TABLE 2:
Plasmids used in this study.
| Plasmid | Description | Reference |
|---|---|---|
| pRS315 | CEN; LEU2 AMP | Sikorski and Hieter (1989) |
| pRS316 | CEN; URA3 AMP | Sikorski and Hieter (1989) |
| pGF-IVL794a | pRS315; prGAL1/10::GFPβ1-9(A)::ADH1(t)::KanR | This study |
| pGF-IVL795b | pRS315; prGAL1/10::GFPβ1-9(B)::ADH1(t)::KanR | This study |
| pGF-IVL553c | pRS315; prNIS1::NIS1::ADH1(t)::KanR | This study |
| pGF-IVL521 | pRS315; prCDC11::HSL1(1-1518)::eGFP::ADH1(t)::KanR | Finnigan et al. (2016) |
| pGF-IVL762d | pRS315; prCDC11::hsl1(611-950 R635A R636A K645A H648A K649A R653A K654A)::eGFP::ADH(1)::KanR | Finnigan et al. (2016) |
| pGF-IVL536e | pRS315; prCDC11::hsl1(611-950; 1245-1518)::eGFP::ADH1(t)::KanR | Finnigan et al. (2016) |
| pGF-IVL1095f | pRS315; prBUD4::BUD4(623-774)::eGFP::ADH1(t)::KanR | This study |
| pGF-IVL1110g | pRS315; prHOF1:: eGFP::HOF1(293-355)::ADH1(t)::KanR | This study |
| pGF-IVL1082 | pRS315; prBUD4::BUD4(623-774)::Linker(33)::GFPβ11::ADH1(t)::KanR | This study |
| pGF-IVL1084 | pRS315; prHOF1::HOF1(293-355)::Linker(33)::GFPβ11::ADH1(t)::KanR | This study |
| pGF-IVL1105 | pRS316; prGAL1/10::GFPβ1-9(A)::ADH1(t)::KanR | This study |
aThe evolved GFPβ1-9 was synthesized as a custom gene using a yeast codon bias. The A version designates the position of the last residue: this version ends with the sequence GPVLLPDNGS (Cabantous et al., 2013).
bA second variant of the GFPβ1-9 was created identical to the A version but ending with the sequence GPVLLP. The prGAL1/10 promoter includes 814 base pairs of 5′ UTR sequence.
cThe endogenous NIS1 promoter includes 576 base pairs of 5′ UTR sequence.
dThe putative NLS signal within the N-terminus of Hsl1(611-950) was experimentally determined to require residues R635 R636 K645 H648 K649 R653 K654 for constructs beginning at residue 611 (Finnigan et al., 2016). Mutation of these residues to alanine eliminates nuclear localization of this Hsl1 fragment.
eThe combination of the central Hsl1(611-950) septin-binding domain and the C-terminal KA1 domain contained within Hsl1(1245-1518) has been shown to be sufficient to efficiently target Hsl1 to the bud neck in vivo (Finnigan et al., 2016).
fThe Bud4(623-774) fragment has been shown to be sufficient to target to the septin collar in vivo (Wu et al., 2015). The BUD4 sequence was amplified from pGF-V416 and assembled by in vivo ligation and homologous recombination. Both N- and C-terminal eGFP fusions to this Bud4 fragment displayed localization to the septin collar.
gThe Hof1(293-355) fragment has been shown to be sufficient to target to the septin collar in vivo (Meitinger et al., 2013). The HOF1 sequence was amplified from pGF-V454 and assembled via in vivo ligation. Only the N-terminal eGFP fusion to this Hof1 fragment displayed localization to the septin collar (unpublished data).