Article |
Address correspondence to John R. Pringle, Dept. of Biology, CB #3280, 421 Fordham Hall, University of North Carolina, Chapel Hill, NC 27599. Tel.: (919) 962-2293. Fax: (919) 962-0320. E-mail: jpringle{at}email.unc.edu
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Abstract |
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Key Words: cell polarity; cell cycle; budding; septins; transmembrane proteins
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Introduction |
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In contrast, the bipolar pattern, as seen in normal diploid cells, appears to depend on persistent markers that are deposited at both the birth-scar-distal and birth-scar-proximal poles of the daughter cell, as well as at the division site on the mother cell (Chant and Pringle, 1995). These markers can direct bud formation to the marked site either in the next cell cycle or in a later one. A screen for mutants defective specifically in bipolar bud-site selection led to the identification of the BUD8 and BUD9 genes, whose mutant phenotypes suggest that they encode components of the markers at the distal and proximal poles of the daughter cell, respectively (Zahner et al., 1996; Harkins et al., 2001). The biochemical properties and localizations of Bud8p and Bud9p are consistent with this hypothesis. Both proteins appear to be integral membrane proteins of the plasma membrane, and each protein consists of a long, heavily glycosylated, NH2-terminal extracytoplasmic domain followed, at the COOH terminus, by two apparent transmembrane domains bracketing a cytoplasmic domain of 40 amino acids. The Bud8p and Bud9p cytoplasmic domains are very similar to each other in sequence and presumably provide the recognition sites for the Rsr1p/Bud2p/Bud5p GTPase signaling module, which appears to transmit the positional information from the axial and bipolar cortical markers to the proteins responsible for cell polarization (Pringle et al., 1995; Roemer et al., 1996b; Chant, 1999; Park et al., 1999; Kang et al., 2001; Marston et al., 2001). As expected (Chant and Pringle, 1995; Amberg et al., 1997), Bud8p appears to be localized primarily to the presumptive bud site, the distal pole of the bud, and the distal pole of the daughter cell (Taheri et al., 2000; Harkins et al., 2001). In addition, although Bud9p has also been reported to localize to the distal pole (Taheri et al., 2000), other data suggest strongly that it actually localizes primarily to the bud side of the mother-bud neck and thence to the proximal pole of the daughter cell (Harkins et al., 2001; this study).
In this study, we investigated when and how the very similar Bud8p and Bud9p proteins are localized to the opposite poles of the cell. We found that Bud8p is delivered to the bud site early in the cell cycle, whereas Bud9p is delivered to the neck just before cell division. The timing and site of localization of each protein appear to depend primarily on when in the cell cycle its gene is transcribed.
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Results |
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As in our previous studies, we observed GFPBud8p primarily at the tips of buds of various sizes and at the distal poles and nascent budding sites on unbudded cells (Fig. 1 A, cells 18). A detectable GFP-Bud8p signal was observed at the bud tip in 54% (n = 254) of budded cells, whereas a detectable signal was present at one pole in 44% (n = 234) of unbudded cells (61% of unbudded daughters; n = 200). Virtually no unbudded cells were observed with GFPBud8p signal at both poles, and the pole with signal was always the distal pole (Fig. 1 A, cells 58). Moreover, the unbudded cells with a GFPBud8p signal were essentially all newborn daughter cells (with no bud scars), a result that was expected given that daughter cells nearly always make their first buds at their distal poles (Chant and Pringle, 1995) and that the Bud8p at that pole appears to pass to the bud during bud emergence (Harkins et al., 2001). In addition, as observed previously, GFPBud8p was sometimes detected at the mother-bud neck (Fig. 1 A, cell 4). However, in contrast to our previous observations, such signal was now observed infrequently (13% of the 254 budded cells counted) and was always on the mother side, rather than the bud side, of the neck, consistent with the absence of unbudded daughter cells with GFPBud8p signal at their proximal poles. A signal was observed at the neck only in large-budded cells (25% of such cells), and was seen only in cells that also had a detectable patch of GFPBud8p at the bud tip.
