Yeast strains, plasmids, and culture

The S. cerevisiae strains and plasmids used in this study are listed in Tables S1 and S2. Yeast cultures were grown at 30°C either in YPD media containing 2% glucose or in synthetic media (SD) with the appropriate supplements. Strains containing the ulp1ts allele were grown at a permissive temperature (25°C) and subsequently shifted to 37°C for 3 h before harvesting for biochemical analysis. For cell growth and drug sensitivity analysis, fivefold serial dilutions of the different strains were spotted on YPD plates without or with HU or MMS at the indicated concentrations and growth at the indicated temperatures.

Chromosomally tagged strains and mutants were constructed using a PCR-based strategy (Longtine et al., 1998). For the systematic KR mutagenesis approach, a Nup60-HA complete KR mutant was chemically synthetized (Genevust) and cloned in SpeI–XhoI sites of the p415 plasmid to generate the p415-nup60-HA-Krall plasmid. A set of yeast plasmids expressing Nup60-HA with different regions of the protein carrying KR mutation was generated using the p415-nup60-HA-KRall as an initial template. The p415-Nup60-HA WT plasmid was obtained by PCR amplification of the genomic DNA fragment encoding Nup60-HA and cloning in SpeI–XhoI sites into p415. Plasmids encoding different Nup60-KR-HA point mutants were constructed from the p415-Nup60-HA using a site-directed mutagenesis kit (QuikChange; Agilent Technologies).

To genomically integrate the Nup60 KR mutations, a PCR fragment was generated containing full-length Nup60-HA (WT, UbKR, or SUMO-KR) followed by the CYC1 terminator, the LEU2 cassette, and 50 nt complementary to the NUP60 locus just downstream of the stop codon. PCR fragments were obtained using plasmid p415-Nup60-HA, p415-Nup60-UbKR-HA, or p415-Nup60-SUMO-KR-HA as a DNA template and the oligonucleotides Int-N60-LEU-F, 5′-ATCAAATAAGCACCGCAAGATATCCTAAAATCGACATCCAATGCATCGTAAATCATTG-3′, and Int-N60-LEU210-R, 5′-GTATTGAGTTGGGCTATACGGTAATTATGTCACGGCTAAAATTTTCATTATTCCTTATCACGTTGAGC-3′. Yeast cells were transformed and selected on restrictive medium plates (DO-LEU). Finally, clones were analyzed by DNA sequencing of full-length NUP60 gene.