Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - mlp1 mlp2 double mutant exhibits a strong genetic interaction with RAD52 and RAD27 deletions.
  A mlp1::HIS3 mlp2::URA3 double mutant, carrying the LEU2 marker under the control of the P2 haploid specific promoter, was mated to the indicated rad::KanMX mutant. After sporulation, random spore analysis was performed by spotting serial dilutions of the cells on the indicated media. The growth of the triple mutant is specifically impaired (bottom panel).
• Supplemental Figure 2 - The MG132 proteasome inhibitor partially restores Ulp1 levels at the nuclear envelope in nup133• and nup60• mutants
  wt, nup133• or nup60• cells expressing the ULP1-GFP fusion were deleted of ERG6 to increase their permeability to MG132. Treatment with the drug (50 µM) was performed for 4 hours at 30°C ("+MG132"). Control cells ("ctrl") were treated with equal amounts of DMSO. GFP and DIC images are shown.
• Supplemental Figure 3 - Nucleocytoplasmic transport in nup• mutants
  A, Steady-state localization of different NLS-GFP reporter constructs expressed in wt, nup133•, nup120• or nup60• cells was observed by fluorescence microscopy. DIC images are also shown. cNLS, classical NLS (Kap95/Kap60-dependent import); pNLS, Pho4 NLS (Kap121-dependent import); rgNLS, Nab2 NLS (Kap104-dependent import). See Supplemental Table 2 for a detailed description of the corresponding plasmids carrying the fusion proteins.
  B, Kinetics of nuclear import in the different nucleoporin mutants. Nuclear import was induced and monitored, as described in Material and Methods. A representative time-course acquisition is shown for a wt strain expressing the cNLS-GFP reporter (time in seconds). Nuclear import rates (arbitrary units) were calculated for the cNLS and pNLS reporter constructs expressed in wt, nup133•, nup120• and nup60• strains. The diagram shows the box-plot compilation of the results obtained for at least 10 independent cells. Nuclear import kinetics are not affected by the nucleoporin deletions except for the import of the pNLS reporter in the nup133• strain.
  C, Fluorescence microscopy analysis of wild type cells expressing either the ULP1-GFP fusion protein at its cognate locus (pULP1-ULP1-GFP), or the GFP-ULP1 construct under the control of the NOP1 promoter (pNOP1-GFP-ULP1). yrb4•N induction was achieved by growing the cells for 4 h in the presence of galactose (yrb4•N GAL). The kap121-34 mutant was observed at 30°C.
• Supplemental Figure 4 - Phenotypic analysis of ULP1-complemented cells.
  A, The wt, nup133• rad27•, and nup60• rad27• strains were transformed with pRS315 (Ø) or pRS315-NOP1prom-GFP-ULP1 (ULP1). Transformants were spotted as 5-fold dilutions on synthetic medium lacking leucine and plates were incubated at 30, 33 or 36°C. For each double mutant, the growth condition at which complementation with Ulp1 is best detected is boxed.
  B, Ulp1 levels were checked by Western-blot analysis using an anti-GFP antibody for strains expressing the indicated constructs. When indicated (+), cells were treated with MG132 (50µM for 4 hs) before protein extraction. The position of full-length GFP-Ulp1 and GFP-•N338-Ulp1 are indicated. An anti-NOP1 antibody was used as a loading control.
  C, wt, nup133• or nup60• cells expressing the RAD52-YFP fusion protein were transformed with either an empty plasmid (pRS315, "Ø") or the ULP1-overexpressing construct (pRS315-NOP1prom-GFP-ULP1, "ULP1") and were observed by epifluorescence microscopy. Rad52-YFP foci occurrence was quantified for each phase of the cell cycle. The error bar represents the standard deviation obtained from three independent experiments, where at least 200 cells were counted for each strain.
  D, Effect of the overexpression of Ulp1 on NHEJ activity. NHEJ activity was monitored as in figure 5C, except that a different synthetic medium required for plasmid maintenance was used. This could account for the less pronounced defects observed in the nup133• and nup60• mutants as compared to figure 5C.
• Supplemental Figure 5 - Specificity of the sumoylated forms detected upon anti-myc immunoprecipitations.
  Extracts from either a wt strain (bearing no myc-tagged gene, "untagged") or a strain expressing a 13myc-tagged protein susceptible to undergo sumoylation ("ORF-MYC", our unpublished work) were processed for immunoprecipitation under denaturing conditions using an anti-Myc antibody. Immune pellets were then analyzed by Western blot using an anti-SUMO antibody. Note the different SUMO pattern obtained with the ORF-MYC strain as compared to the YKU70-MYC strain (see figure 6A), and the absence of signal within the range of Yku70-myc molecular weight in both ORF-MYC and untagged strains (the theoretical position of the Yku70-myc fusion is indicated on the left).
• Supplemental Tables