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First published online 11 March 2008
doi: 10.1242/jcs.015933

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The function of two closely related Rho proteins is determined by an atypical switch I region

¹ Applied Microbiology, Biozentrum Universität Basel, Klingelbergstr. 50-70, 4056 Basel, Switzerland
² Department of Genetics, University of Osnabrück, Barbarastr. 11, 49076 Osnabrück, Germany

View larger version (27K): [in this window] [in a new window]   Fig. 1. Lysis phenotypes of Agrho1a. (A) Genomic organization of RHO1 loci and neighboring genes in S. cerevisiae and A. gossypii. (B) Percent identity of Rho1 proteins derived from comparisons with the needle program of the EMBOSS package (Rice et al., 2000). (C) Representative mycelia of wild type, Agrho1a and Agrho1b. For the deletion mutants, spores from a heterokaryotic mycelium were spotted on medium selective for the marker of the deletion. (D) Hyphal lysis of Agrho1a growing on an AFM agar surface. Still images of individual hyphae that lysed during a time-lapse movie (Movie 1). Images were taken every 5 minutes. Sections of frames from time points just prior to and after lysis are shown. Bar, 25 µm. (E) Quantification of mycelial lysis. The alkaline phosphatase release of wild-type cells was set to 0% and the release of an Agslt2 to 100%. Values are given for at least three individual experiments.

View larger version (46K): [in this window] [in a new window]   Fig. 2. Actin-related phenotypes of Agrho1a. (A) A. gossypii hyphae grown for 20 hours in AFM and stained for filamentous actin with Alexa-Phalloidin. Bar, 10 µm. (B) Growth defect of an Agrho1a strain carrying AgABP140 fused to GFP. The control shows growth behavior without the plasmid. Cells were grown for at least 3 days on solid medium at 30°C. (C) Latrunculin A sensitivity of Agrho1a. A dense spore suspension of wild-type (upper panel) or Agrho1a spores (lower panel) was mixed with AFM-Agar and overlaid on AFM plates. Filter discs were placed on the petri dishes and the indicated amount of Latrunculin A was spotted on discs in a volume of 5 µl DMSO. Plates were then incubated at 25°C for about 36 hours. (D) Quantification of Latrunculin A sensitivity. The diameter of the inhibition zone was measured and plotted against the Latrunculin A concentration.

View larger version (39K): [in this window] [in a new window]   Fig. 3. Localization of AgRho1 proteins. N-terminal fusions of yellow fluorescent protein (YFP) to AgRho1a and cyan fluorescent protein (CFP) to AgRho1b localizing at the tip (A) and at the septum (B). The slides were incubated for 1 hour at the given temperature prior to microscopy. The overlay uses green for YFP-AgRho1a and red for CFP-AgRho1b. Bar, 10 µm (A); 5 µm (B). (C) Colocalization of GFP-AgRho1b with 1,3-β-glucan biosynthesis at the tip. Mycelia that were grown on microscopy slides were incubated with 1 mg/ml Aniline Blue in AFM for visualization of 1,3-β-glucan biosynthesis for 5-10 minutes prior to microscopy. Aniline Blue is shown in red and GFP-AgRho1b in green on the overlay image. Bar, 10 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 4. Complementation of S. cerevisiae rho1 mutants by AgRHO1 genes. (A) Complementation of Scrho1. The open reading frame of ScRHO1 was replaced by either AgRHO1a or AgRHO1b in a diploid strain. After sporulation, the resulting strains were subjected to tetrad analysis. Spore growth is shown for AgRHO1a (left) and AgRHO1b (right). (B) Plasmid loss. Strains were transformed with RHO1L, a plasmid containing the URA3 marker and the ScRHO1 gene with promoter and terminator, and streaked on medium without or with counterselection for URA3 by 5'-fluorootic acid. (C) Two segregants from a tetrad shown in A (right panel) were tested for temperature sensitivity at 39°C in a drop dilution assay. Cultures were grown to an OD600 of 1, the number of cells given below the image was spotted on agar plates and incubated for 3 days. (D) Complementation of Scrho1 temperature-sensitive mutants from different complementation groups by the two AgRHO1 genes. Strains carrying the wild-type ScRHO1 allele or an allele of each Scrho1 complementation group were combined with a Scrho1 deletion (top row), AgRHO1a (middle row) or AgRHO1b (bottom row). A drop dilution assay of cultures grown to OD600=1 was performed. The strains were incubated at the permissive (25°C, left) or the restrictive temperature (39°C, right).

