Supplementary Figure S1. Two models for how nuclear substrates gain access to the ER-embeddedubiquitin ligase Doa10. a. Nuclear substrates such as Mat2 are exported out of Nucleus. b. Doa10 can traffic to INM through the lateral channels of the nuclear pore complex.

Cytoplasm

Nucleus

INM

ONM

INM

ONM

Nucleus

Cytoplasm

a. Nuclear substrate is exported to cytoplasm

b. Doa10 is transported to the INM

MAT2

Doa10

Supplementary Figures and Legends

Supplementary Information

0.1 m

N

NE

0. 06 m

Supplementary Figure S2. Ultrastructural localization of Doa10 to the inner NE. Anti-GFP immunogold EM staining was used to localize Doa10-GFP. Arrowheads mark gold beads. Left: Cell without Nup53 overexpression. Right: Nup53-induced INM lamellae.

His – His – Trp –

GBD

GBD-Stt3

GBD-Doa10

GBD-Doa10

GBD-Doa10

GBD-Doa10

Aeb:UASG

aeB

aeb:UASG

Aeb:UASG sir2

Supplementary Figure S3. Targeted silencing by GBD-Doa10 assayed with serially diluted yeast cells. Proteins were expressed in YSB35 (top three rows); YSB1 (no UASG); YSB41, which has UASG but lacks all three HMR-E elements; and RS1132, a sir2 mutant7 .

HMG1HC nup53∆C

Sec61-GFP

2m

Supplementary Figure S4. Overexpression of Hmg1 and Nup53C fail to induce theta nuclei. Sec61-GFP-expressing cells were transformed with either a high-copy HMG1 or nup53C plasmid and imaged by confocal microscopy.

gal

act

ivit

y (%

)

WT

pom152∆nup188∆

doa10∆

Chase time (min)

0

20

40

60

80

100

0 15 30 60 90

Supplementary Figure S5. Partial impairment of Doa10 import in nup188 and pom152 mutants correlates with a mild defect in the degradation of a nuclear substrate, Deg1-gal. Degradation was measured by gal activity assays of three independent cultures after addition of cycloheximide. Half-lives were significantly longer in nup188 (P<0.01) and pom152 (P<0.05) relative to WT.

N

Cyt

ERCrn1

Doa10

Supplementary Figure S6. Scheme to tether Doa10 at cortical ER sites and prevent INM entry. The actin-binding domain of Crn1, which binds to cortical actin patches, was fused to Doa10 (followed by GFP), yielding Doa10-CNG.

a

gal

act

ivit

y (%

)

Chase time (min)

20

40

60

80

100

0 30 60 900

doa10-pgk1

doa10

doa10-hrd1C

WT

b

15 30 0 15300

Pgk1Ura3-SL17

WTdoa10-pgk1 doa10∆

15 300 15 300

doa10-hrd1C

Supplementary Figure S7. a. Nuclear substrate degradation correlates with Doa10 nuclear entry. Deg1-gal degradation was measured as in Suppl.Fig. 5. b. Cytoplasmic substrate turnover is not impaired by Doa10 nuclear exclusion.