMine | TOXIC | − | N/A |
ENT1/YDL161W | 160P | YeastMine | − | N/A | N/A |
GIC2/YDR309C | 90, 312, 345P | Myr, AbA | − | N/A | N/A |
LSB3/YFR024C-A | 262P | YeastMine | − | N/A | N/A |
PAL1/YDR348C | 49P, 391, 436 | AbA, Casp | ++++ | N/A | − |
SLA1/YBL007C | 445, 447P, 449P, 477 | YeastMine | − | N/A | N/A |
TSC11/YER093C | 19P, 97, 188 | YeastMine | N/A | N/A | N/A |
YHR097C | 58, 288P, 294P | Myr | +++ | N/A | +/− |
YSC84/YHR016C | 274, 374P | 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, 172P | 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, 238P | YeastMine | − | N/A | N/A |
SEG1/YMR086W | 56, 118P, 217, 634, 752P | Myr | + | N/A | N/A |
YDR186C | 334P, 540P, 542P, 620, 715P | 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, 90P | 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 | 1003P | 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 | 608P | 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 | 59P, 82, 91, 171, 275 | Myr, YeastMine | + | N/A | N/A |
SET3/YKR029C | 236, 405, 428 | YeastMine | − | N/A | N/A |
SWI4/YER111C | 816P | Myr, YeastMine | − | N/A | N/A |
VHS2/YIL135C | 316, 318, 325P | 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 | 17P, 217P, 233 | YeastMine | − | N/A | N/A |
KIN1/YDR122W | 652, 791P, 879, 986P | YeastMine | + | N/A | N/A |
KIN2/YLR096W | 665P, 818, 1020 | Myr | − | N/A | N/A |
KSP1/YHR082C | 594, 827P, 884P | Myr, AbA, YeastMine | TOXIC | − | N/A |
NPR1/YNL183C | 125P, 255P, 257P, 317P | YeastMine | ++++ | N/A | − |
SKY1/YMR216C | 383P | Myr, YeastMine | − | N/A | N/A |
YAK1/YJL141C | 128P, 206, 240 | Myr, Casp, YeastMine | − | N/A | N/A |
YPL150W | 371P, 452, 890 | YeastMine | − | N/A | N/A |
Lipid Metabolic Process | |||||
ADR1/YDR216W | 180, 230P, 756 | Myr, AbA | − | N/A | N/A |
CDC1/YDR182W* | 9 | N/A | − | N/A | + |
CKI1/YLR133W | 14P, 25P, 30P | Myr, AbA, YeastMine | − | N/A | N/A |
GPT2/YKR067W | 27, 652 | Myr | +++ | N/A | + |
LAC1/YKL008C* | 23, 24 | Myr, YeastMine | +++ | N/A | + |
LAG1/YHL003C | 24P | Myr, YeastMine | +++ | N/A | + |
LCB3/YJL134W | 16P | Myr, YeastMine | − | N/A | + |
Cellular Ion Homeostasis and Transport | |||||
AVT3/YKL146W | 55, 59P, 172, 174 | Myr, AbA, YeastMine | − | N/A | N/A |
CCH1/YGR217W† | 146, 148, 347 | YeastMine | − | N/A | + |
FPS1/YLL043W | 147, 181, 185, 570P | 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, 250P | Myr, YeastMine | TOXIC | − | N/A |
Translation | |||||
DED1/YOR204W | 84, 576P | 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 | 24P, 337 | Myr, AbA, YeastMine | − | N/A | N/A |
SUI2/YJR007W* | 58 | Myr | − | N/A | N/A |
TEF1/YPR080W* | 72P | 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 | 76P, 193p, 396 | YeastMine | − | N/A | N/A |