View larger version (54K): [in this window] [in a new window]   Fig. 5. An atypical histidine in the switch I region of AgRho1a. (A) Alignment of the switch I region of Rho-type proteins from several model organisms. Non-homologous residues are shaded gray. The residue corresponding to position 39 of AgRho1a is boxed. (B) An A. gossypii mutant with an exchange of histidine 39 for tyrosine (C115T) in AgRho1a can complement the lethality of Agrho1b. Spores from mycelia that are heterokaryotic for the indicated mutation were spotted on plates selective for the markers of the mutations. Plates were incubated for 4 days at 30°C. (C) Growth speed of the mutants shown in B. Identically sized inoculums of homokaryotic mycelia were placed on full medium plates and incubated at 30°C for 4 days. Growth speed was determined as the quotient of mycelial diameter and incubation time. (D) The AgRho1aH39Y mutant is able to complement the deletion of ScRHO1 in S. cerevisiae. Yeast cells were grown at 25°C in liquid medium to OD600=1 and diluted. The indicated cell numbers were spotted on agar plates and were incubated at the indicated temperatures for 2 days. (E) GFP-AgRho1aH39Y displays an altered localization pattern at 25°C compared with GFP-AgRho1a. Growth conditions were identical to Fig. 3. Bar, 10 µm.

View larger version (38K): [in this window] [in a new window]   Fig. 6. Rho proteins and GAP interactions. (A) Two-hybrid analysis of the AgRho1 proteins and potential GTPase-activating proteins. The C-terminal lipid-modification motif of the small GTP-binding proteins was truncated from the coding sequence to allow two-hybrid testing. The glutamate-to-histidine exchange in switch II mimics the GTP-bound state of the proteins. The histidine-to-tyrosine exchange is indicated by H39Y, the reverse mutation in AgRho1b by Y40H. The left panel shows a growth control on medium selective for prey and bait plasmids. The medium in the right panel is also selective for interaction. (B) Pull-down experiment in the presence of aluminium fluoride. Glutathion-S-Transferase (GST) or AgRho1a proteins fused to GST were bound to glutathion-sepharose and washed. The eluted proteins were subjected to western blot analysis. The blot was probed with -GST and -6HIS. (C) The H39Y mutation increases interaction strength of AgRho1a with AgLrg1. The indicated prey/bait combinations from A were additionally tested on medium with different concentrations of 3-aminotriazole to visualize the increase in interaction strength of the AgRho1aH39Y mutant. The first image on the left side is the growth control. (D) Two-hybrid analysis of Rho1 proteins from S. cerevisiae and Kluyveromyces lactis. The procedure used was identical to the one described for A.

View larger version (37K): [in this window] [in a new window]   Fig. 7. GAP activity on AgRho1 proteins. (A) Comparison between GTP-locked Agrho1a and Agrho1b mutants with Aglrg1 and Agsac7. Spores from mycelia that are heterokaryotic for the indicated allele were spotted on medium selecting for the mutation in six-well plates. The plates were incubated at 30°C for 2 days. The speckles come from air bubbles in the agar plates. (B) AgSac7-GAP activity towards AgRho1 proteins. The activities were determined as described in Materials and Methods using 5 µg of each protein or buffer as a control. The optical density at 650 nm was determined against a control reaction with buffer only. (C) Localization of the AgRho1 GAPs. To exclude the possibility that the cytoplasmic signal of AgSAC7-GFP is only background fluorescence, we include a wild-type strain without GFP and performed western analysis using a GFP antibody (left bottom panel).

View larger version (24K): [in this window] [in a new window]   Fig. 8. Model of Rho1 regulatory networks in S. cerevisiae and A. gossypii. A. gossypii homologs of Rho1 regulators known from S. cerevisiae that were not subject of our studies are printed in outline. ScROM1/ScROM2 and ScSAC7/ScBAG7 are `twin-ORFs' with only a single homolog of each pair in A. gossypii.