mapping the functional yeast abc transporter …...department of cell biology, johns hopkins school...

Mapping the functional yeast ABC transporter interactome

Jamie Snider1, Asad Hanif1,2, Mid Eum Lee5, Ke Jin1,2,4, Analyn R. Yu1, Chris Graham1,6,

Matthew Chuk1,2, Dunja Damjanovic1,2, Marta Wierzbicka1, Priscilla Tang1,2, Dina Balderes7,

Victoria Wong1, Matthew Jessulat6, Katelyn D. Darowski1,3, Bryan-Joseph San Luis1,2,4, Igor

Shevelev1, Stephen L Sturley7, Charles Boone1,2,4, Jack F. Greenblatt1,2,4, Zhaolei Zhang1,2,4,

Christian M. Paumi8, Mohan Babu6, Hay-Oak Park5, Susan Michaelis9 and Igor Stagljar1,2,3*

1 Donnelly Centre, University of Toronto, ON, Canada

2 Department of Molecular Genetics, University of Toronto, ON, Canada

3 Department of Biochemistry, University of Toronto, ON, Canada

4 Banting and Best Department of Medical Research, University of Toronto, ON, Canada

5 Department of Molecular Genetics, the Molecular Cellular Developmental Biology Program,

The Ohio State University, Columbus, OH, USA

6 Department of Biochemistry, Research and Innovation Centre, University of Regina, Regina,

SK, Canada

7 Department of Pediatrics, Columbia University Medical Center, New York, NY, USA

8 Graduate Center for Toxicology, University of Kentucky, Lexington, KY, USA

9 Department of Cell Biology, Johns Hopkins School of Medicine, Baltimore, MD, USA

* Corresponding author

Email: [email protected]

Phone: 1-416-946-7828

Fax: 1-416-978-8287

Nature Chemical Biology: doi:10.1038/nchembio.1293

mailto:[email protected]

Supplementary Results


YKR103W YKR104W

Mutant

NFT1

PCR amplify as two fragments.

A

B

C

D

Transform fragments and

digested AMBV plasmid into yeast

AMBV Vector Backbone

‘Full-Length’ NFT1

Homologous recombination.

Supplementary Figure 1: Amplification and cloning of ‘Full-Length’ NFT1 gene. The mutant NFT1 gene present in

S288C contains a stop codon (TAG) at position 1219 (shown as a Red line) resulting in the annotation of NFT1 as two

separate open reading frames in the SGD – YKR103W (Blue) and YKR104W (Purple). To repair the mutation, the

mutant gene was PCR amplified as two separate fragments using overlapping primers (B and C) which corrected the

nonsense mutation and restored the WT ‘TAT’ codon at position 1219 (shown as a Green line). Both terminal primers

(A and D) contained regions homologous to the MYTH AMBV bait vector (shown in Orange). Transformation of both

PCR amplified fragments, alongside appropriately digested MYTH AMBV bait plasmid, into yeast, resulted in

homologous recombination producing ‘Full-Length’ WT NFT1 in the AMBV tMYTH bait vector backbone.


ADP1(tMYTH) NubG

NubI

NubG

NubIBPT1(tMYTH)

NubG

NubIYBT1(iMYTH)

NFT1(tMYTH) NubG

NubI

VMR1(tMYTH) NubG

NubI

NubG

NubI

NubG

NubI

PXA1(iMYTH)

PXA2(iMYTH)

DIC YFP RFP Overlay

Vacuolar

Peroxisomal

Scale bar

Supplementary Figure 2: NubGI test and fluorescence microscopy subcellular localization results for vacuolar and

peroxisomal ABC transporter bait strains used in MYTH screening. Scale bar is 10 mm for NubGI images and 6 μm for

microscopy images.

Scale bar


SNQ2(iMYTH)

YFPDIC Overlay

NubG

NubI

NubG

NubIPDR5(iMYTH)

NubG

NubIPDR15(iMYTH)

NubG

NubIPDR12(iMYTH)

PDR10(tMYTH) NubG

NubI

PDR11(iMYTH)

NubG

NubI

YOR1(iMYTH)

NubG

NubI

YOL075c(tMYTH) NubG

NubI

PDR18(tMYTH) NubG

NubI

AUS1(iMYTH) NubG

NubI

YFPDIC Overlay

STE6(iMYTH) NubG

NubI

Scale bar

Supplementary Figure 3: NubGI test and fluorescence microscopy subcellular localization results of ABC transporter MYTH

baits with predicted plasma membrane localization. Scale bar is 15 mm for NubGI images and 6 μm for microscopy images.

