TABLE 1:
Yeast strains used in this study.
| Strain | Genotype | Reference |
|---|---|---|
| BY4741 | MATa leu2∆ ura3∆ met15∆ his3∆ | Brachmann et al. (1998) |
| BY4742 | MATα leu2∆ ura3∆ met15∆ his3∆ | Brachmann et al. (1998) |
| GFY-42 | BY4741; cdc10∆::CDC10::mCherry::ADH1(t)::SpHIS5 | Finnigan et al. (2015b) |
| GFY-59a | BY4741; cdc11∆::CDC11::mCherry::ADH1(t)::SpHIS5 | This study |
| GFY-1794b | BY4742; cdc10∆::GFPβ10::Linker(32)::CDC10::ADH1(t)::HygR cdc11∆::CDC11::mCherry::SpHIS5 | This study |
| GFY-1570c | BY4741; cdc10∆::CDC10::Linker(33)::GFPβ11::ADH1(t)::NatR | This study |
| GFY-1979d | BY4741; PGK1::Linker(33)::GFPβ11::ADH1(t)::KanR | This study |
| GFY-1983e | BY4741; HSP82::Linker(33)::GFPβ11::ADH1(t)::KanR | This study |
| GFY-2035f | BY4741; CDC19::Linker(43)::GFPβ11::SHS1(t)::KanR | This study |
| GFY-2036f | BY4741; GPP1::Linker(43)::GFPβ11::SHS1(t)::KanR | This study |
| GFY-2043f | BY4741; TPI1::Linker(43)::GFPβ11::SHS1(t)::KanR | This study |
| GFY-1793g | BY4742; cdc3∆::GFPβ10::Linker(18)::CDC3::ADH1(t)::HygR cdc10∆::CDC10::mCherry::SpHIS5 | This study |
| GFY-1851h | BY4741; cdc10∆::CDC10::GFPβ11::ADH1(t)::HygR | This study |
| GFY-1801i | BY4742; cdc10∆::GFPβ10::Linker(5)::CDC10::ADH1(t)::HygR cdc11∆::CDC11::mCherry::SpHIS5 | This study |
| GFY-1798 | BY4742; cdc3∆::GFPβ10::Linker(5)::CDC3::ADH1(t)::HygR cdc10∆::CDC10::mCherry::SpHIS5 | This study |
| GFY-1803 | BY4742; cdc12∆::GFPβ10::Linker(5)::CDC12::ADH1(t)::HygR cdc10∆::CDC10::mCherry::SpHIS5 | This study |
| GFY-1804 | BY4742; cdc11∆::GFPβ10::Linker(5)::CDC11::ADH1(t)::HygR cdc10∆::CDC10::mCherry::SpHIS5 | This study |
| GFY-1807 | BY4742; shs1∆::GFPβ10::Linker(5)::SHS1::ADH1(t)::HygR cdc10∆::CDC10::mCherry::SpHIS5 | This study |
| GFY-1796 | BY4742; cdc10∆::GFPβ10::Linker(18)::CDC10::ADH1(t)::HygR cdc11∆::CDC11::mCherry::SpHIS5 | This study |
| GFY-1793 | BY4742; cdc3∆::GFPβ10::Linker(18)::CDC3::ADH1(t)::HygR cdc10∆::CDC10::mCherry::SpHIS5 | This study |
| GFY-1797 | BY4742; cdc12∆::GFPβ10::Linker(18)::CDC12::ADH1(t)::HygR cdc10∆::CDC10::mCherry::SpHIS5 | This study |
| GFY-1795j | BY4742; cdc11∆::GFPβ10::Linker(18)::CDC11::ADH1(t)::HygR cdc10∆::CDC10::mCherry::SpHIS5 | This study |
| GFY-1806 | BY4742; shs1∆::GFPβ10::Linker(18)::SHS1::ADH1(t)::HygR cdc10∆::CDC10::mCherry::SpHIS5 | This study |
| GFY-1852k | BY4741; cdc10∆::CDC10::Linker(10)::GFPβ11::ADH1(t)::HygR | This study |
| GFY-1853l | BY4741; cdc10∆::CDC10::Linker(20)::GFPβ11::ADH1(t)::HygR | This study |
| GFY-1845 | BY4741; cdc3∆::CDC3::GFPβ11::ADH1(t)::HygR | This study |
| GFY-1848 | BY4741; cdc12∆::CDC12::GFPβ11::ADH1(t)::HygR | This study |
| GFY-1842 | BY4741; cdc11∆::CDC11::GFPβ11::ADH1(t)::HygR | This study |
| GFY-1839 | BY4741; shs1∆::SHS1::GFPβ11::ADH1(t)::HygR | This study |
| GFY-1809 | BY4742; bni5∆::GFPβ10::Linker(18)::BNI5::ADH1(t)::HygR cdc10∆::CDC10::mCherry::SpHIS5 | This study |
| GFY-1572 | BY4741; cdc3∆::CDC3::Linker(33)::GFPβ11::ADH1(t)::NatR | This study |
