Supplementary Figure S1. Two models for how nuclear substrates gain access to the ER-embedded

1. a. Nuclear substrate is exported to cytoplasm Cytoplasm ONM INM Nucleus Doa10 b. Doa10 is transported to the INM MAT2 Cytoplasm ONM INM Nucleus Supplementary Figure S1. Two models for how nuclear substrates gain access to the ER-embedded ubiquitin ligase Doa10. a. Nuclear substrates such as Mat2 are exported out of Nucleus. b. Doa10 can traffic to INM through the lateral channels of the nuclear pore complex. Supplementary Information Supplementary Figures and Legends

2. NE N 0.1 m 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.

3. His – His – Trp – GBD Aeb:UASG GBD-Stt3 GBD-Doa10 aeB GBD-Doa10 aeb:UASG GBD-Doa10 Aeb:UASG sir2 GBD-Doa10 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 .

4. HMG1HC nup53∆C 2m Sec61-GFP Supplementary Figure S4. Overexpression of Hmg1 and Nup53Cfail to induce theta nuclei. Sec61-GFP-expressing cells were transformed with either a high-copy HMG1 or nup53C plasmid and imaged by confocal microscopy.

5. 100 80 60 gal activity (%) doa10∆ 40 pom152∆ 20 nup188∆ WT 0 0 15 30 60 90 Chase time (min) 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-bgal. Degradation was measured by bgal 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.

6. Doa10 Crn1 ER N Cyt 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.

7. 100 80 doa10 doa10- pgk1 60 gal activity (%) 40 WT 20 doa10- hrd1C 0 0 30 60 90 Chase time (min) doa10- pgk1 doa10- hrd1C doa10∆ WT 15 30 0 15 30 0 15 30 0 0 15 30 Ura3-SL17 Pgk1 a b Supplementary Figure S7. a. Nuclear substrate degradation correlates with Doa10 nuclear entry. Deg1-bgal degradation was measured as in Suppl.Fig. 5.b. Cytoplasmic substrate turnover is not impaired by Doa10 nuclear exclusion.