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1 supplementary information Figure S1 (a) Alignment of the Sgf11 amino acid sequences from S. cerevisiae, Yarrowia lipolytica and S. pombe with Sgf73 sequences from S. cerevisiae, S. pombe and Candida albicans. ClustalW1.8 ( was used for alignments. The conserved cysteine and histidine residues in the ZnF1 domain are indicated by arrows. (b) Schematic drawing of the structural organization of Sgf73 and Sgf11. In Sgf73, ZnF1 is located between residues and ZnF2 between residues In Sgf11, ZnF1 is located between residues (c) Analysis of expression of Sgf73-TAP and truncated Sgf73-TAP constructs in whole cell extracts (WCE) by anti-proteina Western blotting. (d) Recombinant proteins used for Ubi-AMC hydrolysis assay and reconstitution assay. Asterisks mark common E.coli contaminants. Lane 3 depicts a reconstituted Sus1-Sgf11 heterodimer. 1

2 supplementary information Figure S2 (a) Affinity-purification of Sus1-TAP from wild-type (WT) or Sgf73 C strains, with both strains expressing Thp1-FLAG. After Sus1-TAP immunodepletion (>90%, data not shown), the supernatant was subjected to Thp1-FLAG immunoprecipitation. Eluates derived from these purifications were analyzed by SDS-PAGE and Coomassie staining and after Western blotting further probed with the indicated antibodies. Filled square indicates Ubp8, open square indicates Thp1. Asterisk marks a contaminant, open circles degradation products of Sac3 as determined by mass spectrometry. (b) Analysis of expression levels of 3HA-Sgf73 and indicated N-terminally truncated 3HA-Sgf73 constructs in whole cell extracts (WCE) derived from Sus1-TAP strains. Whole cell extracts were analyzed by SDS-PAGE and Western blotting using anti-ha antibodies. (c) Affinity-purifications of Ada2-TAP from strains co-expressing 3HA- Sgf73 or indicated N-terminal truncations. Eluates were analyzed by SDS-PAGE and Coomassie staining (upper panel) or Western blotting (lower panel) using anti-ha antibodies. Filled circles mark Ada2-TAP. The indicated bands were identified by mass spectrometry. (d) Expression levels of Sac and Thp1 in the indicated strains. Whole cell extracts were analyzed by SDS-PAGE and anti-myc Western blotting. Asterisk indicates putative Sac3 degradation band. 2

3 supplementary information Figure S3 (a) Anti-Flag immunoprecipitates derived from the indicated strains were analyzed by SDS-PAGE and anti-flag Western blotting to detect unmodified Flag-H2B and ubiquitinated Flag-H2B. (b) Measurement of intranuclear position of the GAL locus. The ygc234 strain (ubp8 ) was transformed with either prs416-ubp8, prs416 or prs416-ubp8c146s. The ubp8c146s mutation replaces a conserved catalytically active residue in the Ubp8 active site. Strains were grown in SC-adenine-uracil medium containing 2% raffinose before dilution and growth in glucose or galactose containing media. After Z-stacks acquisition on an AxioImager Z1 (Carl Zeiss) microscope, cells with the reporter locus positioned in the z-section that cuts through the middle of the nucleus were manually scored for a peripheral or intranuclear position according to the criteria defined 1. N represents total number of cells analyzed in three independent experiments. Error bars indicate standard error of the measurements. (c) Box plot depicting the distribution of RNA-dot fluorescent intensity measurements in the respective strains in three independent experiments. Fluorescent intensity was quantified as the mean intensity of the selected dot area using the public domain NIH Image program ( The ends of the boxes define the 25th and 75th percentiles, with a line at the median and error bars defining the 10th and 90th percentiles. N indicates the number of analyzed cells. GAL1 mrna localization of wt versus ubp8c146s cells as determined by fluorescence in situ hybridization (FISH) with GAL1-specific Cy3-labeled probes (scale bar, 2µm). The GAL1 gene locus was visualized simultaneously by TetO-TetR-GFP and DNA was stained with DAPI. (d) Analysis of GAL1 expression by real-time RT-PCR after 4 hours of galactose induction. Data are presented as GAL1 expression relative to WT with WT expression set as 1. N represents number of independent experiments. Error bars indicate standard error of the measurements. 3

4 supplementary information Figure S4 Full scanned images (a) anti-myc Western Blots (Figure 1b) (b) anti-flag Western Blots (Figure 1c) (c) anti-myc Western Blot (Figure 2a) (d) Coomassie-stained SDS-PAGE used for equalizing the amount of Ubp8 in Ub-AMC assay of Figure 2c. Filled circles indicate bait proteins (e) anti-flag Western Blot (Figure 2d). Asterisks indicate cross-reactivity of the antibody (f) anti-proteina Western Blots (Figure 3f) (g) anti-myc and anti-cbp Western Blots of Figure 3g. 4