| GFY-1571 | BY4741; cdc12∆::CDC12::Linker(33)::GFPβ11::ADH1(t)::NatR | This study |
| GFY-1573 | BY4741; cdc11∆::CDC11::Linker(33)::GFPβ11::ADH1(t)::NatR | This study |
| GFY-1567 | BY4741; shs1∆::SHS1::Linker(33)::GFPβ11::ADH1(t)::NatR | This study |
| GFY-1735 | BY4741; bni5∆::BNI5::Linker(33)::GFPβ11::ADH1(t)::NatR | This study |
| GFY-1899 | BY4742; nis1∆::GFPβ10::Linker(18)::NIS1::ADH1(t)::HygR cdc10∆::CDC10::mCherry::SpHIS5 | This study |
| GFY-1854 | BY4741; nis1∆::NIS1::Linker(33)::GFPβ11::ADH1(t)::HygR | This study |
| GFY-1992m | BY4742; hsl1∆::GFPβ10::Linker(32)::HSL1(1-1518)::ADH1(t)::KanR cdc10∆::CDC10::mCherry::SpHIS5 | This study |
| GFY-1995n | BY4742; hsl1∆::GFPβ10::Linker(32)::HSL1(611-950 R635A R636A K645A H648A K649A R653A K654A K775A E776A N777A R828A L831A)::ADH1(t)::KanR cdc10∆::CDC10::mCherry::SpHIS5 | This study |
| GFY-1997o | BY4742; hsl1∆::GFPβ10::Linker(32)::HSL1(611-950; 1245-1518 R635A R636A K645A H648A K649A R653A K654A K775A E776A N777A R828A L831A)::ADH1(t)::KanR cdc10∆::CDC10::mCherry::SpHIS5 | This study |
| GFY-1846 | BY4741; cdc3∆::CDC3::Linker(10)::GFPβ11::ADH1(t)::HygR | This study |
| GFY-1849 | BY4741; cdc12∆::CDC12::Linker(10)::GFPβ11::ADH1(t)::HygR | This study |
| GFY-1843 | BY4741; cdc11∆::CDC11::Linker(10)::GFPβ11::ADH1(t)::HygR | This study |
| GFY-1840 | BY4741; shs1∆::SHS1::Linker(10)::GFPβ11::ADH1(t)::HygR | This study |
| GFY-1847 | BY4741; cdc3∆::CDC3::Linker(20)::GFPβ11::ADH1(t)::HygR | This study |
| GFY-2044 | BY4741; cdc12∆::CDC12::Linker(20)::GFPβ11::ADH1(t)::HygR | This study |
| GFY-1844 | BY4741; cdc11∆::CDC11::Linker(20)::GFPβ11::ADH1(t)::HygR | This study |
| GFY-1841 | BY4741; shs1∆::SHS1::Linker(20)::GFPβ11::ADH1(t)::HygR | This study |
| GFY-1996 | BY4742; hsl1∆::GFPβ10::Linker(18)::HSL1(611-950; 1245-1518 R635A R636A K645A H648A K649A R653A K654A K775A E776A N777A R828A L831A)::ADH1(t)::KanR cdc10∆::CDC10::mCherry::SpHIS5 | This study |
| GFY-1998 | BY4742; hsl1∆::GFPβ10::Linker(5)::HSL1(611-950; 1245-1518 R635A R636A K645A H648A K649A R653A K654A K775A E776A N777A R828A L831A)::ADH1(t)::KanR cdc10∆::CDC10::mCherry::SpHIS5 | This study |
| GFY-1977p | BY4741; PGK1::eGFP::ADH1(t)::KanR | This study |
| GFY-1981p | BY4741; HSP82::eGFP::ADH1(t)::KanR | This study |
| GFY-2031q | BY4741; TPI1::eGFP::ADH1(t)::HygR | This study |
| GFY-2033q | BY4741; GPP1::eGFP::ADH1(t)::HygR | This study |
| GFY-2034q | BY4741; CDC19::eGFP::ADH1(t)::HygR | This study |
| GFY-1318 | BY4741; CDC10::mCherry::KanR bni5∆::GFP::BNI5::SpHIS5 cdc11∆::CDC11::HygR SHS1 + pJT1520 | Finnigan et al. (2015a) |
aStrain was constructed by integrating CDC11::mCherry::ADH1(t)::SpHIS5 amplified from pGF-IVL1 into cdc11∆::KanR yeast (GFY-150). The strain was selected twice on 5-FOA–containing medium to counterselect for the WT CDC11-expressing covering vector. Unless otherwise noted, all strains were selected on 5-FOA to remove these covering vector(s) before diploid formation.
