Table 1 Yeast strains used in this study
GenotypeDescriptionReference
GFY-138aBY4741; cdc12∆::cdc12(K392N; ∆393-407)::HygR + pJT1622This study
GFY-104bBY4741; cdc12∆::cdc12(K392N; ∆393-407)::mCherry::KanR + pJT1622This study
GFY-58BY4741; cdc11∆::CDC11::mCherry::SpHIS5 + pJT1520Finnigan et al. (2015)
GFY-122BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 + pJT1520Finnigan et al. (2015)
GFY-163BY4741; cdc11∆::KanR shs1∆::HygR + pJT1520Finnigan et al. (2015)
GFY-121BY4741; cdc11∆::cdc11(G29D)::mCherry::KanR + pJT1520Finnigan et al. (2015)
GFY-164BY4741; cdc11∆::CDC11::mCherry::SpHIS5R shs1∆::HygR + pJT1520Finnigan et al. (2015)
GFY-989BY4741; cdc11∆::CDC11::mCherry::SpHIS5 shs1∆::shs1(∆349-551)::eGFP::NatR + pJT1520This study
GFY-87BY4741; cdc10∆::KanR shs1∆::SHS1::eGFP::NatR + pJT2022Finnigan et al. (2015)
GFY-137BY4741; cdc10∆::KanR shs1∆::HygR + pJT2022Finnigan et al. (2015)
GFY-94BY4741; cdc10∆::KanR shs1∆::shs1(∆349-551)::eGFP::NatR + pJT2022Finnigan et al. (2015)
GFY-847cBY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 bni5∆::KanR + pJT1520This study
GFY-1004aBY4741; cdc12∆::cdc12(K392N; ∆393-407)::HygR bni5∆::KanR + pJT1622This study
GFY-140BY4741; cdc10∆::HygR + pJT2022This study
GFY-1005BY4741; cdc10∆::HygR bni5∆::KanR + pJT2022This study
GFY-1108BY4741; cdc11∆::CDC11::mCherry::SpHIS5 shs1∆::shs1(∆349-551)::eGFP::NatR bni5∆::KanR + pJT1520This study
GFY-846BY4741; cdc11∆::CDC11::mCherry::SpHIS5R shs1∆::SHS1::eGFP::NatR bni5∆::KanR + pJT1520This study
GFY-850BY4741; cdc11∆::CDC11::mCherry::SpHIS5R shs1∆::HygR bni5∆::KanR + pJT1520This study
GFY-160BY4741; cdc11∆::CDC11::mCherry::SpHIS5R shs1∆::SHS1::eGFP::NatR + pJT1520Finnigan et al. (2015)
GFY-540BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 shs1∆::shs1(1-348)::cdc11(309-415)::eGFP::NatR + pJT1520Finnigan et al. (2015)
GFY-1100BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 shs1∆::shs1(1-348)::cdc11(309-415)d::eGFP::NatR bni5∆::KanR + pJT1520This study
GFY-542BY4741; cdc11∆::cdc11(1-308)::shs1(349-551)::mCherry::KanR shs1∆::shs1(∆349-551)::eGFP::NatR + pJT1520Finnigan et al. (2015)
GFY-1101BY4741; cdc11∆::cdc11(1-308)::shs1(349-551)::mCherry::KanR shs1∆::shs1(∆349-551)::eGFP::NatR bni5∆::KanR + pJT1520This study
GFY-166BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 shs1∆::HygR + pJT1520Finnigan et al. 2015)
GFY-1167dBY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 bni5∆::HA3::BNI5::NatR + pJT1520This study
GFY-1259BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 bni5∆::HA3::bni5(∆349-448)::NatR + pJT1520This study
GFY-1260BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 bni5∆::HA3::bni5(∆2-300)::NatR + pJT1520This study
GFY-1261BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 bni5∆::HA3::bni5(∆424-448)::NatR + pJT1520This study
GFY-1269BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 bni5∆::HA3::bni5(∆2-50)::NatR + pJT1520This study
GFY-1270BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 bni5∆::HA3::bni5(∆2-100)::NatR + pJT1520This study
GFY-1271BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 bni5∆::HA3::bni5(∆2-150)::NatR + pJT1520This study
GFY-1272BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 bni5∆::HA3::bni5(∆2-250)::NatR + pJT1520This study
GFY-1273BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 bni5∆::HA3::bni5(∆2-350)::NatR + pJT1520This study
GFY-1274BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 bni5∆::HA3::bni5(∆281-448)::NatR + pJT1520This study
GFY-1275eBY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 bni5∆::HA3::bni5(S270D T274D)::NatR + pJT1520This study
GFY-1276BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 bni5∆::HA3::bni5(S346A S350A)::NatR + pJT1520This study
GFY-1277BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 bni5∆::HA3::bni5(S346D S350D)::NatR + pJT1520This study
GFY-1278BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 bni5∆::HA3::bni5(S270A T274A S346A S350A)::NatR + pJT1520This study
GFY-1279BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 bni5∆::HA3::bni5(S270D T274D S346D S350D)::NatR + pJT1520This study