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The absence of detectable GFPBud9p at the neck in small-budded cells suggested that Bud9p is delivered to the neck late in the cell cycle. To investigate this issue further, we first examined exponentially growing cells that had been stained with DAPI to reveal their nuclear morphology. In cells that had not completed anaphase, we never observed GFPBud9p at the neck (Fig. 2 D, cells 1 and 5). In contrast, 58% (n = 207) of cells that had completed anaphase had detectable GFPBud9p at the neck (Fig. 2 D, cells 24, 6, and 7). We then examined cells that had been arrested by a temperature-sensitive mutation in CDC15, whose product is a component of the mitotic exit network that controls exit from mitosis and entry into cytokinesis (Morgan, 1999; Lee et al., 2001; McCollum and Gould, 2001; Menssen et al., 2001). The arrested cells displayed no GFPBud9p at their necks (Fig. 2 E). However, within 45 min of return to permissive temperature, 68% (n = 201) of the cells displayed GFPBud9p at the bud side of the neck (Fig. 2 F). In a parallel experiment, cdc15 cells expressing GFPBud8p showed a broad patch of signal at the distal pole of the bud during arrest at restrictive temperature (Fig. 2 G). Upon return to permissive temperature, no concentration of GFPBud8p was observed at the neck before division (Fig. 2 H), although, as expected, tight patches of GFPBud8p were observed at the nascent bud sites in the following cell cycle (Fig. 2 I). Thus, most or all new Bud9p appears to be delivered to the neck very late in the cell cycle, just before cytokinesis, and the delivery of Bud8p and Bud9p appears to be under different cell cycle control.
Actin and septin dependence of Bud9p localization
As integral-membrane proteins, Bud8p and Bud9p are presumably delivered to the plasma membrane in vesicles derived from the secretory system. Both early in the cell cycle (when Bud8p appears to be delivered) and late in the cell cycle (when Bud9p appears to be delivered), polarized delivery of secretory vesicles to the cell surface appears to depend largely on the actin cytoskeleton (Lew and Reed, 1995; Drubin and Nelson, 1996; Pruyne and Bretscher, 2000a, 2000b). Consistent with this model, we observed previously that GFPBud8p delivery to presumptive bud sites was abolished in cells whose F-actin was ablated by treatment with Latrunculin A (Lat A) (Harkins et al., 2001). Similarly, treatment with Lat A also abolished the delivery of GFPBud9p to the neck late in the cell cycle (Fig. 3 A; in the DMSO-only control, 59% [n = 200] of the cells showed GFPBud9p at the neck), indicating that this delivery is also actin dependent. In addition, experiments with a temperature-sensitive septin mutant indicated that delivery of Bud9p to the neck is also septin dependent (Fig. 3 B). In contrast, delivery of Bud8p to presumptive bud sites and bud tips appeared to be unaffected by disruption of the septin/scaffold (Harkins et al., 2001).
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We next examined the function of the chimeric genes by evaluating their abilities to rescue the bud8 and bud9 mutant phenotypes. As expected, the absence of proximal-pole budding in a bud9/bud9 strain (Fig. 5, A, panel 1, and B, panel 1) could be rescued by a plasmid containing a normal BUD9 gene (Fig. 5 B, panel 2; this budding pattern was essentially the same as that in wild-type cells [Harkins et al., 2001]). In contrast, neither low-copy (unpublished data) nor high-copy (Fig. 5 B, panel 3) BUD8 expressed from its own promoter could rescue the bud9/bud9 mutant phenotype. Remarkably, however, low-copy plasmids expressing either untagged or tagged BUD8 from the BUD9 promoter could rescue proximal-pole budding in the mutant strain (Fig. 5, A, panel 2, and B, panels 4 and 6). In contrast, a plasmid expressing BUD9 itself from the BUD8 promoter could not rescue the mutant phenotype (Fig. 5 B, panel 5). As expected if expression in late G1 is critical for BUD9 function, either BUD8 or BUD9 could partially rescue the phenotype of the bud9/bud9 strain when expressed from the CLN2 promoter but not when expressed from the CLB2 promoter (Fig. 5 B, panels 710).
