Yeast Strains, Reagents, and Genetic Techniques
Cells were grown in YPD media containing 1% bacto-yeast extract, 2% bacto-peptone, and 2% glucose, all from Difco (Sparks, MD). For all assays performed, 25°C was the permissive temperature, whereas 14°C was used as the restrictive temperature. Room temperature (RT) was ~22°C.
Sorbitol, sodium azide (NaN3), N-ethylmaleimide (NEM), β-mercaptoethanol, o-dianasidine, glucose oxidase, peroxidase, Calcofluor, tetramethylrhodamine isothiocyanate (TRITC)-conjugated phalloidin, Triton X-100, and 3-amino-1,2,4-triazole were obtained from Sigma Chemical (St. Louis, MO). Zymolyase (100T) was from Seikagaku (Tokyo, Japan). BSA and yeast nitrogen base were from US Biologicals (Swampscott, MA). Formaldehyde (37%), gluteraldehyde, and Spurr’s resin were from Electron Microscopy Sciences (Ft. Washington, PA).
Transformations for suppression analysis were performed using the lithium acetate method described by Ito et al. (1983)
. Strain crossing, tetrad dissection, diploid sporulation, and mating-type determination were performed as described by Guthrie and Fink (1991)
Generation of Mutations in RHO3
Oligo-directed mutagenesis was performed to create the GTP- and GDP-bound forms of RHO3
, as well as the pool of effector domain mutations (Kunkel et al., 1987
). Fourteen synthetic oligonucleotides were used to change each of the 14 amino acids in the effector domain to other random amino acids. Each oligonucleotide had a mixture of bases at the position where different amino acids were desired and was designed to contain at least 10 bp of homology on both sides of the mixed residue. Resulting mutants were then sequenced to determine the amino acid sequence.
To determine the phenotype of each of the rho3 mutants as the only source of Rho3, we transformed the constructs into a diploid yeast strain heterozygous for a chromosomal deletion of RHO3. BY506 (a/α; GAL+/GAL+; ura3-52/ura3-52; leu2-3112/leu2-3112; his3-Δ200/his3-Δ200; rho3Δ::LEU2/RHO3) has one copy of RHO3 disrupted by the insertion of the LEU2 marker. The plasmids containing the rho3 mutant were cleaved within the URA3 gene to target chromosomal integration at the URA3 locus. The transformants were colony purified and sporulated, and tetrads were dissected with a micromanipulator on YPD plates. The plates were grown at 25°C. The haploid progeny were then analyzed by replica plating for the presence of the integrated mutants (scored as ura+), for the presence of a disrupted copy of rho3 (scored as leu+), and for conditional growth at 37 or 14°C.
Cells were grown in YPD to midlog phase, diluted back, and preshifted to the restrictive temperature for 1 h (or kept at the permissive temperature). From these cultures, two aliquots of 0.8 OD599 U were pelleted. One-half was resuspended in 10 mM NaN3 and kept on ice, while the other one-half was resuspended in low-glucose (0.1%) YPD and shifted to 14°C for 5 or 10 h or to 25°C for 1.5 h. After the shift, the fractions were resuspended in NaN3, and aliquots from both fractions were spheroplasted for 30 min at 37°C in a 1.4 M sorbitol and Tris-buffered solution containing β-mercaptoethanol and 0.1 mg/ml 100T zymolyase. Spheroplasted internal fractions were then resuspended in 0.5% Triton X-100.
All internal and external samples were assayed at 37°C in a sodium acetate and NEM buffer by adding 25 μl of 0.5 M sucrose and then stopping the reaction after 15 min with 150 μl of 0.2 M K2HPO4. The addition of an assay mix containing NEM, o-dianasidine, glucose oxidase, and peroxidase in a 0.1 M KPO4 buffer causes a color change in the presence of glucose. This end-point assay was fully stopped and developed by the addition of 6 N HCl. Spectrophotometric readings were taken at A540, and units of activity were calculated and reported as micromoles of glucose released per minute per OD599 of cells.
Invertase, Bgl2, and Carboxypeptidase Y (CPY) blots
RHO3, rho3-V51, and sec4-P48 cells were grown overnight to midlog phase at 25°C in YPD. Strains were preshifted to the restrictive temperature of 14°C for 1 h before being transferred to YP media containing low (0.1%) glucose for 5 h. In addition, the late-secretory temperature-sensitive mutant sec18-1 was preshifted to 37°C for 1 h before a 3-h shift into low glucose. Whole-cell glass bead lysates were prepared and boiled with 2× sample buffer. Samples were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with affinity-purified α-invertase antibody at 1:200 or affinity-purified α-CPY antibody at 1:1000. For Bgl2 blots, cells were spheroplasted, and the internal and external fractions were separated. Internal fractions were boiled in 2× sample buffer, and the external fractions were boiled in 6× sample buffer. Samples were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with affinity-purified α-Bgl2 antibody at 1:100.