GFY-1286BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 bni5∆::HA3::bni5(∆2-200)::NatR + pJT1520This study
GFY-1287BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 bni5∆::HA3::bni5(∆399-448)::NatR + pJT1520This study
GFY-1288BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 bni5∆::HA3::bni5(S270A T274A)::NatR + pJT1520This study
GFY-162BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 shs1∆::shs1(∆349-551)::eGFP::NatR + pJT1520Finnigan et al. (2015)
GFY-573BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 shs1∆::shs1(∆349-551)::BNI5::eGFP::NatR + pJT1520This study
GFY-646BY4741; cdc11∆::cdc11(∆309-415)::BNI5::mCherry::SpHIS5 shs1∆::shs1(∆349-551)::eGFP::NatR + pJT1520This study
GFY-579BY4741; cdc11∆::cdc11(∆357-415)::BNI5::mCherry::SpHIS5 shs1∆::HygR + pJT1520This study
GFY-157BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 shs1∆::SHS1::eGFP::NatR + pJT1520This study
GFY-911BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 shs1∆::SHS1::BNI5::eGFP::NatR + pJT1520This study
GFY-913BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 shs1∆::shs1(∆349-551)::BNI5::eGFP::NatR + pJT1520This study
GFY-888BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 shs1∆::SHS1::eGFP::BNI5::NatR + pJT1520This study
GFY-890BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 shs1∆::shs1(∆349-551)::eGFP::BNI5::NatR + pJT1520This study
GFY-899BY4741; cdc10∆::KanR shs1∆::SHS1::BNI5::eGFP::NatR + pJT2022This study
GFY-901BY4741; cdc10∆::KanR shs1∆::shs1(∆349-551)::BNI5::eGFP::NatR + pJT2022This study
GFY-903BY4741; cdc10∆::KanR shs1∆::SHS1::eGFP::BNI5::NatR + pJT2022This study
GFY-905BY4741; cdc10∆::KanR shs1∆::shs1(∆349-551)::eGFP::BNI5::NatR + pJT2022This study
GFY-861BY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 shs1∆::shs1(∆349-551)::NatR + pJT1520This study
GFY-1295fBY4741; cdc11∆::cdc11(∆357-415)::mCherry::SpHIS5 shs1∆::shs1(∆349-551)::NanoBody::NatR + pJT1520This study
GFY-851BY4741; cdc11∆::cdc11(∆357-415)::HygR shs1∆::NatR + pJT1520This study
GFY-1291fBY4741; cdc11∆::cdc11(∆357-415)::NanoBody::SpHIS5 shs1∆::NatR + pJT1520This study
GFY-1316gBY4741; CDC10::mCherry::KanR bni5∆::GFP::BNI5::SpHIS5 cdc11∆::CDC11::HygR shs1∆::shs1(∆349-551)::NatR + pJT1520This study
GFY-1318gBY4741; CDC10::mCherry::KanR bni5∆::GFP::BNI5::SpHIS5 cdc11∆::CDC11::HygR SHS1 + pJT1520This study
GFY-1319gBY4741; CDC10::mCherry::KanR bni5∆::GFP::BNI5::SpHIS5 cdc11∆::CDC11::HygR shs1∆::NatR + pJT1520This study
GFY-1360gBY4741; CDC10::mCherry::SpHIS5 bni5∆::GFP::BNI5::NatR CDC11 shs1∆::shs1(1-348)::cdc11(309-415)::HygRThis study
GFY-1315gBY4741; CDC10::mCherry::KanR bni5∆::GFP::BNI5::SpHIS5 cdc11∆::cdc11(∆357-415)::HygR shs1∆::SHS1::NatR + pJT1520This study
GFY-1317gBY4741; CDC10::mCherry::KanR bni5∆::GFP::BNI5::SpHIS5 cdc11∆::cdc11(∆357-415)::HygR shs1∆::shs1(∆349-551)::NatR + pJT1520This study
GFY-1320gBY4741; CDC10::mCherry::KanR bni5∆::GFP::BNI5::SpHIS5 cdc11∆::cdc11(∆357-415)::HygR shs1∆::NatR + pJT1520This study
GFY-1452hcdc10∆::CDC10::mCherry::HygR CDC11 myo1∆::MYO1::GFP::SkHIS3 shs1∆::NatR BNI5This study
GFY-1453hcdc10∆::CDC10::mCherry::HygR CDC11 myo1∆::MYO1::GFP::SkHIS3 shs1∆::SHS1::NatR BNI5This study
GFY-1454hcdc10∆::CDC10::mCherry::HygR CDC11 myo1∆::MYO1::GFP::SkHIS3 shs1∆::shs1(∆349-551)::NatR BNI5This study
GFY-1456hcdc10∆::CDC10::mCherry::NatR CDC11 myo1∆::MYO1::GFP::SkHIS3 shs1∆::shs1(1-348)::cdc11(309-415)::HygR BNI5This study
GFY-1455hcdc10∆::CDC10::mCherry::KanR CDC11 myo1∆::MYO1::GFP::SkHIS3 shs1∆::shs1(1-348)::cdc11(309-415)::HygR bni5∆::NatRThis study
GFY-1457hcdc10∆::CDC10::mCherry::HygR CDC11 myo1∆::MYO1::GFP::SkHIS3 shs1∆::NatR bni5∆::KanRThis study
GFY-1458hcdc10∆::CDC10::mCherry::HygR CDC11 myo1∆::MYO1::GFP::SkHIS3 shs1∆::SHS1::NatR bni5∆::KanRThis study
GFY-1459hcdc10∆::CDC10::mCherry::HygR CDC11 myo1∆::MYO1::GFP::SkHIS3 shs1∆::shs1(∆349-551)::NatR bni5∆::KanRThis study
  • a The cdc12-6 allele, cdc12(K391N ∆392-407), arises from loss of a single adenine base at position 1173; in our construct, the ORF is immediately followed by the ADH1 transcriptional terminator sequence and a HygR drug-resistance marker.

