To construct plasmids containing tagged or mutated alleles of GIN4, single-stranded DNA from plasmid pALTER/GIN4 was used with primer ML62 (which mutates the lysine codon at position 48 to a methionine codon), primer ML63 (which introduces a NotI site just upstream of the GIN4 stop codon), or both, to create plasmids pALTER/gin4^K48M, pALTER/ GIN4NotI, and pALTER/gin4^K48MNotI. NotI fragments containing sequences encoding either a triple hemagglutinin tag (3HA) (Tyers et al., 1993) or glutathione-S-transferase (GST) (provided by D. DeMarini, University of North Carolina, Chapel Hill, NC) were cloned into NotI-digested pALTER/GIN4NotI and pALTER/gin4^K48MNotI to yield plasmids pALTER/GIN4-3HA, pALTER/GIN4-GST, and pALTER/gin4^K48M-3HA. The appropriate XbaI fragments from the pALTER derivatives were then cloned into XbaI-digested YCplac111, YEplac181, or YIplac204 to yield plasmids YCp/gin4^K48M, YEp/gin4^K48M, YCp/GIN4-3HA, YCp/gin4^K48M-3HA, and YIp/GIN4-GST. Before transformation into yeast, YIp/GIN4-GST was digested with ApaI to target integration to the ura3 locus.

To construct plasmid YCpGALGST/GIN4N, which contains the kinase region of GIN4 (codons 1–315) fused to GST-encoding sequences under the control of the GAL1/10 promoter, the PCR product obtained using primers ML67 and ML73 and pALTER/GIN4 as template was digested with BglII and XhoI and cloned into BamHI/SalI-digested YCpGALGST. The GIN4 ORF in YCpGALGST/GIN4N includes eight COOH-terminal non-GIN4-encoded amino acids (LDSSSSLA) followed by a stop codon. To construct plasmid YCpGALGST/GIN4C, which contains the COOH-terminal nonkinase region of GIN4 (codons 295–1,142) fused to GST-encoding sequences under the control of the GAL1/10 promoter, an ~2.2-kb SalI fragment from pEG202/GIN4C (see below) was cloned into SalI-digested YCpGALGST.

Because of the complicated genetics of the original synthetic lethal mutant, we asked if gin4-Δ9 and cdc12 mutations were synthetically lethal in the absence of other known mutations. Strains M-238 (cdc12-6 GIN4) and M-267 (CDC12 gin4-Δ9) were crossed, and 24 tetrads were dissected onto YPD plates containing 1 M sorbitol at 23°C. 94 out of 96 spores were viable. The segregants were scored for gin4-Δ9 (Trp⁺) and cdc12-6 (inviability at 37°C) and streaked on YPD plates to assay viability at 30°C. As expected, all 22 wild-type segregants and all 25 gin4-Δ9 single-mutant segregants were viable at 30°C, and cdc12-6 single-mutant segregants were mostly viable at this temperature (22 viable out of 24 total). In contrast, all 23 cdc12-6 gin4-Δ9 double-mutant segregants were inviable at 30°C. The synthetic lethality was confirmed by showing that a low-copy plasmid containing either GIN4 or CDC12, but not a control plasmid, could rescue the viability of cdc12-6 gin4-Δ9 segregants at 30°C (Fig. ​(Fig.11 C). cdc12-6 gin4-Δ9 double-mutant strains grew slowly even at 23°C (Fig. ​(Fig.11 C) and displayed a morphology very similar to that of a cdc12-6 single-mutant strain at restrictive temperature (Fig. ​(Fig.11 D) (Hartwell, 1971; Adams and Pringle, 1984); as expected from their abnormal morphology, the cdc12-6 gin4-Δ9 cells grown at 23°C also lacked detectable septin localization to the mother–bud necks (data not shown). A low-copy plasmid containing either GIN4 or CDC12 restored both normal cell morphology (Fig. ​(Fig.1,1, E and F) and septin localization to the necks (data not shown). These data suggest that deletion of GIN4 exacerbates the functional defect of the mutant Cdc12p and hence that Gin4p normally has a positive role in septin function.

