1. 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. Cytoplasm Nucleus INM ONM INM ONM Nucleus Cytoplasm a. Nuclear substrate is exported to cytoplasm b. Doa10 is transported to the INM MAT 2 Doa10 Supplementary Figures and Legends Supplementary Information

2. 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.

3. His – His – Trp – G BD G BD -Stt3 G BD -Doa10 Aeb:UAS G aeB aeb:UAS G Aeb:UAS G sir2 Supplementary Figure S3. Targeted silencing by G BD -Doa10 assayed with serially diluted yeast cells. Proteins were expressed in YSB35 (top three rows); YSB1 (no UAS G ); YSB41, which has UAS G but lacks all three HMR-E elements; and RS1132, a sir2 mutant 7.

4. HMG1 HC nup53C Sec61-GFP 2 m Supplementary Figure S4. Overexpression of Hmg1 and Nup53 C fail 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. gal activity (%) WT pom152 nup188 doa10 Chase time (min) 0 20 40 60 80 100 015306090 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.

6. N Cyt ER Crn1 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.

7. a b 1530 01530 0 Pgk1 Ura3-SL17 WT doa10- pgk1 doa10 15300 1530 0 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.