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Given the results just described, it was surprising that the reciprocal experiments presented a more complicated picture. As expected, the absence of distal-pole budding in a bud8/bud8 strain (Fig. 6, A, panel 1, and B, panel 1) could be rescued by a plasmid containing a normal BUD8 gene (Fig. 6 B, panel 2; this budding pattern was nearly the same as that in wild-type cells [Harkins et al., 2001]). In contrast, neither low-copy (unpublished data) nor high-copy (Fig. 6 B, panel 3) BUD9 expressed from its own promoter could rescue the bud8/bud8 mutant phenotype. However, although a plasmid expressing BUD8 from the BUD9 promoter also rescued distal-pole budding only poorly (Fig. 6 B, panel 5), plasmids expressing BUD9 from the BUD8 promoter (with either the BUD8 or BUD9 downstream sequences) were only slightly more effective (Fig. 6, A, panel 2, and B, panels 4 and 6). Moreover, although as expected, a plasmid expressing BUD8 from the CLB2 promoter rescued distal-pole budding well (Fig. 6 B, panel 7), and a plasmid expressing BUD9 from the CLN2 promoter showed no rescue (Fig. 6 B, panel 10), a plasmid expressing BUD9 from the CLB2 promoter showed only partial rescue of distal-pole budding (Fig. 6 B, panel 9) and was actually somewhat less effective than a plasmid expressing BUD8 from the CLN2 promoter (Fig. 6 B, panel 8). Although these results are difficult to interpret in detail, they do suggest that effective Bud8p function requires some aspects of the Bud8p protein that are not fully shared by Bud9p.
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Functional dissection of Bud8p
In an attempt to determine what portion(s) of Bud8p are necessary to have effective Bud8p function, we constructed several genes expressing chimeric Bud8p/Bud9p proteins from the BUD8 promoter and tested their abilities to rescue distal-pole budding in a bud8/bud8 mutant strain. Interestingly, a protein comprised of the extracytoplasmic domain of Bud8p and the transmembrane and cytoplasmic domains of Bud9p rescued the bud8/bud8 mutant phenotype essentially as well as did full-length Bud8p (Fig. 7 A; compare Fig. 6 B, panel 2), whereas the reciprocal construct rescued no better than did the expression of full-length Bud9p from the BUD8 promoter (Fig. 7 B; compare Fig. 6 B, panel 4). Thus, the transmembrane and cytoplasmic domains of Bud8p and Bud9p appear to be interchangeable, and the special feature(s) of Bud8p appear to lie in the extracytoplasmic domain. Analysis of additional chimeras suggested that both the NH2-terminal and COOH-terminal halves of the Bud8p extracytoplasmic domain contribute to Bud8p function (Fig. 7, C and D; compare and A and B, respectively) and that a region near the middle of the domain is important (Fig. 7 E) but not sufficient (Fig. 7 F) for effective Bud8p function.
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Discussion |
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The timing of gene expression appears to be a primary determinant of Bud8p and Bud9p localization and function: when Bud8p is expressed from the BUD9 promoter, it localizes as if it were Bud9p and appears fully competent to provide Bud9p (but not Bud8p) function. Moreover, when Bud9p is expressed from the BUD8 promoter, it localizes as if it were Bud8p and is unable to provide Bud9p function. However, under these conditions, the Bud9p localization signal appears weak, and the protein at the distal pole is only partially effective in providing Bud8p function. Thus, there appear to be features of the Bud8p polypeptide itself that are important for its efficient delivery, stability, and/or function at the distal pole. Although these features have not yet been defined in detail, experiments with chimeric proteins indicate that they lie primarily in the central and NH2-terminal portions of the extracytoplasmic domain. Because it seems likely that the extracellular domains of Bud8p and Bud9p must interact with components of the cell wall in order to maintain the localization of the proteins, it is possible that the special feature(s) of Bud8p allow it to interact effectively with some (unknown) component of the wall at the distal pole.