5 Supplementary Table 1 : Saccharomyces cerevisiae strains, plasmids and primers Yeast Strain Genotype Source SGF73-TAP ADA2-TAP SUS1-13MYC Mat a, his3δ1 leu2δ0 met15δ0 ura3δ0 SGF73 -TAP::HIS3 MX6 Mat a, ade2, arg4, leu2-3,112, ura3-52, trp1-289, ADA2- TAP::K.l. URA3, SUS1-13MYC::KanMX4 Open Biosystems Ref 2 ADA2-TAP SUS1- Mat a, ade2, arg4, leu2-3,112, ura3-52, trp1-289, ADA2-13MYC ubp8δ TAP::K.l. URA3, SUS1-13MYC::KanMX4 ubp8δ::natnt2 ADA2-TAP SUS1- Mat a, ade2, arg4, leu2-3,112, ura3-52, trp1-289, ADA2-13MYC sgf73δ TAP::K.l. URA3, SUS1-13MYC::KanMX4 sgf73δ::natnt2 SGF TAP MATa; ura3-52; trp1d2; leu2-3,112; his3-11; ade2-1; can1-100 SGF73 aa1-114-tap ::TRP1 SGF TAP MATa; ura3-52; trp1d2; leu2-3,112; his3-11; ade2-1; can1-100 SGF73 aa1-104-tap ::TRP1 SGF TAP MATa; ura3-52; trp1d2; leu2-3,112; his3-11; ade2-1; can1-100 SGF73 aa1-96-tap ::TRP1 SGF TAP MATa; ura3-52; trp1d2; leu2-3,112; his3-11; ade2-1; can1-100 SGF73 aa1-92-tap ::TRP1 SGF TAP MATa; ura3-52; trp1d2; leu2-3,112; his3-11; ade2-1; can1-100 SGF73 aa1-53 ::TRP1 Y131 MATa his3-11,-15 leu2-3,112 trp1-1 ura3-1 ade2-1 hta- Ref 3 1htb1::LEU2 hta2-htb2:: + prs426-hta1-htb1 YZS277 MATa his3-11,-15 leu2-3,112 trp1-1 ura3-1 ade2-1 hta- Ref 3 1htb1::LEU2 hta2-htb2:: + prs413-flag-htb1-k123r- HTA1 YZS276 MATa his3-11,-15 leu2-3,112 trp1-1 ura3-1 ade2-1 hta- Ref 3 1htb1::LEU2 hta2-htb2:: + prs413-flag-htb1-hta1 YZS276 ubp8δ MATa his3-11,-15 leu2-3,112 trp1-1 ura3-1 ade2-1 hta- 1htb1::LEU2 hta2-htb2:: + prs413-flag-htb1-hta1 ubp8δ::natnt2 YZS276 sgf73δ MATa his3-11,-15 leu2-3,112 trp1-1 ura3-1 ade2-1 hta- 1htb1::LEU2 hta2-htb2:: + prs413-flag-htb1-hta1 sgf73δ::natnt2 YZS276 SGF73ΔC MATa his3-11,-15 leu2-3,112 trp1-1 ura3-1 ade2-1 hta HA 1htb1::LEU2 hta2-htb2:: + prs413-flag-htb1-hta1 SGF73ΔC HA::natNT2 THP1-TAP SUS1- Matα, his3, leu2, trp1, ura3, THP1-TAP::TRP1, SUS1- Ref 2 13MYC MYC::HIS3 THP1-TAP SUS1- Matα, his3, leu2, trp1, ura3, THP1-TAP::TRP1, SUS1-13MYC sgf73δ MYC::HIS3 sgf73δ::natnt2 SAC3-GFP Matα ade2-1; ade3; his3-11,15; ura3-52; leu2-3,112; trp1- Ref 3 1; SAC3-GFP::TRP1 SAC3-GFP ubp8δ Matα ade2-1; ade3; his3-11,15; ura3-52; leu2-3,112; trp1-1; SAC3-GFP::TRP1 ubp8δ::natnt2 SAC3-GFP sgf73δ Matα ade2-1; ade3; his3-11,15; ura3-52; leu2-3,112; trp1-1; SAC3-GFP::TRP1 sgf73δ::natnt2 SAC3-GFP sus1δ Matα ade2-1; ade3; his3-11,15; ura3-52; leu2-3,112; trp1-1; SAC3-GFP::TRP1 sus1δ::natnt2 THP1-GFP Matα, his3; ura3; leu2; trp1; THP1-GFP::TRP1 Ref 4 THP1-GFP ubp8δ Matα, his3; ura3; leu2; trp1; THP1-GFP::TRP1 ubp8δ::natnt2 THP1-GFP sgf73δ Matα, his3; ura3; leu2; trp1; THP1-GFP::TRP1 sgf73δ::natnt2