Note that cells expressing Aus1p baits were grown under anaerobic conditions, as described in the Online Methods section.

Scale bar


WT

(A

ero

bic

)

55

170

WT

(A

na

ero

bic

)

Au

s1p-C

ub-L

exA

-VP

16 (

Ae

robic

)

Au

s1p-C

ub-L

exA

-VP

16 (

An

aero

bic

)

Pd

r11p-C

ub-L

exA

-VP

16 (

Ae

robic

)

Pd

r11p-C

ub-L

exA

-VP

16 (

An

aero

bic

)

α-Hexokinase

α-VP16

Supplementary Figure 4: Expression of Aus1p and Pdr11p under aerobic and anaerobic growth conditions

monitored by Western blot. MYTH-tagged Aus1p and Pdr11p protein levels were measured using antibody directed

against the VP16 moiety of the MYTH tag. WT cells, not expressing MYTH-tagged protein, were used as a negative

control. Hexokinase levels were monitored in all cases to ensure equal loading.


VC

-AU

S1

VN-YDR476C

VN-YDR307W

VN-NSG2

VN-HXT5

VN-GUP2

Scale bar

AD

P1

-VC

BP

T1

-VC

VN-YDL085C-A

VN-COF1

VN-PFY1

[+]

[+]

[+]

[+]

[-]

VN-ERG27 [+]

VN-SMF2 [-]

VN-SOP4 [-]

[+]

[+]

[+]

Supplementary Figure 5: BiFC validation of selected interactions identified in MYTH screening. Left panels – YFP

channel. Right panels – DIC channel. Scale bar is 5 μm. Images marked with a ‘*’ were obtained via confocal microscopy

as described in the Online Methods.

*

*

*

See Fig. 1d for ADP1-VC

+ VN-ORM2.


PD

R5

-VC

VN-ORM2

PDR5-VN

NCR1-VN

[+]

[+]

[-]

PD

R5

-VC

VN-SMF2

VN-MCH5

VN-YOL075C

VN-SEO1

VN-NCR1

VN-YER097W

[+]

[+]

[+]

[+]

[-]

[-]

VN-ERG27

VN-ORM2 VN-ERG28

VC

-PD

R10

[+]

[+] [-]

VC

-PD

R10

VN-GUP2

VN-AGP1

VN-PTR2

[-]

[+]

[+]

Scale bar

Supplementary Figure 6: BiFC validation of selected interactions identified in MYTH

screening. Left panels – YFP channel. Right panels – DIC channel. Scale bar is 5 μm. .

Images marked with a ‘*’ were obtained via confocal microscopy as described in the Online

Methods.

VN-YOR215C [+]

See Fig. 3b for VC-PDR10

+ VN-PDR12.


+ VN-ZRC1.

* *

*

*

*

See Fig. 1d for PDR5-VC

+ VN-RAS2.


PD

R1

2-V

C

VN-PDR5

VN-YBR056W

VN-GTT1

[+]

[+]

[+]

PD

R1

5-V

C

VN-PTR2

VN-YER097W

VN-YOR215C [+]

[-]

[-]

Scale bar





Images.

*


VN-PDR11

PDR11-VN

VN-OSW5

OSW5-VN

VC

-PD

R18

SN

Q2

-VC

VN-PDR5

VN-CCC1

VN-AVT3

VN-SOP4

NCR1-VN

[+]

[-]

[-]

[-]

[-] [-]

[-]

[-]

[-]

VN-YGL081W

VM

R1

-VC

[-]

VN-SSM4

VN-ZRC1

[-]

[-]

Scale bar

Supplementary Figure 8: BiFC

validation of selected interactions

identified in MYTH screening. Left

panels – YFP channel. Right panels –

DIC channel. Scale bar is 5 μm.

See Fig. 3b for SNQ2-VC

+ PDR5-VN.


COF1-VN

YB

T1

-VC VN-GYL1

VN-YER121W

[+]

[+]

[+]

YC

F1

-VC

VN-BMH1

VN-ADH1

COF1-VN

VN-RAS2

VN-YCK3 [+]

[+]

[-]

[-]

[-]

VC

-YO

L075

C

VN-ZRC1

VN-ORM2

VN-YNL305C

[+]

[-]

[-]

Scale bar




See Fig. 3b for VC-YOL075C

+ VN-PDR5.

