Conserved residues differentially regulate association of Sus1 with SAGA or TREX-2. a, multiple sequence alignment of Sus1 from representative species including fungi, protozoa, plants and metazoa. The alignment was generated with ClustalW2, and conserved residues were shaded using JalView. b, secondary structure prediction for Sus1 was calculated from PsiPred. For schematic depiction of Sus1 mutational strategies, mutant alleles are numbered (sus1ΔN, sus1–1 to sus1–12, sus1ΔC). All Sus1 charged residues are indicated in red. Residues mutated according to the clustered charge to alanine algorithm are shaded in gray, and gray arrowheads denote sequence changes. Open boxes/open arrowheads depict (partially) conserved Sus1 residues/sequence changes with a predicted localization within protein loops. N- and C-terminal truncations are indicated. Positions of sus1–10, sus1–11 and sus1–12 mutations are also indicated in a. c, affinity purification of genomically expressed Sus1-TAP from wild-type (WT), sus1–10, sus1–11, and sus1–12 strains. Eluates were analyzed by SDS-PAGE (4–12% gradient gels, MOPS buffer) and Coomassie staining. Indicated bands were assigned according to their molecular mass (15). Filled circles designate Ubp8 and open circles designate Thp1. Asterisk indicates a contaminant, eEF-1α, which runs slightly below Thp1. Ubp8, Thp1, and eEF-1α were determined by mass spectrometry. Eluates (TAP) were analyzed by Western blotting using anti-Sac3 and anti-Cdc31 antibodies. Sus1-TAP expression levels (WCE) were determined by anti-ProtA detection (ProtA is part of the TAP tag) and normalization with Arc1 (a yeast cytosolic marker protein) levels. d, Thp1-TAP affinity purification from the indicated sus1 mutant allele backgrounds. Indicated Coomassie-stained bands were determined by mass spectrometry. Asterisks label contaminants of the purification (from top to bottom: keratin, Eno2, and Tdh3). FLAG-tagged Sus1 and Cdc31 were immunodetected by anti-FLAG and anti-Cdc31 antibodies, respectively.

Sus1 mutant proteins bind to Sac3 and Sgf11 with different affinities in vitro. Recombinant GST-Sac3-(573–805)-Cdc31 complex or GST-Sgf11 was immobilized on GSH beads and incubated with the indicated recombinant 6xHis-Sus1 wild-type (WT) or mutant proteins (input). Proteins bound to the beads were eluted with GSH and analyzed by SDS-PAGE (12% gel, MES buffer) and Coomassie staining. Asterisks indicate Sac3 and Sgf11 degradation products, as determined by mass spectrometry. Note that GST-Sac3-(573–805) harbors the entire CID, which binds to both Cdc31 and Sus1.

Uncoupling of Sus1 from Sac3 causes TREX-2 mislocalization and mRNA export defects in vivo. a, growth analysis of SUS1-TAP wild-type (WT) and mutant cells on rich medium (YPD) at 30 and 37 °C. Cell density was normalized, and 10-fold serial dilutions were prepared and then plated. Plates were incubated for 2 days. b, subcellular localizations of Sac3-GFP in the indicated wild-type and mutant sus1 strains are shown. Fluorescence microscopic and Nomarski photographs of representative cells grown at 30 °C are shown (scale bar, 2 μm). c, analysis of nuclear mRNA export in the indicated wild-type and sus1 mutant strains. Exponentially growing cells were shifted to 37 °C and grown for 2 more hours before detection of poly(A)⁺ RNA by fluorescent in situ hybridization with Cy3-labeled oligo(dT) probes. DNA was stained with 4,6-diamidino-2-phenylindole. Numbers indicate the percentage of cells (∼200 cells counted in each case) that exhibit an apparent mRNA export defect. Scale bar, 2 μm. d, analysis of global histone H2B ubiquitin levels. Anti-FLAG-H2B immunoprecipitates derived from SUS1-TAP cells, sus1Δ cells, and the indicated mutants. Recovered proteins were analyzed by SDS-PAGE and Western blotting using an anti-FLAG antibody to detect unmodified FLAG-H2B and ubiquitinated FLAG-H2B.

sus1 mutant alleles differentially perturb the genetic interactions of SUS1. The yra1Δsus1Δ double mutant strain expressing both the wild-type YRA1 allele (URA3 plasmid) and yra1ΔRBD allele (TRP1 plasmid) were transformed with the wild-type SUS1 and the indicated sus1 mutant alleles on LEU2 plasmids. Growth was followed on SDC-Trp-Leu plates and on 5-FOA plates after shuffling out the URA3 cover plasmid. The ada3Δsus1Δ strain was transformed both with a URA3 plasmid containing wild-type SUS1 and the indicated sus1 mutant alleles on LEU2 plasmids. Growth was followed on SDC-Leu plates and on 5-FOA plates after shuffling out the URA3 cover plasmid. Cell density was normalized, and 10-fold serial dilutions were prepared and then plated. Plates were incubated for 2–3 days at 30 °C.