To test this hypothesis further, we asked if overexpression of GIN4 could rescue the temperature-sensitive lethality of a cdc12-6 strain. As expected, cells of strain M-238 containing a control plasmid were viable at 23°C but inviable at 32° or 37°C, and cells containing a CDC12 plasmid were viable at all three temperatures (Fig. ​(Fig.33 A). In contrast, cells containing either a low-copy or a high-copy GIN4 plasmid were able to grow at 32°C although not at 37°C (Fig. ​(Fig.33 A), and the low-copy GIN4 plasmid even restored nearly normal morphology to the cells grown at 32°C (Fig. ​(Fig.3,3, B–D). (Because a high-copy GIN4 plasmid itself causes morphological defects [see below], it could not be tested for rescue of the cdc12-6 morphological defect.)

In many unbudded cells, Gin4p was visualized as a single ring in the cell cortex (Fig. ​(Fig.55 E, cells 1, 2, and 6). These rings appear to correspond to incipient bud sites, as they were always located at a cell pole with polarized F-actin (as detected by staining with rhodamine phalloidin; data not shown), and they corresponded precisely to rings of septin proteins (Fig. ​(Fig.5,5, E and F, cells 1, 2, and 6) that had the appearance (diameter and brightness) of septin rings marking incipient bud sites rather than of those marking previous division sites (Kim et al., 1991; Ford and Pringle, 1991). Consistent with this interpretation, when such cells had two rings of septin proteins, the Gin4p ring always (40 out of 40 cells observed) corresponded to the septin ring that appeared to be at the incipient bud site (Fig. ​(Fig.5,5, E and F, cell 6); unbudded cells with two rings of Gin4p were not observed. Thus, unlike the septins, which typically remain visible at the division site for some time after cytokinesis (Kim et al., 1991; Ford and Pringle, 1991), Gin4p seems to disappear from the neck/division site at about the time of cytokinesis. Gin4p appears to arrive at the incipient bud site in late G1, coincident with the arrival of the septins. Every unbudded cell identified as having a ring of Gin4p-3HA had a corresponding ring of Cdc11p (50 out of 50 cells observed), and nearly every unbudded cell identified as having a ring of Cdc11p at the incipient bud site had a corresponding ring of Gin4p-3HA (48 out of 50 cells observed). (The cells with a ring of Cdc11p at the incipient bud site but no corresponding ring of Gin4p-3HA might have lost the GIN4-3HA plasmid before immunofluorescence staining.)

Like the septins, Gin4p was localized to the mother–bud neck throughout the budded phase of the cell cycle (Fig. ​(Fig.5,5, A, B, and E). However, in budded cells, the septins are consistently visualized on both sides of the neck as an apparent double ring (Haarer and Pringle, 1987; Kim et al., 1991; Ford and Pringle, 1991) (Fig. ​(Fig.55 C; Fig. ​Fig.55 F, cells 3–5), whereas Gin4p was visualized as either an apparent single ring, (Fig. ​(Fig.55 A, cells 1, 2, 4, and 6; Fig. ​Fig.55 B) or an apparent double ring (Fig. ​(Fig.55 A, cells 3, 5, and 7; Fig. ​Fig.55 E, cells 3–5). The “single rings” of Gin4p were located in the middle or on the mother side of the neck, within the region occupied by the septins, and the “double rings” precisely colocalized with the septins. Careful focusing up and down revealed that Gin4p in double rings was, like the septins, in fact present as a continuous band throughout the neck region, at least until very late in the cell cycle; thus, the single ring versus double ring appearance seems simply to reflect differences in the width of the band of Gin4p along the mother–bud axis rather than a discrete difference in protein organization. Both “single rings” and “double rings” of Gin4p were observed at all stages in the cell cycle, but there was a sharp increase in the frequency of cells with “double rings” at about the time of the G2/M transition (data not shown).