* *

*

*


VN-YCK1

VN-RAS2

VN-YPL199C

VN-YCK2

VN-GYL1

YO

R1

-VC

VN-YDL173W

VN-YGL082W

YO

R1

-VC

[+]

[+] [-]

[+]

[+]

[+]

[-]

Scale bar




*

*

*

*


PX

A1

-VC

VN-AHP1

VN-GRX1

VN-YPL225W

VN-ARF1

Diploid of BY4741 (Control);

no VN or VC fusions

[+]

[+]

[+]

[-]

VN-AHP1

VC

-PX

A2

VN-YPL225W

VN-ARF1

VN-GRX1

VN-BMH1

VN-ADH1

[-]

[-]

[-]

[-]

[-]

[-]

Scale bar

Supplementary Figure 11: BiFC

validation of selected interactions

identified in MYTH screening. Left

panels – YFP channel. Right panels

– DIC channel. Arrows in PXA1-VC

point to positive punctate signal

corresponding to interaction at

peroxisomes. Scale bar is 5 μm.


Rgt2

p-T

AP

Erg

28p-T

AP

Pdr10p-HA

160

180

160

180

WCL

(α-HA)

IP

(α-HA)

Rgt2

p-T

AP

Pdr5

p-T

AP

Ncr1p-HA

Rgt2

p-T

AP

Yer0

97w

p-T

AP

Pdr5p-HA

125

145

125

145

165

175

165

175

Rgt2

p-T

AP

Pdr1

8p-T

AP

Osw5p-HA

Rgt2

p-T

AP

Snq2p-T

AP

Avt3p-HA

Rgt2

p-T

AP

Ycf1

p-T

AP

Adh1p-HA

Rgt2

p-T

AP

Ycf1

p-T

AP

Cof1p-HA

15

25

60

80

30

40

15

25

15

25

60

80

30

40

15

25

Rgt2

p-T

AP

Yor1

p-T

AP

Gyl1p-HA

Rgt2

p-T

AP

Ycf1

p-T

AP

Ras2p-HA

Rgt2

p-T

AP

Yol0

75cp-T

AP

Ynl305cp-HA

WCL

(α-HA)

IP

(α-HA)

Rgt2

p-T

AP

Pxa1p

-TA

P

Arf1p-HA

Pxa2p

-TA

P

Rgt2

p-T

AP

Pxa2p

-TA

P

Ahp1p-HA

Rgt2

p-T

AP

Pxa2p

-TA

P

Grx1p-HA

Rgt2

p-T

AP

Pxa2p

-TA

P

Adh1p-HA

25

35

25

35

35

45

15

25

15

25

10

18

30

40

25

35

25

35

35

45

15

25

15

25

10

18

30

40

Supplementary Figure 12: Co-immunoprecipitation validation of MYTH interactions which could not be confirmed by BiFC. Sizes indicated at top and bottom of boxes were

estimated from MW markers on blots and are given in kDa. Rgt2p-TAP was used as a negative control. Interactions which were not successfully validated by Co-IP are listed

in the box below the images. Four interactions (Pdr11p-Pdr18p, Pdr15p-Yer097wp, Ssm4p-Pdr18p and Bmh1p-Pxa2p) could not be tested by Co-IP due to technical issues associated with strain generation.

Interactions Not Succesfully Validated by Co-IP: Aus1p-Gup2p, Adp1p-Sop4p, Adp1p-Smf2p, Pdr10p-Gup2p, Snq2p-Ccc1p, Snq2p-Sop4p, Snq2p-Ncr1p,

Vmr1p-Ygl081wp, Yol075cp-Zrc1p, Yor1p-Ygl082wp and Pxa2p-Ylp225wp

72

55

Rgt2

p-T

AP

Pdr1

5p-T

AP

Ptr2p-HA

72

55


Zrt1p-HA

34

45

34

45

WCL(α-HA)

IP(α-HA)

Rgt

2p-T

AP

Pdr5

p-TA

P

Pdr1

5p-T

AP

Rgt

2p-T

AP

Pdr1

8p-T

AP

Zrc1p-HA

40

55

40

55

Rgt

2p-T

AP

Pdr1

0p-T

AP

Zrt1p-HA

34

45

34

45

Supplementary Figure 13: Co-immunoprecipitation validation of ABC transporter interactions with Zrc1p and Zrt1p.Sizes indicated at top and bottom of boxes were estimated from MW markers on blots and are given in kDa. Rgt2p was used as a negative conttrol.


