Strains, media, growth conditions, genetic methods, and DNA manipulation. Standard methods were used. Yeast strains, plasmids and DNA primers are listed in Tables 1 and 2. All gene deletions were made precisely from the ATG to stop of the open reading frame using the oligonucleotide primers listed in Table 2. All were confirmed by PCR initially and confirmed by PCR in segregants of crosses involving two or more gene deletions.

Identification and characterization of RAX2. To isolate mutations which revert the bipolar budding pattern of the axl1 mutant to random or axial, transposon mutagenesis was used (1). A Tn3-LEU2 transposon mutagenized genomic library were transformed into an axl1::HIS3 ace2 mutant strain (CPY21) exhibiting a bipolar budding pattern. Approximately 40,000 transformants were screened for a difference of colony morphology (2). One mutant, HS42, exhibited an axial or random budding pattern. This mutant was found to have transposon insertion at 299,174 on chromosome XII within the ORF YLR084C. YLR084C was named as RAX2.

Calcoflour staining of bud scars and GFP imaging. Bud scars and birth scars were stained with Calcoflour (3). To observe division scars and Rax2-GFP, cells were lightly patched to the appropriate media. Induction and repression experiments were performed by patching exponentially growing cells from one plate type to another. For staining, cells from the patch were picked by toothpick, swirled in 2 µl of 0.1% Calcoflour on a slide, and pressed relatively hard with a cover slip. Immediate observation by fluorescence microscopy was performed. To view all the GFP patches on the cell surface, focusing up and down during observation was often necessary.

Cell counting method (4). For each strain, at least 100 cells were counted for each category or each time point. Data presented are the summary of the observations of two independent strains. The proximal and distal poles were assigned by locating the birth scar (proximal pole). Bud scars were scored as proximal (p) if they touched or overlapped the birth scar. Bud scars close to the distal pole were scored as distal (d) if they were within a bud scar's radius of the exact pole. Any bud scar that did not touch either pole was scored as medial (m). A three-bud-scar pattern was scored axial (a) if bud scars were connected in a chain emanating from the birth scar. A pattern was scored as bipolar (b) if all the bud scars were at the distal pole, the proximal pole (and did not satisfy axial criteria), or distributed at both poles. A cell was scored as random (r) if one or more scars were in the cell's midsection (not at either pole).

Construction of strains carrying RAX2-GFP. The GFP(S65T)-HIS3M×6 fragment of plasmid pFA6a-GFP(S65T)-HIS3M×6 (5) was amplified with sequences immediately flanking the stop codon of RAX2 using primers pTC17 and pTC18. The amplified DNA was purified and transformed into wild-type strain HAB251-15B. Correct transformants, confirmed by diagnostic PCR, all showed Rax2-GFP signal.

Construction of GAL1-RAX1. RAX1was amplified using primers pTC23 and pTC24. The PCR fragment was cut with Not I, purified, and ligated into the Not I site of pRS316-GAL (E. Bi, University of Pennsylvania Medical School). Constructs of the correct orientation were transformed into a rax1 RAX2-GFP homozygous diploid. Transformants were placed on a glucose plate overnight, then were patched in parallel to a glucose and a galactose plate. After growth, Rax2-GFP signals could be seen on the galactose plate but not on the glucose plate.

Construction of GAL1-RAX2-MYC. A Myc tag was fused to the C-terminus of RAX2 by the same method above, using plasmid pFA6a-13Myc-TRP1, primers pTC17 and pTC18 for PCR. Primers pTC21 and pTC22 were used to amplify the RAX2-Myc fragment with Hind III sites introduced at the fragment ends. This fragment was cut then ligated into the Hind III site of plasmid YEp181-Gal. Constructs were checked for right orientation and transformed into rax2/rax2 strain. Transformants were first grown in glucose-containing medium, then in raffinose-containing medium overnight and induced in galactose media for 2 hours. Myc signal could be detected only in the culture from galactose media, not from glucose or raffinose media.

