Schematic overview of the construction and subsequent screening of carboxy-terminal YFP fusions. Primers for PCR amplification were designed with 5′-att sites such that resulting PCR products could be cloned into the pDEST-vYFP vector by phage λ-based recombination. The resulting gene fusion carries a 10-codon linker between the target open reading frame and YFP. Subsequent screening steps are as indicated, with a diagrammatic representation of a protein differentially localized to the nucleus under conditions of filamentous growth.

Phenotypic analysis of kinase deletion mutants in the filamentous Σ1278b genetic background. Each haploid deletion mutant was assayed for surface-spread filamentation (A), cell morphology (B), and invasive growth (C). Surface-spread filamentation was assayed on SLAD medium (see Materials and Methods) supplemented with 1% butanol. Cells from these colonies were inoculated into a small volume of water for DIC microscopy. Invasive growth was assayed on YPD medium as described in Materials and Methods. The Σ1278b background strain Y825 served as a wild-type control for these filamentous growth assays.

The observed nuclear translocations are dependent upon the kinase activity of Fus3p, Ksp1p, Kss1p, Sks1p, and Tpk2p, respectively. Each indicated kinase-vYFP fusion was imaged in a strain carrying the kinase-dead allele listed to the left. For example, the subcellular distribution of Ksp1p and Sks1p is shown in a haploid fus3Δ strain carrying the fus3-K42R kinase-dead allele on a low-copy plasmid. DAPI-stained and DIC images are as described previously. Note the mixed distribution of each kinase over the cytoplasm and nucleus upon loss of the indicated kinase activity. Images of these kinases in the kinase-dead background strains under vegetative growth conditions are presented in Supplemental Figure SF6. Bar, 3 μm.

Subcellular localization of the yeast kinome during filamentous growth. This diagram summarizes the localization of 125 protein kinases, constituting the yeast kinome, under conditions of filamentous growth. In total, 119 kinases do not shift localization during the transition to filamentous growth, and the localization of these kinases is tallied in the top left. For simplicity, Yck3p, which localizes to the vacuole, is included in the cytoplasmic compartment. The six kinases that shift localization between vegetative and filamentous growth conditions are boxed in red, and the pathway context of each kinase is shown. Flo8p and Ste12p are included as representative targets of Tpk2p and Kss1p, respectively. Speculative and unknown interactions are indicated with question marks.

Differential localization of the protein kinases Bcy1p, Fus3p, Ksp1p, Kss1p, Sks1p, and Tpk2p. Kinase-vYFP fusions were visualized by fluorescence microscopy (left) under vegetative growth conditions and during filamentous growth. Yeast cells were stained with the DNA-binding dye DAPI (middle) to visualize the nucleus and mitochondria. The yeast cell shape and vacuoles were imaged by differential interference contrast (DIC) microscopy (right).

Interdependent localization of yeast kinases during filamentous growth. (A) Each kinase-vYFP fusion was visualized by fluorescence microscopy under filamentous growth conditions in a haploid strain of Σ1278b deleted for the indicated kinase. DAPI-stained and DIC images are provided. Images of the kinase-vYFP fusions under vegetative growth conditions are presented in Supplemental Figure SF2. The mixed distribution of these kinases over the cytoplasm and nucleus signifies a loss of nuclear localization under filamentous growth conditions upon deletion of the indicated kinase gene. Only kinases exhibiting a loss of nuclear localization are shown here. (B) Matrix of kinase localizations in gene deletion backgrounds under conditions of filamentous growth. The results corresponding to the images shown in part A are boxed in red. Images of nuclear-localized kinase-vYFP fusions unaffected by the indicated gene deletions are presented in Supplemental Figure SF3. (C) Localization-based regulatory relationships among yeast kinases. Each forward pointed arrow indicates that the given kinase is required for the wild-type localization of the subordinate kinase; for example, Fus3p is required for the wild-type nuclear localization of Sks1p during filamentous growth. Reciprocal relationships between kinases are indicated by the circular arrows in the left column. (D) The network of regulated protein localization between Bcy1p, Fus3p, Ksp1p, Kss1p, Sks1p, and Tpk2p is illustrated here; arrows are drawn as described for C. Double-sided arrows indicate that the localization of the given kinase pair is reciprocally affected under filamentous growth conditions.

Tagging Ksp1p with a NES abolishes filamentous growth. (A) Schematic diagram of the NES-tagged form of Ksp1p, where the NES (encoding LQLPPLERLTLD) is fused at the carboxy terminus of the Ksp1p-vYFP chimera. As visualized by fluorescence microscopy, addition of the NES prevents the butanol-induced nuclear translocation of Ksp1p. DIC and vYFP images are shown. Bar, 3 μm. (B) Phenotypic analysis of NES-tagged Ksp1p. For comparison, we have included a haploid ksp1Δ strain carrying a centromeric plasmid with the KSP1-vYFP fusion under transcriptional control of the native KSP1 promoter; this strain exhibits wild-type filamentous growth properties. Addition of the NES results in decreased filamentous growth, evidenced in a loss of surface-spread filamentation, rounded cell morphology, and decreased invasive growth. Bar, 3 μm.