Wide-Field Fluorescence and Imaging
For actin staining, cells were grown overnight in YPD media to ~0.5 OD599. They were then either shifted to the restrictive temperature of 14°C for 5 h or kept at the permissive temperature before fixative (formaldehyde to 3.7%) was added directly to the media. A second round of fixation was performed in KPO4 buffer (pH 6.5), and the cells were transferred to a sorbitol buffer for overnight storage at 4°C.
The next day cells were permeabilized for 10 min in 0.1% Triton X-100 and washed twice with PBS. Cells were resuspended in 100 μl of PBS and stained in the dark for 25 min with 35 μl of 3.3 μM TRITC-phalloidin dissolved in methanol. Cells were washed six times with PBS and then resuspended in the appropriate volume of mounting media (90% glycerol with 4′,6-diamidino-2-phenylindol to visualize DNA and o-phenylenediamine to retard photobleaching). To study the deposition of chitin in the bud scars, cells from the same round of fixation were not permeabilized, and 3 μl of 1 mg/ml Calcofluor was added instead of labeled phalloidin. Stained cells was viewed on a Nikon Eclipse E600 microscope (Garden City, NY); images were captured with a Princeton Instruments charge-coupled device camera (Trenton, NJ) and Metamorph Imaging software (Universal Imaging, West Chester, PA).
Thin-Section Electron Microscopy
Cells were grown overnight in YPD media to midlog phase and diluted back the next day, and then one-half of the culture was shifted to the restrictive temperature 14°C and grown for 5 h to an OD599 of 0.8. Ten optical density units were harvested on a 115-ml, 0.45-μm Nalgene (Rochester, NY) filter, washed with 0.1 M cacodylate, and resuspended in a 0.1 M cacodylate and 3% gluteraldehyde fixative solution. Cells were then allowed to fix for 1 h at RT and then overnight at 4°C. Cells were spheroplasted for 40 min at 37°C in a KPO4-buffered solution of 0.3 mg/ml zymolyase and then washed with cold cacodylate. Then 1.5 OD U was pelleted in a 1.7-ml microfuge tube and incubated on ice for 1 h at RT with a solution of 2% osmium tetroxide and 0.1 M cacodylate. Pellets were washed three times each with dH2O and incubated in a 2% aqueous solution of uranyl acetate for 1 h at RT in the dark. Each pellet was rinsed twice with dH2O and taken through the following 10-min ethanol dehydration steps: 50, 70, 85, and 95% and then four rinses in 100% ethanol. The pellet was rinsed once with 100% acetone and then covered with a 50% acetone and 50% Spurr’s resin mixture. This remained at RT for 5 h, the acetone and Spurr’s resin mixture was replaced with 100% Spurr’s resin, and these remained at RT overnight. The 100% Spurr’s resin was changed the next day, and the pellets were baked in Spurr’s resin for 24 h at 80°C. Thin sections were cut and layered onto an uncoated copper grid and poststained with lead citrate and uranyl acetate before viewing. Cells were viewed on a JEOL 100CXII Electron microscope (Tokyo, Japan) and photographed at 60 kV.
The constructs were prepared by a recombinational cloning method (Hudson et al., 1997
) in which the indicated Rho3 fragments were PCR amplified with 70 bp extensions on each end. These extensions then directed the in vivo incorporation of the fragment into a Nco
II-digested pOBD.CYH vector. Rho3 and activated Rho3-V25 were amplified by PCR, whereas the Rho3-V25,V51 double mutant was generated by fusion PCR, and all were confirmed by DNA sequence analysis. The C-terminal CAAX box was deleted by placing a termination codon in place of the cysteine residue. The GAL4AD
constructs were made in a similar way and inserted into the activating domain vector pOAD by recombinational cloning. All constructs were tested to verify that they did not interact when paired with the opposing empty vector. Transformants were shown to express the expected size of the GAL4BD-RHO3
fusion by Western blotting and then transformed with the GAL4AD-MYO2
constructs. Constructs were transformed into the strain PJ694α that has the HIS3
gene as a reporter (James et al., 1996
). To reconfirm that similar amounts of Rho3 fusion proteins were present, immunoblotting of the two-hybrid transformants was performed on whole-cell lysates as described above. Blots were probed with affinity-purified Rho3 antibodies and detected with 125