SSD1/YDR293C | 164P, 482P, 503 | Myr, AbA, YeastMine | TOXIC | − | N/A |
Golgi Vesicle Transport | |||||
BRE5/YNR051C | 398P | Myr, YeastMine | +++ | N/A | − |
EXO84/YBR102C | 76, 313, 494, 554 | YeastMine | − | N/A | N/A |
MUK1/YPL070W | 173, 184P, 185P | Myr | +++ | N/A | +/− |
RGP1/YDR137W | 220, 364P, 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 | 277P, 410, 728 | YeastMine | N/A | N/A | N/A |
DNA Replication | |||||
CDC13/YDL220C | 314, 333P | YeastMine | TOXIC | − | N/A |
CTI6/YPL181W | 155, 216P | Myr, YeastMine | − | N/A | N/A |
RIM4/YHL024W | 93, 429, 525, 607 | YeastMine | − | N/A | N/A |
Endocytosis | |||||
ALY2/YJL084C | 166P, 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, 228P, 385 | Myr, YeastMine | − | N/A | N/A |
HER1/YOR227W | 28P, 102p, 157P | Myr, AbA | TOXIC | − | + |
YSP2/YDR326C | 326, 518, 1237 | Myr, YeastMine | +++ | N/A | + |
RNA Catabolic Process | |||||
JSN1/YJR091C | 174, 275P, 600 | YeastMine | − | N/A | N/A |
PUF2/YPR042C | 55, 143, 246, 902 | Myr | N/A | N/A | N/A |
Cytokinesis | |||||
CYK3/YDL117W | 159, 207P, 746 | AbA, YeastMine | +++ | N/A | − |
Chromosome Segregation | |||||
DSN1/YIR010W | 240, 250P | YeastMine | − | N/A | N/A |
Peroxisome Organization | |||||
PEX31/YGR004W | 432P | YeastMine | ++ | N/A | + |
Pseudohyphal Growth | |||||
PAM1/YDR251W | 471, 553P, 625 | Myr, AbA, Casp, YeastMine | − | N/A | N/A |
Response to Starvation | |||||
ATG21/YPL100W | 191, 237P | 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 PGAL1-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 PGAL1-CCH1 vector failed.
Saccharomyces cerevisiae strains used in this study
Strain | Genotype | Source/reference |
---|---|---|
BY4741 | MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0 | Research Genetics, Inc. |
BY4742 | MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0 | Research Genetics, Inc. |
yAM135-A | BY4741 Ypk1(L424A)::URA3-ypk2Δ::KanMX4 | This study |
JTY6142 | BY4741 ypk1Δ::KanMX4 | Research Genetics, Inc. |
yAM120-A | BY4741 ypk2Δ::KanMX4 | This study |
yAM159-A | BY4741 3xFLAG-Lag1::LEU2 | This study |
yAM163-A | BY4741 3xFLAG-Lag1(S23A S24A)::LEU2 | This study |
yAM165-A | BY4742 3xHA-Lac1::HIS3 | This study |
yAM166-A | BY4742 3xHA-Lac1 (S23A S24A)::HIS3 | This study |
JTY5574 | BY4741 cna1Δ::KanMX4 cna2Δ::KanMX4 | M.S. Cyert, Stanford Univ. |
YDB379 | BY4741 Ypk1-3xFLAG::natNT2 | J.S. Weissman, Univ. of California, San Francisco |
yAM205-A | BY4742 Lac1::LEU2 Lag1::LEU2 | This study |
yAM207-B | BY4742 Lac1(S23A S24A)::LEU2 Lag1(S23A S24A)::LEU2 | This study |
yAM210 | BY4742 Lac1(S23E S24E)::LEU2 Lag1(S23E S24E)::LEU2 | This study |
yGT12 | BY4742 LYS2+ Lac1::LEU2 Lag1::LEU2 lcb3Δ::natNT2 | This study |
yGT13 | BY4742 LYS2+ Lac1(S23A S24A)::LEU2 Lag1(S23A S24A)::LEU2 lcb3Δ::natNT2 | This study |