SGT2

SEC27

TPM2

SBP1

YLR361C-A

TMA22 HYP2

YLR257W

TIF2MYO1

KEM1

VBA4

CPR6

TPI1

SSB1

FBA1

TRX2

CCT3SSB2

STM1CKA2

EGD1NUM1

IPL1

PSA1

HAL5

BPT1

ASC1

VMA10

FAB1

YDL085C-A

TUS1

TIF3 ARC1

GTT1

VPS9

LSM4

GUS1LSM5

STE6

PFY1

EGD2

FPS1

TRP5FRE1

FRT2YNL010W RNR4

GOR1

GAS4

ZUO1

FPR4SMD1 CAM1

PXA2

SIN3HOM2

VMR1SNC2

CKA1

CKB1

DED81CDC28

YCF1

TMA7

MLP2

SOD1 ADH1

KIN4SNU13

BMH1 PAB1 EFB1

CDC19

GPM1

CKB2

TFG1

GTS1

YCK3

RGC1

RHO1 FAS2

PHO81

ATX1

DAL81

GRX1

ARF1

YMR099C

PIL1

CYS4

MOG1

PMA1 NCE102

HXT7 RAX2

FET4YLR050C TMN3

FUN26

FLC2

SCP160

USE1

ADP1

YJR096W YDR524C-B

BSD2 VMA16

MRL1

OTU1 SUR7

YND1 SUR4

PMT1

GLE1

MCH5

HKR1

DPM1

SMF2

NCR1PDR5VPS55

SNQ2

CCW12

CSM3 MCM5PUP1

WHI3

CCC1

PGI1DUR3

AVT3

TLG1

TPO3

VTC1HLJ1

URN1YGR026WECM27

TMS1

AIM4YGR266W

YAL019W-A

NRT1

FUN12

PRY3

UTP10YAP3SER1

SEO1

PAN5

OYE3 QCR8

MLS1

HXT16HSP82

ERP3

THI7

PMT7

YNL305CPGA2

YOR192C-C

HXT3

CUE4

COQ2

ERD1

HTZ1 FPR2

TSC13

ZRC1

AUS1

ENO2

SBH1

VTC2

HAP1

YKE2

CRN1

ABP1

UBC1

DAD4

FMP32

LEU4

AML1

PNC1DPP1

SAM3

SHO1

YER121W

RRP12

PMC1

YKU80

YCR061WSNA4

TVP15

KEX2

FTH1

NOP1

PRO2

YBT1

KES1

SSC1

YOL075C

KAR2

TEF1

SUI1

SBA1

ADE2

YPL225W

PXA1

AHP1

SHE2MSL1IPP1

NSG2THI80

GPM3

NPT1

TUM1

YMR155WYHL008C

YIL060WYMR196W

YDR476CIFH1

NPL3 YJR015W

MTC4

ERG20 SYN8 ADE12

SPT14

CFT2 GAS5

ECM33 RFS1

LRE1HPF1

PAR32 YGR250C

ATP1

SSO2

YCK1

YGL082W

VPH1

TPD3

VMA2

PPZ1

PST2

GPA2

MEP3

RPL4A

RPL24ARPL27A

RPL5

YOR1YPL199C

RPL10

RPL30YBR184W

MAK21

PBN1

YJL070C

VMA6

RPL7B

RPS1A

IRC5

AGC1

SYP1

GIN4

MID2

OSH7

RPP0

MAD2

QDR3 FTR1

VHT1

DYN3

KRE27FAA3

FUI1

WSC2

SNA3

HYR1

RIM1

TUB2

SLM1

WSC3GPX2

ENA2

PDR12 YVH1 YDL156WRCK1

SOP4

TPO1

HNM1

HXT5DIP5QDR2

TPO2

NFT1 YGL081W PDC1

ALK1

SEC72

RIB4 PRO3

PDR18CSG2

RTN1

HEK2

FLC1

PDR10OSW5

ERG9 YCK2

COS1

CBR1

GYL1

PDR11

SMT3

PGK1

PTR2

BZZ1POM33

ERG11

SSM4

COS8

COF1

LSM8

YNL208W

YDR056C PHO86

GUP2

YNR062CGET2

ZRT1

RPS24B

MRH1

IFA38

AGP1

SDS23FMP45

UIP3

SPT8

TPO4SFK1TOR2

TOR1

MUP1TCB3

PLM2

PDR15

YER097W AIM41

HSP30

HXT1

OST2ERG27

ERG28

ORM2VMA3

YBR056WITR1 UBI4

NAB2 RAS2

Human disease gene ortholog

Has human ortholog

No human ortholog

Bait

Membrane protein as prey

Soluble protein as prey

Conserved & novel

Conserved but not in MYTH dataset

Conserved in both MYTH & literature databaseNot ConservedOrthologs in human have experimental PPIOrthologs in human don’t have experimental PPI