Consistent with the evidence for a role of cell cycleregulated expression, the BUD8 and BUD9 mRNAs show distinct peaks in abundance during the cell cycle (Cho et al., 1998; Spellman et al., 1998; this study). For several reasons, these peaks are likely to reflect cell cycleregulated transcription. First, this is the most common mechanism for cell cycle regulation of transcript levels (Spellman et al., 1998). Second, consistent with the apparent times of transcription of the two genes, the BUD9 upstream sequences contain potential binding sites for the MBF and SBF transcription factors, whereas the BUD8 upstream sequences contain potential binding sites for the MCM1 + SFF transcription factor (Spellman et al., 1998). Finally, despite the complications that might result from using promoters of different inherent strengths, expression of either Bud8p or Bud9p from the CLN2 promoter, which is known to drive G1 transcription (Breeden, 1996; Spellman et al., 1998), can provide partial Bud9p function, whereas expression of either protein from the CLB2 promoter, which is known to drive G2/M transcription (Spellman et al., 1998), can provide partial Bud8p function.
However, these results also raise an important question: Why are the BUD8 and BUD9 mRNAs expressed so far in advance of when the corresponding proteins appear to arrive at the cell surface? The experiments involving block and release by a temperature-sensitive mutation affecting the mitotic exit network are particularly intriguing: in the cdc15-arrested cells, GFPBud9p is not detectable at the cell surface, and GFPBud8p is present only as a diffuse patch at the distal pole of the bud (as would be expected from its appearance in large-budded cells during an unperturbed cell cycle). Upon release by return to permissive temperature, GFPBud9p appears at the bud side of the neck before cell division, whereas no detectable new delivery of GFPBud8p to the cell surface occurs until the beginning of the next cell cycle, when it appears at the nascent bud site in the usual way. The several possible explanations for these observations include cell cycleregulated delays in mRNA translation (conceivably coupled to a localization of the mRNAs themselves) or in the delivery of Bud8p- and Bud9p-containing secretory vesicles to the cell surface. We cannot yet discriminate between these (or perhaps other) explanations. In further studies of this issue, it will be necessary also to explain how starved cells (which are arrested in G0/G1) (Pringle and Hartwell, 1981) can deliver new Bud8p to the nascent bud site upon refeeding (Harkins et al., 2001) without passing through the stage of the cell cycle in which BUD8 appears to be transcribed.
A concern in these studies was whether a lag in the development of fluorescence by the GFP-tagged proteins might complicate the interpretation of the results. That is, it seemed possible that Bud8p and/or Bud9p might be translated and delivered to the plasma membrane immediately after the corresponding mRNA was expressed, but that the fluorescence of the tagged proteins might become detectable only later. However, such a model is difficult to reconcile with several features of the available data. First, to explain the appearance of new GFPBud8p at the nascent bud site just at the time of bud emergence, we would need to assume that protein synthesized in G2/M of one cell cycle could be delivered at that time to the precise spot that would be used for budding in the next cell cycle. This seems quite unlikely, particularly in a case like that of the random-budding rsr1 mutant. Second, the absence in the cdc15-arrested cells of any spot of GFPBud8p resembling those seen at new bud sites also appears to argue against the model, as does the appearance of GFPBud8p at the nascent bud site when starved cells are refed (Harkins et al., 2001). Third, the absence of GFPBud9p at the neck of cells arrested at the cdc15 block for 34 h (about twice the length of the normal cell cycle) is also difficult to reconcile with the model. Although this experiment could be misleading if the GFP has greater difficulty in achieving a fluorescent state at 37°C, we also observed that cells arrested in G2/M at 23°C by nocodazole and benomyl had no detectable GFPBud9p at the neck even after 3 h of incubation (unpublished data). Thus, it seems safe to conclude that there really is a substantial delay between the expression of the BUD8 and BUD9 mRNAs and the arrival of the proteins at the cell surface.
