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First published online 23 December 2002
doi: 10.1242/jcs.00266

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Krh1p and Krh2p act downstream of the Gpa2p G subunit to negatively regulate haploid invasive growth

Montserrat Batlle^*, Ailan Lu, David A. Green, Yong Xue and Jeanne P. Hirsch

Brookdale Department of Molecular, Cell, and Developmental Biology, Mount Sinai School of Medicine, New York, NY 10029, USA
^* Present address: Servei de Cardiologia, Escala 1, planta 6, Hospital Clínic de Barcelona, Villaroel 170, Barcelona 08036, Spain
Present address: Applied Biosystems, 35 Wiggins Avenue, Bedford, MA 01730, USA

View larger version (83K): [in a new window]   Fig. 1. Effect of krh1 krh2 mutations on FLO11 induction, invasive growth and cell morphology. (A) RNA was isolated from wild-type strain SKY763 (lane 1), krh1 strain SKY.k1-2B (lane 2), krh2 strain SKY.k2-4C (lane 3), krh1 krh2 strain HS182-1D (lane 4), gpa2 strain BS13B (lane 5) and krh1 krh2 gpa2 strain HS183-3D (lane 6). The RNA was transferred to nylon membrane, hybridized with a FLO11 probe, and rehybridized with an ACT1 probe. The relative amount of FLO11 RNA, normalized to ACT1 RNA, is shown below each lane. (B) Wild-type strain SKY763 (WT), krh1 strain SKY.k1-2B (krh1), krh2 strain SKY.k2-4C (krh2), krh1 krh2 strain HS182-1D (krh1 krh2), gpa2 strain BS13B (gpa2), krh1 krh2 gpa2 strain HS183-3D (krh1 krh2 gpa2), flo11 strain HS184-5A (flo11), and krh1 krh2 flo11 strain HS197-13D (krh1 krh2 flo11) were patched onto YEPD/2.5% agar medium, incubated for 4 days at 25°C, and photographed before (Total growth) and after (Invasive growth) rubbing the surface of the plate with a glass rod under a stream of water. (C) Wild-type strain BS1B (KRH1 KRH2) and double mutant strain HS154-3D (krh1 krh2) were diluted 1:20 from an overnight saturated culture into fresh YEPD medium and incubated at 30°C with shaking for 2 days. (D) Wild-type strain BS1B (KRH1 KRH2) and double mutant strain HS154-3D (krh1 krh2) were patched onto YEPD plates and incubated at 30°C for 5 days. Magnification: upper panel, 1x; lower panel, 25x.

View larger version (14K): [in a new window]   Fig. 2. Effect of krh1 krh2 mutations on heat shock sensitivity and sporulation. (A) Wild-type strain W3031B (open bar), krh1 strain G102-5C (filled bar), krh2 strain G103-4A (shaded bar), and krh1 krh2 strain H147-3C (hatched bar) were grown to saturation for 2 days in synthetic complete medium, incubated at 50°C for 20 minutes, and diluted and plated to determine the percentage survival. Values shown are the mean and standard deviation from three independent experiments. (B) The following strains were incubated in sporulation medium for 3 days and the percentage sporulation was determined by visual inspection: krh1 krh2 diploid strain H174 containing plasmids YEp181-FLKRH1 and YEp112-KRH2 (open bar, n=2); strain H174 containing plasmids YEplac181 and YEp112-KRH2 (filled bar, n=5); strain H174 containing plasmids YEp181-FLKRH1 and YEplac112 (shaded bar, n=2); and strain H174 containing plasmids YEplac181 and YEplac112 (hatched bar, n=5). Values shown are the means from independent experiments.

View larger version (22K): [in a new window]   Fig. 3. Relationship between KRH1 and KRH2 and GPA2. Wild-type strain BS1B (lanes 1,2) and krh1 krh2 strain HS154-3D (lanes 3,4) transformed with either plasmid pG2CT-112.2 (lanes 2,4) or vector YEplac112 (lanes 1,3) were grown to log phase and RNA was isolated and hybridized as described in the legend to Fig. 1A. (B) Wild-type haploid strain W3031B (WT) and double mutant strain H147-3C (krh1 krh2) transformed with either plasmid pG2CT-112.2 (GPA2*) or YEplac112 (vector) were grown to saturation for 2 days in synthetic dropout medium lacking tryptophan, incubated at 50°C for 20 minutes, and diluted and plated to determine the percent survival. Values for YEplac112 are represented by the open bars; values for pG2CT-112.2 are represented by the filled bars. Values shown are the mean and standard deviation from three independent experiments. Note that the krh1 krh2 strain is more heat-shock sensitive when grown in medium lacking tryptophan than when grown in complete medium (Fig. 2A).

View larger version (28K): [in a new window]   Fig. 4. Relationship between KRH1 and KRH2 and SCH9. (A) Wild-type strain W3031B, sch9 strain B14-53B, krh1 krh2 strain H147-3C, and krh1 krh2 sch9 strain H148-6A, all transformed with vector YEplac112, were grown to saturation for 2 days in synthetic dropout medium lacking tryptophan, incubated at 50°C for 20 minutes, and diluted and plated to determine the percentage survival. Value for W3031B is represented by the open bar; value for B14-53B is represented by the filled bar; value for H147-3C is represented by the shaded bar; value for H148-6A is represented by the hatched bar. Values shown are the mean and standard deviation from three independent experiments. (B) Strains with the following genotypes were streaked out for single colonies onto YEPD medium and grown for 2 days at 30°C: H147-3C (krh1 krh2), H152-28A (gpa2 krh1 krh2 sch9) and H137-22A (gpa2 sch9).

View larger version (70K): [in a new window]   Fig. 5. Relationship between KRH1 and KRH2 and TPK2. (A) RNA from wild-type strain BS1B transformed with YEplac112 (lane 1), tpk2 strain BS3B transformed with YEplac112 (lane 2), krh1 krh2 strain HS154-3D (lane 3), and krh1 krh2 tpk2 strain HS161-4C (lane 4) was isolated and hybridized as described in the legend to Fig. 1A. (B) RNA from wild-type strain BS1B (lanes 1,2) and tpk2 strain BS3B (lanes 3,4) transformed with either plasmid pG2CT-112.2 (lanes 2,4) or vector YEplac112 (lanes 1,3) was isolated and hybridized as described in the legend to Fig. 1A. (C) krh1 krh2 strain HS154-3D and krh1 krh2 tpk2 strain HS161-4C were diluted 1:20 from an overnight saturated culture into fresh YEPD medium and incubated at 30°C with shaking for 2 days. (D) RNA from wild-type strain BS1B (lanes 1,2) and krh1 krh2 strain HS154-3D (lanes 3,4) transformed with either plasmid YEpTPK2.2 (lanes 2,4) or vector YEp351 (lanes 1,3) was isolated and hybridized as described in the legend to Fig. 1A.

View larger version (55K): [in a new window]   Fig. 6. Repeat sequences in Krh1p and Krh2p. The six repeated sequences from Krh1p and Krh2p were aligned based on the double glycine present in each repeat. The invariant double glycine is highlighted in black. Other conserved residues that are present in at least four repeat sequences are highlighted in gray. The kelch repeat consensus sequence from Adams et al. (Adams et al., 2000) is shown below. The Krh1p and Krh2p protein sequences were derived from the nucleotide sequence of the genes from the 1278b strain background, which differs from that in the Saccharomyces Genome Database by about 1% at the nucleotide level.