Supplementary Figure 14: Integrated ABC interactome showing conservation of proteins in humans and association with known humandisease. Node color is used to indicate if a protein has a human ortholog (blue), has a human ortholog associated with human disease (red) or lacks an identifiable human ortholog (grey). Interactions between two proteins with a human ortholog are termed ‘conserved’and are connected by lines colored to indicate if the interaction is unique to our MYTH screen (red line), previously reported but not detectedby MYTH (yellow line) or previously reported and confirmed by our MYTH screen (blue line). Conserved interactions are connected by dashed lines if they have been experimentally validated in humans, or solid lines if they have not been experimentally validated in humans.Non-conserved interactions are connected by solid grey lines.


YBT1

ENO2KAR2

YOL075C

CBR1

ZRC1

PXA1

HAP1

VTC2

SSM4

GUP2

OSW5

SBH1

SBA1

TPI1

COF1

KEM1

CPR6

SSB1

SSC1

AML1

DAD4

PRO2

CRN1

LEU4

PNC1

YKE2

UBC1

FMP32

ABP1

SUI1

AHP1ATX1YPL225W

TEF1ADE2

GRX1

MOG1

FPR2

YER121W

HXT3

CUE4

TSC13

PGA2

NOP1

YNL305C

YOR192C-C

COQ2

PGI1

SSO2

CSG2

YBR056W

YDL156W

TUB2

YGL082W

PDR12RAS2

ERG28

MRH1

YNL208W

YDR056C

PDR15

FLC1

POM33ORM2

ERG27

PHO86VBA4 AGP1

YNR062CGET2

VPS55

HLJ1

VTC1

TLG1

SNQ2

CCC1NCR1

AVT3

QDR2SYN8

THI7HXT16

YDR476C

QCR8

YMR155W

YHL008C

YJR015W

MTC4

YND1

YAL019W-ASUR4TMN3

FUN12YGR026W

SER1

AIM41

YLR050CCCW12

YER097W

ZRT1

PDR5

OST2

PMT7

HXT5

AUS1ITR1

NSG2IFH1

SPT14

VMA3ERP3

DPM1

SOP4

YDR524C-B

ADP1SMF2

VMA16 FUN26MCH5

SEO1

PMA1

GLE1

SSB2STM1

YCF1

EGD1VMA10

SNU13

FAS2

MYO1

TPM2YLR257W

TIF2FPR4

YLR361C-A

HYP2

CAM1

YMR099C

SEC27

ARF1

SGT2

SBP1

HOM2

CYS4

DAL81

ARC1

TIF3

EGD2

BMH1

PAB1

SOD1

GTT1

ADH1

EFB1

PHO81

GPM1

CDC19

ASC1 YCK3

TFG1

PXA2

SIN3

TMA22ZUO1

YOR1YCK1

YPL199C

YCK2

IRC5YBR184W

PAR32 YJL070C

HPF1

LRE1

GAS5

ECM33

VMR1

STE6

VPS9

FRT2

GAS4

NFT1 TMA7

FPS1YGL081W

GUS1

PDC1TRP5

RNR4

PGK1

PRO3

PDR10RTN1

YNL010W

DED81

GYL1

PDR11

ERG9

COS1

PTR2

YDL085C-A

COS8

PFY1

PDR18

TRX2

BPT1

FBA1

PIL1

Cell Cycle, Growth & DivisionCell WallCytoskeletonGeneral RegulationMetabolismNuclear FunctionOtherProtein Degradation, Folding & ModificationProtein Synthesis & RibosomeRNA Processing / RegulationStress ResponseTransport, Trafficking & SecretionUnknown

Bait

Membrane protein as prey

Soluble protein as prey

Supplementary Figure 15: ABC transporter interactome showing only interactions identified in our MYTH screen. Individual nodesare colored according to functional classification and are assigned a shape based on whether they are bait/prey and localized to the membrane or soluble fractions, as described in the legend.


Cell Cycle Growth & Division (5%) Cell Wall

(2%)Cytoskeleton

(3%)DNA Replication, Maintenance &

Repair(1%)

General Regulation

7%

Metabolism15%

Nuclear Function1%

Other1%

Protein Degradation,

Folding & Modification

6%

Protein Synthesis & Ribosome

7%

RNA Processing & Regulation (5%)

Stress Response5%

Transport, Trafficking &

Secretion26%

Unknown16%

Supplementary Figure 16:

Functional distribution of nodes

in the integrated ABC

transporter interactome.