6 THP1-GFP sus1δ Matα, his3; ura3; leu2; trp1; THP1-GFP::TRP1 sus1δ::natnt2 SUS1-TAP Mat α, ade2, his3, leu2, trp1, ura3, SUS1-TAP::TRP1 Ref 2 SUS1-TAP ubp8δ Mat α, ade2, his3, leu2, trp1, ura3, SUS1-TAP::TRP1 Ref 5 ubp8δ::kanmx4 SUS1-TAP sgf73δ Mat α, ade2, his3, leu2, trp1, ura3, SUS1-TAP::TRP1 sgf73δ::natnt2 SUS1-TAP SGF73- Mat α, ade2, his3, leu2, trp1, ura3, SUS1-TAP::TRP1 13MYC SGF73 13MYC::HIS3 MX6 SUS1-TAP SGF73ΔC Mat α, ade2, his3, leu2, trp1, ura3, SUS1-TAP::TRP MYC SGF73ΔC MYC::HIS3 MX6 SUS1-TAP SGF73ΔC Mat α, ade2, his3, leu2, trp1, ura3, SUS1-TAP::TRP MYC SGF73ΔC MYC::HIS3 MX6 SUS1-TAP SGF73ΔC Mat α, ade2, his3, leu2, trp1, ura3, SUS1-TAP::TRP MYC SGF73ΔC MYC::HIS3 MX6 SUS1-TAP SGF73ΔC Mat α, ade2, his3, leu2, trp1, ura3, SUS1-TAP::TRP MYC SGF73ΔC MYC::HIS3 MX6 SUS1-TAP SGF73ΔC Mat α, ade2, his3, leu2, trp1, ura3, SUS1-TAP::TRP MYC SGF73ΔC MYC::HIS3 MX6 SUS1-TAP 3HA- Mat α, ade2, his3, leu2, trp1, ura3, SUS1-TAP::TRP1 SGF73 P ADH::natNT2 3HA-SGF73 SUS1-TAP 3HA-ΔN1- Mat α, ade2, his3, leu2, trp1, ura3, SUS1-TAP::TRP1 104 SGF73 P ADH::natNT2 3HA-ΔN1-104 SGF73 SUS1-TAP 3HA-ΔN1- Mat α, ade2, his3, leu2, trp1, ura3, SUS1-TAP::TRP1 226 SGF73 P ADH::natNT2 3HA-ΔN1-226 SGF73 SUS1-TAP 3HA-ΔN1- Mat α, ade2, his3, leu2, trp1, ura3, SUS1-TAP::TRP1 272 SGF73 P ADH::natNT2 3HA-ΔN1-272 SGF73 SUS1-TAP THP1- Mat α, ade2, his3, leu2, trp1, ura3, SUS1-TAP::TRP1 FLAG THP1-FLAG::kanMX6 SUS1-TAP THP1- Mat α, ade2, his3, leu2, trp1, ura3, SUS1-TAP::TRP1 FLAG SGF73ΔC MYC THP1-FLAG::kanMX6 SGF73ΔC MYC::HIS3 MX6 ygc105 Mat a, leu2, his3, trp1, ade2, ura3 LEU2::TetR-GFP::leu2, Ref 1 (teto*112)-nat::intergal1-fur4, pasz11ade2::gfp- Nup49 ygc234 Mat a, leu2, his3, trp1, ade2, ura3 LEU2::TetR-GFP::leu2, (teto*112)-nat::intergal1-fur4, pasz11ade2::gfp- Nup49 ubp8δ::his3 MX6 ygc244 Mat a, leu2, his3, trp1, ade2, ura3 LEU2::TetR-GFP::leu2, (teto*112)-nat::intergal1-fur4, pasz11ade2::gfp- Nup49 sgf73δc MYC::HIS3 MX6 ADA2-TAP 3HA- Mat α, ade2, his3, leu2, trp1, ura3, ADA2-TAP::TRP1 SGF73 P ADH::natNT2 3HA-SGF73 ADA2-TAP 3HA-ΔN1- Mat α, ade2, his3, leu2, trp1, ura3 ADA2-TAP::TRP1 104 SGF73 P ADH::natNT2 3HA-ΔN1-104 SGF73 ADA2-TAP 3HA-ΔN1- Mat α, ade2, his3, leu2, trp1, ura3, ADA2-TAP::TRP1 272 SGF73 P ADH::natNT2 3HA-ΔN1-272 SGF73 ADA2-TAP 3HA- SGF73 SUS1-13myc Mat α, ade2, his3, leu2, trp1, ura3, ADA2-TAP::TRP1 P ADH::natNT2 3HA-SGF73 SUS1-MYC::HIS3 MX6 ADA2-TAP 3HA-ΔN1-104 SGF73 SUS1-13myc Mat α, ade2, his3, leu2, trp1, ura3 ADA2-TAP::TRP1 P ADH::natNT2 3HA-ΔN1-104 SGF73 SUS1-13MYC::HIS3 MX6

