Strains and plasmids
Deletion of MLP1 and MLP2 and GFP tagging were performed by homologous recombination in W303
haploid cells 45 using PCR products. The HIS5 gene from S. pombe was amplified by PCR using
primers with 3’ sequences specific for HIS5 sequence and 5’ tails homologous to upstream
/downstream sequences of the MLP1 and MLP2 open reading frames. HIS5 PCR amplifications were
made using primers #91 5‘-TAA CAT TAT ATC AGG GTG AAT ATT ACT GAC AAA AAT AAT AAC TTA
AGT CTT CTT TAT AAT ATG ATG ACC CAT ACG ATG TTC CTG ACT ATG and #92 5’-TAG GGC
AGA ATG AAG CTC CTC CAC ATT GAA AAA GGT TTA GTT TGT ATT GAT CCC TTG TTT TTA CTA
GTC GAC GGT ATC GAT AAG CTT to delete MLP1 and #89 5’-AGT GGA AGT TTA CCA AAA GAA
ATT TAA GGC GAA AGA ACA CTG GGC GGA AGC AAA CCG GCA ACC CAT ACG ATG TTC CTG
ACT ATG and #90 5’-GAC ATT AGT GAC ATT TAA AAT ATG TAG ATG TTT CAT ATT TAT ATA ATT
ACA TTG TTT AAT ATT ACA GTC GAC GGT ATC GAT AAG CTT to delete MLP2 (strain YVG3). The
haploid double mutant (strain YVG12) was generated by crossing of single mutants, and subsequent
sporulation and dissection. MLP2 deletion with TRP1 (K. l) was performed using #89 and #90 primers
to amplify from a plasmid containing TRP1 instead of HIS5. All deletions were confirmed by PCR.
The C-terminal GFP tagging of Mlp1p (strain YVG31) and Mlp2p (strain YVG36) were also performed
by homologous recombination with PCR products containing the GFP gene and the HIS5 marker.
Primers #29 5’-AAT GAG TCA AAA AAG ATC AAG ACT GAA GAT GAG GAA GAA AAA GAA ACC
GAT AAG GTG AAT GAC GAG AAC AGT ATA ATT GAA GGT AGA GGT GAA GCT CAA AAA CTT
ATT and #92 5’-TAG GGC AGA ATG AAG CTC CTC CAC ATT GAA AAA GGT TTA GTT TGT ATT
GAT CCC TTG TTT TTA CTA GTC GAC GGT ATC GAT AAG CTT were used for Mlp1p tagging.
Primers #28 5’-GAG AGC GGT ACA TCT TCT GAT CCA GAC ACC AAA AAG GTT AAA GAG AGT
CCA GCA AAT GAT CAA GCT TCC AAC GAG ATT GAA GGT AGA GGT GAA GCT CAA AAA CTT
ATT and #90 5’-GAC ATT AGT GAC ATT TAA AAT ATG TAG ATG TTT CAT ATT TAT ATA ATT ACA
TTG TTT AAT ATT ACA GTC GAC GGT ATC GAT AAG CTT were used for Mlp2p tagging. The
expression of the GFP fusion proteins was confirmed by western-blot analysis with a polyclonal
antibody against GFP (Interchim, ref: A6455). The C-terminal tagging of Mlp2p with IgG binding
repeats of Staphylococcus aureus Protein A (strain YVG189) was performed with PCR products
containing the pA gene and the HIS5 marker using the primers #28 and #90. The expression of the
protein-A fusion protein was confirmed by western-blot analysis using a polyclonal antibody against pA
(Sigma, ref: P3775).
The Nup145606-1341 mutant in MLP1-GFP (Strain YVG105) and in MLP2-GFP (strain YVG106)
was generated by in-frame insertion of a myc epitope followed by a STOP through homologous
recombination in the genomic NUP145 copy at a position corresponding to amino acid 606 using the
TRP1 marker of K. lactis. The primers #105 5’-AGG GAA ATG AAC ATA ATA TCC TAT AAT CCC TTT
GGC GGG ACT TGG ACT TTC AAA GTC AAT CAT TTT TCC GGT TCT GCT GCT AGT and #106 5’GTC AAA TTC TTT AGA AGT GGC AAA CAC AGA ACG TAG TGA AGA CCC AGC TAA AAT TAA TTG
ACC TCG AGG CCA GAA GAC were used to amplify a myc-TRP1 fragment from pUC19-myc9TRP1(K.l) plasmid 46.