The overlapping localization of Gin4p and the septins suggested that Gin4p localization may be septin dependent. To test this, a temperature-sensitive cdc12 septin mutant expressing Gin4p-GST and harboring either a control plasmid or a low-copy CDC12 plasmid was grown to exponential phase at 23°C, shifted to 37°C for 45 min, and stained with antibodies to GST or antibodies to Cdc3p. Both Gin4p-GST and Cdc3p remained detectable at the neck in cells containing the CDC12 plasmid (Fig ​(Fig6,6, A and B), but both proteins were lost from the neck in cells containing the control plasmid (Fig. ​(Fig.6,6, C and D). Thus, Gin4p localization to the neck indeed appears to be septin dependent.

Immunofluorescence staining revealed that gin4-Δ9 cells grown at various temperatures were also frequently abnormal in septin organization. In both diploid and haploid mutant strains, a fraction of cells (ranging from ~61% at 23°C to ~10% at 37°C) displayed approximately normal septin staining (Fig. ​(Fig.88 E, cell 3; compare to the wild-type cells in the inset and in Fig. ​Fig.5,5, C and F) (Table ​(TableV).V). However, other cells displayed one of several aberrant staining patterns. First, and most strikingly, many cells displayed a set of five to eight parallel “bars” of septin staining running through the neck along the mother–bud axis (Fig. ​(Fig.88 E, cells 1 and 2; Fig. ​Fig.88 F) (Table ​(TableV).V). Second, many cells displayed a band of septin staining in the neck whose endpoints were less well defined (“fuzzier”) than those in wild-type cells (Fig. ​(Fig.88 E, cell 4) (Table ​(TableV).V). Often (as in Fig. ​Fig.88 E, cell 4), it appeared that fuzzy bands of septin staining might actually be poorly resolved sets of bars. (Similarly, the higher ratio of fuzzy bands to bars scored in haploid cells, relative to diploid cells, may reflect, at least in part, a lower resolution of structures in the smaller necks of haploid cells.) Both fuzzy and bar septin staining patterns were observed both in morphologically abnormal cells and in cells of relatively normal morphology, suggesting that the abnormalities in septin organization are not simply a consequence of abnormal cell morphology. Necks with either fuzzy or bar septin staining appeared to contain amounts of septins comparable to those in wild-type necks, as judged by the intensity of immunofluorescence staining; thus, the absence of Gin4p appears to affect the organization of septins at the neck rather than their ability to localize to that part of the cell. Third, in some cells, septins were not detectable at the neck (Fig. ​(Fig.8,8, E, cell 5, and G, arrow); such cells were rare at 23°C but more common at higher temperatures (Table ​(TableV),V), and their overall morphology typically resembled that of temperature-sensitive septin mutants incubated at restrictive temperature (Hartwell, 1971; Adams and Pringle, 1984). The absence of detectable septin staining indeed appeared to correlate with a defect in cytokinesis: the cytoplasm appeared to be continuous through necks that were devoid of septin staining, and microtubules could span them (Fig. ​(Fig.88 H); moreover, the two or more nuclei in such cells were typically synchronized in the cell cycle (Fig. ​(Fig.88 I and data not shown). Occasionally, even necks with septin staining appeared to fail in cytokinesis or cell separation (Fig. ​(Fig.88 E, cell 6); in these cells, septin staining persisted at the neck(s) as if septin disassembly had been abnormally delayed.

Remarkably, however, in most cases, gin4-Δ9 bud necks with fuzzy or bar septin staining appeared to be competent to undergo cytokinesis. In gin4-Δ9/gin4-Δ9 diploid cells, at each temperature examined, the fraction of multibudded and/or multinucleate cells (indicative of failures of cytokinesis) was much less than the fraction of cells with fuzzy or bar septin staining (Table ​(TableVV).