  • b The mCherry tag is fused in-frame after the K391N substitution within cdc12-6.

  • c For all yeast deleted for BNI5, a bni5∆::KanR strain was first constructed by amplifying a knockout cassette PCR fragment using the KanR cassette with primer tails with homology to the immediate 5′ and 3′ UTR following the BNI5 ORF. Subsequent deletions of BNI5 used PCR fragments generated from this strain with ∼500 base pairs of flanking sequence.

  • d Construction of vectors containing the native BNI5 promoter, any epitope tag or fluorescent tag, and the full-length WT BNI5 gene resulted in extremely low plasmid yields from bacteria (TOP10 Escherichia coli; <5% yield compared to other vectors) using standard plasmid isolation kits. Therefore, to confirm that the desired Bni5-containing constructs had been properly generated, diagnostic PCR was first performed and then DNA sequence analysis was carried out on the PCR product (produced using a high-fidelity polymerase) containing the entire ORF including its junctions to both the promoter and terminator. The issue of low plasmid yield from bacteria did not occur for any of the truncated forms of BNI5 or for constructs expressing BNI5 from different promoters.

  • e A modified Quikchange PCR strategy (Zheng et al. 2004) was used on the gene within a TOPO-II vector prior to integration into the genome. Successive rounds of Quikchange were used to introduce multiple mutations.

  • f The 117-residue anti-GFP nanobody (sequence: MQVQLVESGGALVQPGGSLRLSCAASGFP-VNRYSMRWYRQAPGKEREWVAGMSSAGDRSSYEDSVKGRFTISRDDARNTVYLQMNSLKP-EDTAVYYCNVNVGFEYWGQGTQVTVSSK) is derived from a single-chain Camelidae heavy chain antibody selected for very high affinity binding to GFP (Rothbauer et al. 2006; Kubala et al. 2010) and is encoded by a synthetic sequence using yeast codon bias (GenScript, Piscataway, NJ).

  • g The CDC10::mCherry::KanR or CDC10::mCherry::SpHIS5 alleles were integrated over a WT CDC10 by amplifying 500 nucleotides of 5′ and 3′ UTR surrounding the CDC10 locus from genomic DNA from strain GFY-42 or GFY-1258.

  • h These strains do not contain a covering plasmid expressing any WT septin. To integrate MYO1::GFP at its endogenous locus, a PCR fragment containing 1183 base pairs of the C terminus of MYO1, the GFP tag, the ADH1 transcriptional terminator sequence, and SkHIS3 MX cassette, along with 1000 bases of 3′ UTR region were PCR amplified from genomic DNA (from JTY4510) and directly transformed into the appropriate strains. Integrations were confirmed using multiple diagnostic PCRs both upstream and downstream of the integrated fragment.