Strains used in this study are listed in Table S1 . Homothallic (h90) strains able to switch mating type or 1:1 mixtures of heterothallic h+ × h− cells (also called P × M cells) were used as indicated. Minimal sporulation liquid (MSL) media with or without nitrogen (MSL+N and MSL−N) liquid or agar were used to grow and mate the cells, respectively (Egel et al., 1994 (link)). All live-cell imaging was performed on cells placed on MSL−N with 2% electrophoresis-grade agarose pads, covered with a coverslip sealed with VALAP (1:1:1 Vaseline/lanolin/paraffin).
Genes were tagged at their endogenous genomic locus at their 3′ end, yielding C-terminally tagged proteins. This was achieved by PCR amplification of a fragment from a template plasmid with primers carrying 5′ extensions corresponding to the last 80 nucleotides of the ORF and the first 80 nucleotides of the 3′UTR, which was transformed and integrated in the genome by homologous recombination, as previously described (Bähler et al., 1998 (link)). For tagging of genes with sfGFP, a pFA6a-sfGFP-kanMX plasmid was used as a template. sfGFP was amplified from pMaM4 (a plasmid provided by M. Knop, University of Heidelberg, Heidelberg, Germany; containing yeast codon-optimized sfGFP), with primers osm2680 (5′-ccTTAATTAActccaagggtgaagagctatttac-3′; PacI site uppercase) and osm2681 (5′-aGGCGCGCCcttataaagctcgtccattccg-3′; AscI site uppercase), digested with AscI and Pac1 and ligated to similarly treated pSM674 (pFA6a-EGFP-kanMX6; described in Bähler et al., 1998 (link)). The sfGFP replaced EGFP, resulting in pFA6a-sfGFP-kanMX6 (pSM1538). We then used this vector as template for PCR-based targeted tagging of fus1, agn2, eng2, and exg3 (Bähler et al., 1998 (link)).
To yield Pmap3-driven fluorescent reporters, the map3 promoter region was amplified from genomic DNA with primers osm935 (5′-cccCTGCAGaagcatgcacgctgctcac-3′; PstI site uppercase) and osm936 (5′-agaGTCGACggtaaactcaacgtataag-3′; SalI site uppercase), digested with Pst1 and SalI, and ligated to similarly treated pSM242 (pRIP42:GFP; an integrative plasmid containing GFP under control of nmt41 promoter and a ura4+ selection marker), replacing the nmt41 promoter and yielding plasmid pSM793 (pRIP-Pmap3:GFP, ura4+). To generate a red reporter, the tdTomato tandem repeat was amplified from pFA6a-tdTomato-kanMX with primers osm944 (5′-aatGGATCCatggtgagcaagggcgaggaggtc-3′; BamHI site uppercase) and osm945 (5′-ttaCCCGGGcttgtacagctcgtccatgc-3′; XmaI site uppercase), digested with BamHI and XmaI, and ligated to similarly treated pSM793, yielding plasmid pSM1709 (pRIP-Pmap3:tdTomato; ura4+). Plasmids were linearized with NruI and integrated at the map3 promoter in h90 cells.
Genes were tagged at their endogenous genomic locus at their 3′ end, yielding C-terminally tagged proteins. This was achieved by PCR amplification of a fragment from a template plasmid with primers carrying 5′ extensions corresponding to the last 80 nucleotides of the ORF and the first 80 nucleotides of the 3′UTR, which was transformed and integrated in the genome by homologous recombination, as previously described (Bähler et al., 1998 (link)). For tagging of genes with sfGFP, a pFA6a-sfGFP-kanMX plasmid was used as a template. sfGFP was amplified from pMaM4 (a plasmid provided by M. Knop, University of Heidelberg, Heidelberg, Germany; containing yeast codon-optimized sfGFP), with primers osm2680 (5′-ccTTAATTAActccaagggtgaagagctatttac-3′; PacI site uppercase) and osm2681 (5′-aGGCGCGCCcttataaagctcgtccattccg-3′; AscI site uppercase), digested with AscI and Pac1 and ligated to similarly treated pSM674 (pFA6a-EGFP-kanMX6; described in Bähler et al., 1998 (link)). The sfGFP replaced EGFP, resulting in pFA6a-sfGFP-kanMX6 (pSM1538). We then used this vector as template for PCR-based targeted tagging of fus1, agn2, eng2, and exg3 (Bähler et al., 1998 (link)).
To yield Pmap3-driven fluorescent reporters, the map3 promoter region was amplified from genomic DNA with primers osm935 (5′-cccCTGCAGaagcatgcacgctgctcac-3′; PstI site uppercase) and osm936 (5′-agaGTCGACggtaaactcaacgtataag-3′; SalI site uppercase), digested with Pst1 and SalI, and ligated to similarly treated pSM242 (pRIP42:GFP; an integrative plasmid containing GFP under control of nmt41 promoter and a ura4+ selection marker), replacing the nmt41 promoter and yielding plasmid pSM793 (pRIP-Pmap3:GFP, ura4+). To generate a red reporter, the tdTomato tandem repeat was amplified from pFA6a-tdTomato-kanMX with primers osm944 (5′-aatGGATCCatggtgagcaagggcgaggaggtc-3′; BamHI site uppercase) and osm945 (5′-ttaCCCGGGcttgtacagctcgtccatgc-3′; XmaI site uppercase), digested with BamHI and XmaI, and ligated to similarly treated pSM793, yielding plasmid pSM1709 (pRIP-Pmap3:tdTomato; ura4+). Plasmids were linearized with NruI and integrated at the map3 promoter in h90 cells.