To ask if the role of Gin4p in promoting normal septin organization at the neck depends on the Gin4p kinase activity, we examined cells of a gin4Δ strain that had been transformed with plasmids expressing either Gin4p-3HA or Gin4p^K48M-3HA. Although the former plasmid restored septin organization as effectively as did a plasmid expressing normal Gin4p, the plasmid expressing Gin4p^K48M-3HA only partially restored septin organization (Table ​(TableV).V). Similar results were obtained with a strain (M-692) expressing only Gin4p^K48A-3HA. Thus, the Gin4p kinase activity appears to promote, but not to be absolutely essential for, normal septin organization at the neck.

To investigate further the functional properties of the abnormally organized septins, we examined the localization and function of Bni4p and Bud4p, two proteins that localize to the neck in a septin-dependent manner (see Introduction). In wild-type cells, Bni4p is visualized as a sharp band on the mother-cell side of the neck (DeMarini et al., 1997) (Fig. ​(Fig.99 A). In contrast, in gin4-Δ9 cells, Bni4p was typically visualized as a fuzzy band spanning the neck (Fig. ​(Fig.99 B, arrowheads) (Table ​(TableV)V) or as a set of bars similar to the bars of septin staining (Fig. ​(Fig.99 B, arrows) (Table ​(TableV);V); moreover, in cells grown at 37°C, Bni4p was often undetectable (Table ​(TableV).V). At all temperatures tested, aberrant Bni4p organization was detected more frequently than was aberrant septin organization (Table ​(TableV),V), suggesting that subtle alterations of septin organization could have more pronounced (and/or more easily detected) effects on Bni4p localization or organization. Remarkably, the abnormally organized Bni4p appeared to be functional in assembling components of the chitin synthase III complex: at 23° or 30°C, most gin4-Δ9 cells displayed either fuzzy or bar Bni4p staining (Table ​(TableV),V), but nearly all cells deposited chitin primarily at the neck (Fig. ​(Fig.99 H) (Table ​(TableVI).VI). Strikingly, like Bni4p itself, the chitin was typically present on both sides of the neck (Fig. ​(Fig.99 H) (Table ​(TableVI),VI), rather than being restricted to the mother-cell side of the neck as in wild-type cells (Fig. ​(Fig.99 G) (Table ​(TableVI).VI).

Similar results were obtained with Bud4p. In wild-type cells, Bud4p localizes to the neck from G2 until the end of the cell cycle in a tight band that is symmetric on the mother and bud sides of the neck (Sanders and Herskowitz, 1996). In contrast, in haploid and diploid gin4-Δ9 cells, Bud4p was often visualized in fuzzy or bar patterns (Fig. ​(Fig.99 C and data not shown) whose frequencies were similar to those observed for Cdc11p (Table ​(TableV).V). The abnormally organized Bud4p appeared to be largely functional in axial bud-site selection: at 30°C, ~36% of the cells displayed Bud4p in a fuzzy or bar pattern (Table ​(TableV),V), whereas only ~6% of the cells budded in nonaxial patterns as judged by Calcofluor staining of bud scars (see Materials and Methods). Similarly, at 34°C, ~61% of the cells displayed Bud4p in a fuzzy or bar pattern (Table ​(TableV),V), whereas only ~15% of the cells budded in nonaxial patterns.

Interestingly, in cells undergoing polarized morphogenesis (shmooing) in response to mating pheromone, the septins are also typically visualized either as a fuzzy band (Kim et al., 1991; Ford and Pringle, 1991) or as a set of bars parallel to the projection axis (Fig. ​(Fig.9,9, D and E). This septin localization corresponds to that of the broad and somewhat fuzzy band of chitin deposited at the bases of the shmoo projections (Schekman and Brawley, 1979; Kim et al., 1991; Konopka et al., 1995) (Fig. ​(Fig.99 I), suggesting that in shmooing cells, as in vegetative cells (DeMarini et al., 1997), the septins serve as a template for the localization and/or assembly of Chs3p (Santos and Snyder, 1997) and the other components of the chitin synthase III complex. These observations raise the intriguing possibility that the absence of Gin4p in mutant vegetative cells might result in an altered organization of the septins that mimics what occurs normally in shmooing cells. Consistent with this hypothesis, Gin4p was not detectable by immunofluorescence in shmooing cells (Fig. ​(Fig.99 F), although the protein could be detected by Western blotting in such cells (Altman and Kellogg, 1997).