The C-terminal myc-tagging of Hdf1p and Rad50p were generated by in-frame insertion of a mycepitope, through homologous recombination, in the genomic copy just before the stop codon using the
TRP1 marker of K. lactis. Primers #40 5’-ATA AAA GAA GAA AAG AAG CCC TTT GAT AAA AAG
CCG AAA TTC AAT ATA ATT GAA GGT AGA TCC GGT TCT GCT GCT AGT and #41 5’-GAT TTA
GAG TTA CAA CTA TAG ATT ACT GTC GTG CAT AAA ACC TCG AGG CCA GAA GA were used to
amplify a myc-TRP1 fragment from pUC19-myc9-TRP1(K.l) plasmid and tagged Hdf1p. Primers #54
5’-GAG AGA TGA TAG GCA AAA ATC ACA AAT TGA GTG GGT CGA TAT TAA CAG AGT CAC TTA
TTC CGG TCC TGC TAG T and #55 5’-TAA GCA ACA GAA GCG TTA TCA CAA ACT CGT CGA CGA
AGT ATT TCA GCA CCT CTA ATT ACC TCG AGG CCA GAA GAC TAA were also used to amplify this
fragment for tagging of Rad50p. The expression of the myc-fusion proteins was confirmed by westernblot analysis using a monoclonal antibody against myc (9E10).
HDF1 null strains were generated through homologous recombination of the TRP1 gene removing the
entire gene of HDF1. Primers #103 5’- ACT GTT CTA GTT TTC AAC AGT AAA GCT ATG ATT TGT
TAA GTG ACT CTA AGC CTG ATT TTA AAA CGG GAA TAT TCC GGT TCT GCT GCT AGT and
#104 5’- ATC TTG CTA ATA GTT GTA CAG TAC AAC GTT TAG CAC GAC AAA AGT TCT TAA TAA
TAA ATA ACC TCG AGG CCA GAA GAC were used to amplify the myc-TRP1 fragment from pUC19myc9-TRP1(K.l) plasmid and deleted HDF1. The deletions were confirmed by PCR.
The MLP1 gene was subcloned from cosmid pUKG096 47 generously provided by Bernard Dujon
(Pasteur Institute, Paris, France). A 7.3Kb NdeI-NcoI fragment containing the MLP1 ORF was cloned
instead of URA3 into a pRS316 vector. It was then excized by the same set of restriction enzymes,
blunted and recloned into the unique SmaI site of pRS315 and pHT4467. The plasmid containing a
GFP tagged version of MLP1 was constructed on the basis of the plasmid described above by
replacing the 3’ end of the gene from the XhoI site by the same fragment of the gene in fusion with
GFP after amplification by PCR from the tagged genomic copy of MLP1 (details upon request).
The N-terminal GFP tagging of Rap1p (strain YVG165) was performed by homologous recombination
with PCR products containing the RAP1 promotor, theGFP gene and the TRP1 marker. The resulting
fusion protein deleted for its 234 first amino acids was found to be fonctional and correctly localized.
Yeast strains
Strain name
Genotype
Derivation
W303
MATa, leu2-3,112, his3-11,15, trp1-1, can1-100,
ade2-1, ura3-1
W303, MATa, trp1
BMA64, MATa, mlp1::HIS5
BMA64, MATa, mlp2::HIS5
BMA64, MATa, mlp1::HIS5, mlp2::HIS5
BMA64, MAT, ade3::TRP1, mlp1::HIS5
BMA64, MATa, MLP1-GFP::HIS5
BMA64, MATa, MLP2-GFP::HIS5
YVG31, NUP145∆606-1341-myc9::TRP1
YVG36, NUP145606-1341-myc9::TRP1
MAT, leu2-3,112, his3-11,15, trp1-1, can1-100,
ade2-1, ura3-1, adh4::URA3
YG59, mlp1::HIS5
YG59, mlp2::HIS5
YG59, mlp2::HIS5, mlp1::TRP1
YG59, yku70::TRP1
YG59, NUP145606-1341-myc9::TRP1
BMA64, MATa, Yku70-myc9::TRP1
YVG3, Yku70-myc9::TRP1
BMA64, MATa/, mlp1::HIS5/mlp1::HIS5,
mlp2::HIS5/mlp2::HIS5
BMA64, MATa, Yku70::TRP1
BMA64, MATa, NUP145606-1341-GFP::TRP1
BMA64, MATa, MLP2-pA::HIS5, Yku70myc9::TRP1
BMA64, MATa, Rad50-myc9::TRP1
BMA64, MATa, GFP-∆1-234RAP1::TRP1
YVG165, NUP145606-1341-pA::HIS5
Thomas and
Rothstein, 1989
BMA 64
YVG1
YVG3
YVG12
YVG19
YVG31
YVG36
YVG105
YVG106
YG59
YVG117
YVG120
YVG127
YVG185
YVG186
YVG155
YVG156
YVG160
YVG159
YVG179
YVG189
YVG161
YVG165
YVG170
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deletion were performed with HIS5 gene from S. pombe and TRP1 gene from K. lactis
complementing his3 and trp1 mutated strains.