It has long been known that many yeast genes are expressed in a cell cycleregulated manner (Johnston, 1990; Breeden, 1996), and recent microarray experiments have indicated that the total of such genes is in the range of 416 (Cho et al., 1998) to 800 (Spellman et al., 1998). However, the functional significance of this regulation has been tested in relatively few cases, and in many cases in which tests have been done, eliminating the cell cycle regulation of transcription has had little or no effect on cell viability or function (Johnston, 1990; Breeden, 1996; Igual et al., 1996; Detweiler and Li, 1997). Thus, BUD8 and BUD9 provide important examples of cases in which the timing of transcription does appear to be critical for protein localization and function. Another important example, and an interesting comparison to the case of BUD8 and BUD9, is provided by Axl2p/Bud10p, an integral-membrane component of the cortical marker used in axial budding (Halme et al., 1996; Roemer et al., 1996a). Altering the normal late-G1 expression of AXL2 results in a severe disruption of protein localization and function, apparently because expression of the AXL2 mRNA is quickly followed by translation and the delivery of vesicles containing the newly made Axl2p to wherever the general secretory vesicle traffic of the cell is directed at that time in the cell cycle (Lord et al., 2000). Nonetheless, the transport of Axl2p to the cell surface appears to involve distinctive features, because it is affected much more than general secretory traffic by mutations affecting the ER-membrane protein Erv14p (Powers and Barlowe, 1998). Another interesting comparison is provided by the proteins involved in mating-type switching: cell cycle regulation of transcription of the HO gene is part of the complex mechanism (involving also the asymmetric localization of the mRNA for the transcriptional repressor Ash1p) by which the HO protein is localized specifically to the mother cell (Bobola et al., 1996; Sil and Herskowitz, 1996; Long et al., 2001; Maxon and Herskowitz, 2001; and references cited therein). It will be interesting to see if there are many other examples in which protein localization (and hence function) depends on the timing of gene expression, and, if so, which types of mechanisms are involved.
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Materials and methods |
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To synchronize cells late in the cell cycle using the cdc15-2 temperature-sensitive mutation, a culture growing exponentially at 23°C was shifted to 37°C and incubated for 3 to 4 h, at which point 98% of the cells had large buds. To release cells from the arrest, they were collected by centrifugation at 650 g for 5 min and resuspended in fresh medium at 23°C.
Plasmids used in this study are listed in Table II and/or described in the Supplemental materials and methods (available at http://www.jcb.org/cgi/content/full/jcb.200107041/DC1). Escherichia coli strains DH12S and DH5 (Life Technologies) were used for plasmid maintenance by standard procedures (Sambrook et al., 1989). Standard methods of DNA manipulation were used (Sambrook et al., 1989; Ausubel et al., 1995). PCR was performed using Taq DNA polymerase (Promega). Other enzymes were purchased from New England Biolabs, and oligonucleotide primers (Integrated DNA Technologies) are listed in Table III (Supplemental materials and methods, available at http://www.jcb.org/cgi/content/full/jcb.200107041/DC1). DNA sequencing was performed by the UNC-Chapel Hill Automated DNA Sequencing Facility.
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To construct a strain expressing the HA-BUD8 coding region from the chromosomal BUD9 promoter, plasmid YIpPBUD9HA-BUD8 was digested with HindIII (cutting the plasmid once, in the BUD9 upstream sequence) and transformed into bud9-1 strain LSY87. A Ura+ transformant showing restoration of bipolar budding was designated LSY305. PCR using DNA from LSY305 as template and primers LS157 and LS156 (one upstream of the HindIII site in the BUD9 upstream sequences, the other within the BUD8 ORF) confirmed that LSY305 had the desired structure at the BUD9 locus.
RNA methods
Yeast total RNA was isolated by the hot-phenol extraction method as described by Leeds et al. (1991). For Northern blotting, samples of total RNA were denatured by formamideformaldehyde treatment fractionated on 1% agarose/formaldehyde gels (Ausubel et al., 1995), and then transferred to Hybond-N+ membranes (Amersham Pharmacia Biotech). Hybridization of DNA probes to these RNA blots was essentially as described by Sambrook et al. (1989). To generate probes specific for BUD8, BUD9, CLN2, CLB2, HA, or ACT1, primer pairs LS93 and LS94, LS95 and LS96, LS112 and LS113, LS114 and LS115, LS158 and LS159, and LS183 and LS184, respectively, were used to amplify DNA fragments by PCR from templates YCpBUD8, YCpBUD9, pGAL-CLN2 (a gift from S. Reed via D. Lew [Duke University, Durham, NC]), pGAL-CLB2 (a gift from S. Reed via D. Lew), YCpHA-BUD9, and pRS316-GAL1-ACT1 (Liu et al., 1992), respectively. To allow normalization of the amounts of RNA per lane, a preliminary blot was probed for ACT1, and the signals were quantitated using a Storm 840 Phosphorimager and Imagequant 5.0 software (Molecular Dynamics).