WT

snq2

pdr5

yor1

pdr18

pdr5 snq2

pdr18 pdr5

pdr18 snq2

yor1 pdr5

yor1 snq2

yor1 pdr18

0 mM 0.15 mM 0.2 mM

Supplementary Figure 17: Growth of ABC deletion strains challenged with the Snq2p transport substrate benomyl. Ten-

fold serial dilutions of each strain were spotted onto SD-Complete media containing benomyl at the indicated concentrations

and grown at 30C for 3 days. Scale bar is 20 mm.

Benomyl

Scale bar


WT

adp1

aus1

bpt1

ykr103w

pdr10

pdr11

pdr12

pdr18

pdr5

pxa1

pxa2

snq2

vmr1

ybt1

pdr15

ycf1

ykr104w

yor1

0 mM 0.2 mM

Benomyl

Supplementary Figure 18: Growth of ABC deletion strains challenged with the Snq2p transport substrate benomyl. Ten-fold

serial dilutions of each strain were spotted onto SD-Complete media containing 0 or 0.2 mM benomyl and grown at 30C for

3 days. Scale bar is 20 mm.

Scale bar


WT

snq2

pdr5

yor1

snq2 yor1

0 µM 1 µM 2 µM

pdr5 snq2

pdr5 yor1

Cycloheximide

Supplementary Figure 19: Growth of selected ABC single and double deletion strains challenged with the Pdr5p

transport substrate cycloheximide. Ten-fold serial dilutions of each strain were spotted onto SD-Complete media

containing 0, 1 or 2 µM cycloheximide and grown at 30C for 3 days. Scale bar is 15 mm.

Scale bar


WT

adp1

aus1

bpt1

ykr103w

pdr10

pdr11

pdr12

pdr18

pdr5

pxa1

pxa2

snq2

vmr1

ybt1

pdr15

ycf1

ykr104w

yor1

yol075c

0 µM 1 µM 5 µM

Cycloheximide

Supplementary Figure 20: Growth of ABC deletion strains challenged with the Pdr5p transport substrate cycloheximide. Ten-

fold serial dilutions of each strain were spotted onto SD-Complete media containing 0, 1 or 5 µM cycloheximide and grown at

30C for 3 days. Scale bar is 20 mm.

Scale bar


0 mM 0.2 mM

WT

adp1

aus1

bpt1

ykr103w

pdr10

pdr11

pdr12

pdr18

pdr5

pxa1

pxa2

snq2

vmr1

ybt1

pdr15

ycf1

ykr104w

yor1

0.4 mM

2,3,5-triphenyltetrazolium chloride (TTZ)

Supplementary Figure 21: Growth of ABC deletion strains challenged with the Pdr5p transport substrate 2,3,5-

triphenyltetrazolium chloride (TTZ). Ten-fold serial dilutions of each strain were spotted onto SD-Complete media containing 0,

0.2 or 0.4 mM TTZ and grown at 30C for 3 days. Scale bar is 15 mm.

Scale bar


α-VP16

(Snq2p-CYT) α-Hexokinase

-ve WT pdr5 yor1 pdr18kDa -ve WT pdr5 yor1 pdr18kDa

Supplementary Figure 22: Full images of Snq2p-CYT and hexokinase control Western Blots.

250

100

130

70

55

35

250

100

130

70

55

35


size bar: 3µm size bar: 1µm

YF

PC

FP

YF

P+

CF

P

size bar: 3µm

16X zoomed images

of boxed regions

size bar: 1µm

Supplementary Figure 23: Co-localization of the bimolecular fluorescent complex of VC-PDR10 and VN-ZRC1 with (a) the plasma membrane marker

CFP-Cdc42 (2 views) and (b) the endoplasmic reticulum marker Sec63-mCherry. For YFP+CFP and YFP+RFP overlay images YFP signal is shown in

Green, while CFP and RFP signals are shown in Red.