The protein kinase activity of Gin4p appears to be important for its positive role in septin function, as kinase-dead mutants of Gin4p can rescue neither the cdc12 single mutant at intermediate temperatures nor the synthetic lethality of the gin4 cdc12 double mutant, and strains expressing only a kinase-dead allele have many cells with conspicuously abnormal septin organization (Table ​(TableV).V). Moreover, the two-hybrid interaction observed was between the COOH-terminal region of Cdc3p and the kinase domain of Gin4p; this interaction appeared to depend on the presence of an intact kinase domain, as a construct lacking only the NH₂-terminal 17 amino acids of Gin4p failed to interact (Table ​(TableIV;IV; note the caveat that negative results in two-hybrid assays can have a variety of causes). As two-hybrid interactions have been detected between other protein kinases and their substrates (Yang et al., 1992; Staudinger et al., 1995; Cook et al., 1996) and Cdc3p appears to be phosphorylated in vivo (Healy, A.M., and J.R. Pringle, unpublished results; Reed, S.I., personal communication), it is possible that the COOH-terminal region of Cdc3p is a substrate of Gin4p; phosphorylation of the predicted coiled-coil domain in this region of Cdc3p might regulate possible homotypic interactions, interactions with other septins, or interactions with septin-associated proteins. However, further studies will be required to test these possibilities and to identify other possible substrates of Gin4p.

However, several lines of evidence suggest that Gin4p does not function only at the G2/M transition. First, consistent with the presence in its upstream region of apparent MCB (at −310 to −303 and −287 to −280) and SCB (at −249 to −243) sequences (Koch and Nasmyth, 1994; Breeden, 1996), GIN4 appears to be transcribed differentially early in the cell cycle (Okuzaki et al., 1997), like other genes whose products function in late G1 or S phase. (In this regard, however, we also note that a plasmid-borne GIN4 that has only 109 bp of upstream sequence, and hence lacks the MCB and SCB sequences, is able to provide GIN4 function; see plasmid pΔ110-1, Fig. ​Fig.11 A.) Second, GIN4 was also identified on the basis of the synthetic lethality of gin4 cln1 cln2 cells (Benton et al., 1997), suggesting a role for Gin4p early in the cell cycle when the Cdc28p/G1 cyclin complexes are active. Finally, Gin4p is already colocalized with the septins before bud emergence, and the abnormal organization of the septins in gin4Δ cells is evident even in cells with tiny buds, implying that Gin4p is involved in septin organization from the beginning of the cell cycle.

We thank D. Kellogg for plasmids and valuable discussions; J. Frazier (University of California, San Francisco, CA), C. Field (Harvard University Medical School, Boston, MA), H. Nojima, S. Tanaka, and D. Okuzaki (all three from Osaka University, Osaka, Japan) for valuable discussions; S. Hanks for valuable discussions and for performing the phylogenetic analysis shown in Fig. ​Fig.2;2; D. Levin (Johns Hopkins University, Baltimore, MD) for strains, plasmids, and helpful comments; E. Bi (University of Pennsylvania, Philadelphia, PA), D. DeMarini, and C. De Virgilio (Botanisches Institut, Universität Basel, Basel, Switzerland) for antibodies, plasmids, and valuable discussions; P. Hieter for a plasmid library; S. Sanders (Massachusetts Institute of Technology, Cambridge, MA) for antibodies; other members of our laboratory for encouragement and valuable discussions; and S. Whitfield (University of North Carolina, Chapel Hill, NC) for expert assistance with the illustrations.

This work was supported by a National Institutes of Health (NIH) grant to J.R. Pringle (GM-31006), by funds from the RJEG Trust, and by an NIH postdoctoral fellowship to M.S. Longtine (GM-15766).