Construction of GAL1-BUD10-MYC. A Myc tag was fused to the C-terminus of BUD10 using plasmid pFA6a-13Myc-TRP1 as template and primers pTC25 and pTC26. The BUD10-Myc fragment was amplified using primers pTC27 and pTC29 with the addition of Not I site at each end. The PCR product was cut, then ligated into the Not I site of pRS316-GAL. Constructs with the right orientation were transformed into wild-type cells (HAB251-15B). Transformants were turned on under the same condition as used for RAX2-Myc and the signal was detected.

Construction of a BUD8-MYC strain. Plasmid pFA6a-13Myc-TRP1 and primers pTC30 and pTC31 were used to produce a fragment for chromosomal tagging of BUD8. The fragment was transformed into wild-type cells and correct transformants were detected by PCR. All such transformants exhibited a Bud8-Myc signal by immunofluorescence.

Immunofluorescence. Standard indirect immunofluorescence was used to visualize Bud8-Myc. Cells were stained with mouse anti-Myc antibody (Babco) and a secondary CY3-conjugated goat anti-mouse antibody (Jackson Immunologicals). DNA was stained by Hoechst 33342 (Molecular Probes). Samples were observed and photographed by an epifluorescence microscope.

Western blotting. To prepare detergent soluble and insoluble membrane fractions, the following method was used. Cells were grown in glucose containing minimum media until mid-log phase, washed and transferred to raffinose media overnight. Cells were then washed and grown in galactose media for 2 hours. Cells were washed and placed in glucose medium. Samples were collected at the indicated times following this transfer. Cells were pelleted, washed and resuspended in PBS with 1 mM EDTA buffer (Mallinckrodt) containing protease inhibitors and then vortexed with glass beads for five 3-min pulses. The suspension was spun at 4000g for 5 min at 4°C. Supernatant was transferred to Beckman ultracentrifuge tubes prechilled in ice and spun at 65,000g for 20 min at 4°C. The resulting pellet was resuspended in PBS containing 1% Nonidet P-40 (Sigma) and protease inhibitors, dispersed by pipetting up and down, and incubated on ice for 20 min. The suspension was spun at 65,000g for 20 min at 4°C. The resulting supernatant is the detergent soluble membrane fraction, from which protein concentration was measured using Coomassie plus protein assay reagent (Pierce), and the pellet is the detergent insoluble membrane fraction. Samples were boiled with sample buffer for 5 min before loading to a 10% SDS-PAGE gel. Equal volumes of samples were loaded. Immunoblots were performed by standard methods. Mouse monoclonal anti-Myc antibody (Babco) was used at dilution of 1:2000, and the secondary goat anti-mouse HRP antibody (Jackson Immunologicals) was used at the same dilution. Bands were visualized by Supersignal reagent (Pierce).

Northern blotting. Cells were grown under the same condition and samples were collected in the same way as in preparing protein extract. The hot phenol method (6) was used to extract yeast RNA. Standard methods were employed to perform Northern blotting (7). Equal amounts of RNA from each time point were loaded in a 1% agarose/formaldehyde gel. The RNA samples were then transferred to Zeta Probe membrane (Bio-Rad). A 2.6-kb fragment from full-length RAX2 was used as a probe and labeled with [a-32p] using the Prime-a-gene labeling system (Promega). Prehybridization, hybridization, and washing of the membrane were performed according to manufacturer's specifications.

References

1. N. Burns et al., Genes Dev. 8, 1087 (1994).

2. A. Fujita et al., Nature 372, 567 (1994).

3. J. R. Pringle, Methods Enzymol. 194, 732 (1991).

4. J. Chant, J. R. Pringle, J. Cell Biol. 129, 751 (1995).

5. M. S. Longtine et al., Yeast 14, 653 (1998).

6. K. Köhrer, H. Domdey, Methods Enzymol. 194, 398 (1991).

7. J. Sambrook, E. F. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1994).

Supplemental Figure 1. The effects of rax2 mutations on bud site selection in haploid cells. Scoring was performed exactly as described for Fig. 1B of the report. See Web table 1 for strains used.

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