16X zoomed images

of boxed regions

YF

PR

FP

YF

P+

RF

P

15X zoomed images

of boxed regions

size bar: 3µmsize bar: 1µm

a b


α-VP16 (Zrt1p-CYT)

-ve

WT

pdr5

yo

r1

pdr1

8

α-Hexokinase

-ve

WT

pdr5

yo

r1

pdr1

8

-ve

WT

pdr5

yo

r1

pdr1

8

α-VP16 (Zrc1p-CYT) α-Hexokinase

-ve

WT

pdr5

yo

r1

pdr1

8

α-VP16 (Zrt1p-CYT)

-ve

WT

pdr5

yo

r1

pdr1

8

α-Hexokinase-v

e

WT

pdr5

yo

r1

pdr1

8

-ve

WT

pdr5

yo

r1

pdr1

8

α-VP16 (Zrc1p-CYT)

-ve

WT

pdr5

yo

r1

pdr1

8

α-Hexokinase

a c

b d

Supplementary Figure 24: Full images of Zrt1p-CYT, Zrc1p-CYT and hexokinase control Western Blots. (a) and (b) show blots performed on protein

extracts from cells grown under conditions of zinc limitation. (c) and (d) show blots performed on protein extracts isolated from cells grown under zinc-

replete conditions. Unlabelled lanes in (a) were not used in generation of figures. The ‘*’ in (a) and (c) identify the bands presented in the cropped image.

2501007055

35

25

15

2501007055

35

25

15

kDa kDa

2501007055

35

25

15

kDa250

1007055

35

25

15

kDa

1751309570625142

kDa

29

2214

17513095

706251

42

kDa

29

2214

1751309570625142

kDa

29

22

14

1751309570625142

kDa

29

22

14

**


1.0

0.5

zrt1WT

Fra

ction o

f C

ontr

ol

1.5

2.0

2.5

3.0

155 18 5

zrt1

15

zrt1

18

zrt1

0.0

Supplementary Figure 25: Analysis of zinc uptake in Y7092 WT and deletion strains. Values represent the average of three replicates (n=3) and are

expressed relative to WT. Error bars indicate standard deviation. Zinc uptake is increased ~2.7 fold in the pdr15 strain relative to WT (two-tailed t-test, p-

value = 1.6x10-3), and decreased ~0.5 fold in pdr5 zrt1 (two-tailed t-test, p-value = 2.9x10-4) and pdr18 zrt1 (two-tailed t-test, p-value = 6.5x10-5) double deletion strains.


Supplementary Table 1: List of primers (shown 5’ to 3’) used in generation of iMYTH and tMYTH ABC

transporter baits. Primer sequence in uppercase corresponds to ABC transporter gene-specific

sequence.

Standard Name

Systematic Name tMYTH / iMYTH

Tagging Primer Sequence

ADP1 YCR011C tMYTH F: tgcacaatatttcaagctataccaagcatacaatcaactccaagcaacacaATGGGAAGTCATCGACGTTATC

R: tcgacggtatcgataagcttgatatcgaattcctgcagatCTTTTGTTCCACAACTATCCAC

AUS1 YOR011W iMYTH F: ATCACCAAAGTAATTCCACACAGAGGGAAGAAGCCTGTACAGAACatgtcgggggggatccctcc

R: GTTGTACAAAGGACTCTTTCAATTGTTTAGTCAGTCATTGGTGACactatagggagaccggcaga

BPT1 YLL015W tMYTH F: tgcacaatatttcaagctataccaagcatacaatcaactccaagcaacacaATGTCTTCACTAGAAGTGGTAG

R: tcgacggtatcgataagcttgatatcgaattcctgcagatTTTCAAATACCCACCTTTCTC

NFT1 YKR103W/YKR104W tMYTH

A: tgcacaatatttcaagctataccaagcatacaatcaactccaagcaacacaATGATAAAAAATGGTACATGCCCC

B: CCCCGCTAGCCCCGCATCCAAATATGAGGCCTTCATTATGATC

C: GATCATAATGAAGGCCTCATATTTGGATGCGGGGCTAGCGGGG

D: tcgacggtatcgataagcttgatatcgaattcctgcagatTCTTTTATTATCGAATGAGAC

PDR10 YOR328W tMYTH F: tgcacaatatttcaagctataccaagcatacaatcaactccaagcaacacaATGTTGCAAGCGCCCTCAAG

R: tcgacggtatcgataagcttgatatcgaattcctgcagatTTTCTTTAATTTTTTGCTTTTC

PDR11 YIL013C iMYTH F: ATGAGGCGGTCCAAAACATCTCATAATCCAAACGAACAAAGCGTAatgtcgggggggatccctcc

R: TCCGAAAAATGATTATAGATTAATTTAGAATATGATTTGGGAATAactatagggagaccggcag

PDR12 YPL058C iMYTH F: ATTTTCCAAACAGTTCCAGGTGACGAAAATAAAATCACGAAGAAAatgtcgggggggatccctcc