bStrain was constructed by integrating the tagged CDC10 allele (from pGF-IVL824) into cdc10∆ yeast (GFY-1603). The GFPβ10 sequence is MDLPDDHYLSTQTILSKDLN (Cabantous et al., 2013). The 32-residue linker sequence is DVGGGGSEGGGSGGPGSGGEGSAGGGSAGGGS. CDC11 was tagged with mCherry by amplifying the entire locus from chromosomal DNA from GFY-59 and transforming into GFY-1643. N-terminally tagged proteins were constructed using this strategy unless otherwise noted. All of the flexible linker sequences (N- or C-terminal) were modeled as previously described (Cabantous et al., 2013).
cStrain was constructed by integrating the tagged CDC10 allele (from pGF-IVL810) into cdc10∆ yeast (GFY-140). The GFPβ11 sequence is EKRDHMVLLEYVTAAGITDAS (Cabantous et al., 2013). The 33-residue linker is DYKDDDDKGSGAGGSPGGGSGGSGSSASGGSTS. C-terminally tagged strains were constructed using this strategy unless otherwise noted.
dStrain was constructed by creating an integrating vector containing the entire PGK1 ORF fused to the C-terminal tag including the drug cassette and, finally, 491 base pairs of 3′ UTR (pGF-IVL1054). The entire cassette was amplified and transformed into BY4741 yeast.
eStrain was constructed by first creating an integrating vector containing 471 base pairs of the HSP82 ORF fused to the C-terminal tag including the drug cassette, and finally, 500 base pairs of 3′ UTR (pGF-IVL1056). The entire cassette was amplified and transformed into BY4741 yeast.
fThese strains were constructed by amplifying the C-terminal cassette (from pGF-V763) including a slightly larger linker sequence (including an upstream GRRIPGLINP) with short 30–base pair oligonucleotide tails to each locus of interest. We used 469 base pairs of SHS1 3′ UTR sequence as the terminator, and the promoter of CCW12 (992 base pairs) replaced the Ptef sequence of the MX cassette. Strains were confirmed via diagnostic PCR and DNA sequencing of the full junction of the gene with the C-terminal tag.
gThe 18-residue linker sequence is DVGGGGSEGGGSGGPGSG.
hThere is no linker between the C-terminus of CDC10 and the GFPβ11 sequence.
iThe 5-residue linker has the sequence DVGGG.
jStrains with an N-terminal GFPβ10 tag with a 32-residue linker appended to CDC11 were lethal.
kThe 10-residue linker has the sequence GSSASGGSTS.
lThe 20-residue linker has the sequence GSPGGGSGGSGSSASGGSTS.
mThe tagged HSL1 construct was integrated into hsl1∆::HygR yeast (GFY-1902) by amplifying two PCR fragments that contained overlapping sequence within the HSL1 ORF from plasmid pGF-IVL1034.
nThis Hsl1 construct has a putative NLS sequence mutated to alanine to prevent nuclear import, as well as the KEN box (residues 775–781) and D-box (residues 828–836) destruction motifs to prevent APC-dependent degradation.
oThis Hsl1 construct contains both the septin-binding domain (residues 611–950) and sequence containing the C-terminal KA1 domain (residues 1245–1518), both of which are required for optimal localization of Hsl1 to the septin collar in vivo (Finnigan et al., 2016).
pStrains were constructed similar to GFY-1979 and GFY-1983 using full-length eGFP and confirmed via DNA sequencing.
qStrains were constructed similar to GFY-2035 using eGFP::ADH1(t)::HygR as template DNA. Proper integration was confirmed via DNA sequencing.