yGT14 | BY4742 LYS2+ Lac1(S23E S24E)::LEU2 Lag1(S23E S24E)::LEU2 lcb3Δ::natNT2 | This study |
yAM168 | BY4741 3xHA-Lac1::HIS3 3xFLAG-Lag1::LEU2 | This study |
yAM184 | BY4741 3xHA-Lac1(S23A S24A)::HIS3 3xFLAG-Lag1(S23A S24A)::LEU2 | This study |
yAM192-A | BY4741 MET15+ 3xHA-Lac1(S23E S24E)::HIS3 3xFLAG-Lag1(S23E S24E)::LEU2 | This study |
yKL4 | BY4741 TOR2+::Hygr | Kristin Leskoske, this lab |
yKL5 | BY4741 Tor2(L2178A)::Hygr | Kristin Leskoske, this lab |
Plasmids used in this study
Plasmid | Description | Source/reference |
---|---|---|
pGEX6P-1 | GST tag, bacterial expression vector | GE Healthcare, Inc. |
pGEX4T-1 | GST tag, bacterial expression vector | GE Healthcare, Inc. |
YCpLG | CEN, LEU2, PGAL1 vector | (Bardwell et al., 1998) |
BG1805 | 2 µm, URA3, PGAL1, C-terminal tandem affinity (TAP) tag vector | Open Biosystems, Inc. |
pRS313 | CEN, HIS3, vector | (Sikorski and Hieter, 1989) |
pRS316 | CEN, URA3, vector | (Sikorski and Hieter, 1989) |
pRS416 | CEN, URA3, vector | (Sikorski and Hieter, 1989) |
pBC111 | CEN, LEU2, vector | (Iida et al., 2007) |
CHp282 | pRS416 PMET25-GFP | Chau Huynh, this laboratory |
pLB215 | pRS416 PMET25-Ypk1 | (Niles et al., 2012) |
pAX53 | pRS416 PMET25-Ypk1(K376A) | This study |
pAX50 | BG1805 Ypk1(L424A) | This study |
pFR203 | pGEX4T-1 Orm1(1-85) | (Roelants et al., 2011) |
pBT6 | pGEX6P-1 Fps1(1-255) | This study |
pBT7 | pGEX6P-1 Fps1(531-669) | This study |
pBT12 | pGEX6P-1 Smp1 | This study |
pAX55 | pGEX6P-1 Lcb3(1-79) | This study |
pAX56 | pGEX6P-1 Cdc1(1-41) | This study |
pAX58 | pGEX6P-1 Her1(1-224) | This study |
pAX59 | pGEX6P-1 Rts3 | This study |
pAX62 | pGEX6P-1 Fkh1 | This study |
pAX63 | pGEX6P-1 Yhp1 | This study |
pAX66 | pGEX6P-1 YNR014W | This study |
pAX67 | pGEX6P-1 YHR097C | This study |
pAX94 | pGEX6P-1 Mds3(545-1016) | This study |
pAX131 | pGEX4T-1 Lac1(1-76) | This study |
pAX132 | pGEX4T-1 Lac1(1-76)(S23A S24A) | This study |
pFR291 | pGEX4T-1 Lag1(1-80) | This study |
pAX133 | pGEX4T-1 Lag1(1-80)(S23A S24A) | This study |
pAX134 | pGEX6P-1 Muk1(1-305) | This study |
pAX215 | pGEX6P-1 Cyk3 | This study |
pAX223 | pGEX6P-1 Gpt2(1-35) | This study |
pAX224 | pGEX6P-1 Gpt2(570-743) | This study |
pAX225 | pGEX6P-1 Bre5 | This study |
pAX226 | pGEX6P-1 Npr1(1-437) | This study |
pAX227 | pGEX6P-1 Pal1 | This study |
pAX228 | pGEX6P-1 Ysp2(97-665) | This study |
pAX229 | pGEX6P-1 Ysp2(1072-1282) | This study |
pAX230 | pGEX6P-1 Atg21 | This study |
pAX231 | pGEX6P-1 Pex31(250-462) | This study |
pAX136 | pRS313 PLAC1-3xHA-Lac1 | This study |
pFR273 | pRS316 PYPK1-Ypk1(D242A) | (Roelants et al., 2011) |
pAX250 | pRS313 PTPI1-GFP-Atg8 | This study |
pBCT-CCH1H | pBC111 PTDH3-Cch1 | (Iida et al., 2007) |