R: ACTCACGAGTGGGATAGAAATGAAATTCTTTTCTTTTAAATGGTAactatagggagaccggcag

PDR15 YDR406W iMYTH F: AGGGTACCCAAGAAGAACGGTAAGATTTCCGAAAAACCCAAGAAGatgtcgggggggatccctcca

R: CTATATAGAATATAGATAATATAAAACGAAAAGAGCCTGATGTTGactatagggagaccggcaga

PDR18 YNR070W tMYTH F: tgcacaatatttcaagctataccaagcatacaatcaactccaagcaacacaATGGAATGCGTTTCAGTAGAAG

R: tcgacggtatcgataagcttgatatcgaattcctgcagatAATGAAACCGAAGTTTCTCCATAAG

PDR5 YOR153W iMYTH F: TGGTTAGCAAGAGTGCCTAAAAAGAACGGTAAACTCTCCAAGAAAatgtcgggggggatccctcca

R: GATGCCTATAAAAAAAAAGTACCGATGAGATAACCTAGGAATAAAactatagggagaccggcaga

PXA1 YPL147W iMYTH F: TGGGAAGATGAGAGGACGAAGCTACGGGAAAAGCTTGAAATTATTatgtcgggggggatccctcca

R: CGTGGATTCATCAAGTGAGATATATATATATATGTTCATATTTGTactatagggagaccggcaga

PXA2 YKL188C iMYTH F: TTGAACAAAAAAGTTAAAACAAAAAAGGAAGAAGGAAAGGAGAGGatgtcgggggggatccctcca

R: AATTATATATAGGAAAGTGTTTATTTGCATAAAAAGGGAAAAATAactatagggagaccggcaga

SNQ2 YDR011W iMYTH F: GTATCTATACTCAATAAAATTAAAAACATAAGGAAAAAGAAGCAGatgtcgggggggatccctcca

R: AAACTTTTTTACTCAACAAGACTTGACTTTTGAAAACTACGAGAGactatagggagaccggcaga

STE6 YKL209C iMYTH F: AATAATCGCGGGGAATTATTCCAAATTGTTTCCAACCAAAGCAGTatgtcgggggggatccctcca

R: GTCTCGAATATTTGAGTATGTTTTAGTTTTTTGTTTTATATTTTCactatagggagaccggcaga

VMR1 YHL035C tMYTH F: tgcacaatatttcaagctataccaagcatacaatcaactccaagcaacacaATGGGAACGGATCCCCTTATTATC

R: tcgacggtatcgataagcttgatatcgaattcctgcagatTTTCATCATCTTACTTGATT

YBT1 YLL048C iMYTH F: TTGGCTAAAAAAGCCTTTGTGGAAAAATTGAACTCTAAAAAGGACatgtcgggggggatccctcca

R: ATTTATAGTACGTGAACATGTGTGCGTATATACATATATATATATactatagggagaccggcaga

YCF1 YDR135C iMYTH F: TTGTTCTATTCACTGTGCATGGAGGCTGGTTTGGTCAATGAAAATatgtcgggggggatccctcca

R: TAAGCCATTATCATCGTTGCCTTCATTATATCTTTTTATTGCTGGactatagggagaccggcaga

YOL075C YOL075C tMYTH F: tgcacaatatttcaagctataccaagcatacaatcaactccaagcaacacaATGTCACAGCAGGAGAATGGTG

R: tcgacggtatcgataagcttgatatcgaattcctgcagatCCATTTTATCCACTCCAATTT

YOR1 YGR281W iMYTH F: TGTTCTAGATCTGGTATTGTGGAAAATGATTTCGAGAACAGAAGTatgtcgggggggatccctcca

R: ATGTATAAATATATATTTCTAGAATGAAAAAGGACCGAAGGCGTTactatagggagaccggcaga


Supplementary Table 2: List of primers (shown 5’ to 3’) used in quantitative real time PCR

experiments.

Standard

Name Systematic Name F Primer R Primer

ACT1 YFL039C AGTGTGATGTCGATGTCCGT TGACCTTCATGGAAGATGGA

SNQ2 YDR011W CTTGTTGGTGAGGTTGGTTG GGCCCATGAAGATTGAGAAT

ZRC1 YMR243C AAAGCGGGAATAACGATTTG GATTGTGGCAACACTTCACC

ZRT1 YGL255W TCCTGCCATTATGCTAACGA CAGCTGCAGTGTTTCTCACA


mapping the functional yeast abc transporter …...department of cell biology, johns hopkins school...

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