7 ADA2-TAP SAC3-13myc THP1-13myc Mata, ade2, arg4, leu2-3,112, ura3-52, trp1-289, ADA2- TAP::K.l. URA3, SAC3-13MYC::TRP THP1-13 MYC::KanMX4 ADA2-TAP SAC3-13myc THP1-13myc ubp8δ ADA2-TAP SAC3-13myc THP1-13myc SGF73ΔC HA Mata, ade2, arg4, leu2-3,112, ura3-52, trp1-289, ADA2- TAP::K.l. URA3, SAC3-13MYC::TRP THP1-13MYC::KanMX4 ubp8δ::natnt2 Mata, ade2, arg4, leu2-3,112, ura3-52, trp1-289, ADA2- TAP::K.l. URA3, SAC3-13MYC::TRP THP1-13MYC::KanMX4 SGF73ΔC HA::natNT2 BY4741 MATa; his3δ1; leu2δ0; met15δ0; ura3δ0 EUROSCARF sgf73δ MATa; his3δ1; leu2δ0; met15δ0; ura3δ0 sgf73δ::kanmx4 EUROSCARF sacδ MATa; his3δ1; leu2δ0; met15δ0; ura3δ0 sac3δ::kanmx4 EUROSCARF thp1δ MATa; his3δ1; leu2δ0; met15δ0; ura3δ0 thp1δ::kanmx4 EUROSCARF ubp8δ MATa; his3δ1; leu2δ0; met15δ0; ura3δ0 ubp8δ::kanmx4 EUROSCARF sus1δ MATa; his3δ1; leu2δ0; met15δ0; ura3δ0 sus1δ::kanmx4 EUROSCARF sgf73δ sacδ MATa; his3δ1; leu2δ0; met15δ0; ura3δ0 sgf73δ::kanmx4 sac3δ::natnt2 sgf73δ thp1δ MATa; his3δ1; leu2δ0; met15δ0; ura3δ0 sgf73δ::kanmx4 thp1δ::natnt2 sgf73δ ubp8δ MATa; his3δ1; leu2δ0; met15δ0; ura3δ0 sgf73δ::kanmx4 ubp8δ::natnt2 sgf73δ sus1δ MATa; his3δ1; leu2δ0; met15δ0; ura3δ0 sgf73δ::kanmx4 sus1δ::natnt2 Plasmids pet24d-gst-tev-sgf pet24d-gst-tev-ubp8 pet24d-gst-tev-sus1 pproexhtb-6xhis-tev-sgf11 prs416-ubp8 prs416-ubp8c146s Source RT-PCR Primers GAL1 FOW GAL1 REV GAL1 TaqMan Probe TGAAGAGTCTCTCGCCAATAAGAA TCGCGAGAACAATTCAAGGA AGGGCTTTAGTGTTGACGATGTCGCACA

8 Supplementary References 1. Cabal, G. G. et al. SAGA interacting factors confine sub-diffusion of transcribed genes to the nuclear envelope. Nature 441, (2006). 2. Rodriguez-Navarro, S. et al. Sus1, a Functional Component of the SAGA Histone Acetylase Complex and the Nuclear Pore-Associated mrna Export Machinery. Cell 116, (2004). 3. Robzyk, K., Recht, J. & Osley, M. A. Rad6-dependent ubiquitination of histone H2B in yeast. Science 287, (2000). 4. Fischer, T. et al. The mrna export machinery requires the novel Sac3p-Thp1p complex to dock at the nucleoplasmic entrance of the nuclear pores. EMBO J. 21, (2002). 5. Köhler, A. et al. The mrna export factor Sus1 is involved in Spt/Ada/Gcn5 acetyltransferasemediated H2B deubiquitinylation through its interaction with Ubp8 and Sgf11. Mol Biol Cell 17, (2006).

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