Staining and microscopy
To visualize bud scars and birth scars for the determination of budding patterns, exponentially growing cells were suspended in water containing 1 µg/ml Calcofluor as described by Zahner et al. (1996) and examined on a Nikon Microphot SA microscope using an Apo 60X/1.40 NA oil-immersion objective. To visualize GFPBud8p or GFPBud9p in living cells, cells were grown to exponential phase in the dark, collected by centrifugation at 23°C, and suspended in water or in water containing 1 µg/ml Calcofluor. To visualize nuclear DNA, the exponentially growing cells were stained by adding DAPI (Sigma-Aldrich) from a 1-mg/ml stock solution in water to a final concentration of 1 µg/ml and continuing to incubate the culture for 90 min before resuspending the cells in water for examination. To prepare slides for time-lapse microscopy, cells growing exponentially in SC-Leu medium were pipetted onto slabs of 2x SC medium (like SC but with all ingredients at twice normal concentration; 2% glucose as carbon source) containing 25% gelatin, and coverslips were applied and sealed with Valap (Salmon et al., 1998). All images for figures were collected using Metamorph software (Universal Imaging Corporation) with a Nikon Eclipse 600 FN microscope equipped with a Hamamatsu ORCA-2 CCD camera and an Apo 100X/1.40 NA oil-immersion objective. Exposure times were set automatically and averaged 6 s for GFP images except for those of cells expressing GFPBud9p from YEpPBUD8GFP*BUD9, which averaged
15 s. Exposure times for Calcofluor images averaged
0.2 sec.
Online supplemental material
The online supplement (available at http://www.jcb.org/cgi/content/full/jcb.200107041/DC1) contains the details of plasmid constructions and a table of oligonucleotide primers used.
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Footnotes |
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N. Pagé's present address is Swiss Institute for Experimental Cancer Research, 1066 Epalinges/VD, Switzerland.
* Abbreviations used in this paper: GFP, green fluorescent protein; Lat A, Latrunculin A.
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Acknowledgments |
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This work was supported by National Institutes of Health Grant GM31006.
Submitted: 10 July 2001
Revised: 18 January 2002
Accepted: 23 January 2002
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References |
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---|
Amberg, D.C., J.E. Zahner, J.W. Mulholland, J.R. Pringle, and D. Botstein. 1997. Aip3p/Bud6p, a yeast actin-interacting protein that is involved in morphogenesis and the selection of bipolar budding sites. Mol. Biol. Cell. 8:729753.[Abstract]
Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl. 1995. Current Protocols in Molecular Biology. John Wiley & Sons, New York.
Bender, A., and J.R. Pringle. 1989. Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSR1. Proc. Natl. Acad. Sci. USA. 86:99769980.[Abstract]
Bi, E., and J.R. Pringle. 1996. ZDS1 and ZDS2, genes whose products may regulate Cdc42p in Saccharomyces cerevisiae. Mol. Cell. Biol. 16:52645275.[Abstract]
Breeden, L. 1996. Start-specific transcription in yeast. Curr. Top. Microbiol. Immunol. 208:95127.[Medline]
Chant, J., and I. Herskowitz. 1991. Genetic control of bud site selection in yeast by a set of gene products that constitute a morphogenetic pathway. Cell. 65:12031212.[Medline]
Chant, J., and J.R. Pringle. 1995. Patterns of bud-site selection in the yeast Saccharomyces cerevisiae. J. Cell Biol. 129:751765.[Abstract]
Chant, J., M. Mischke, E. Mitchell, I. Herskowitz, and J.R. Pringle. 1995. Role of Bud3p in producing the axial budding pattern of yeast. J. Cell Biol. 129:767778.[Abstract]
Chen, T., T. Hiroko, A. Chaudhuri, F. Inose, M. Lord, S. Tanaka, J. Chant, and A. Fujita. 2000. Multigenerational cortical inheritance of the Rax2 protein in orienting polarity and division in yeast. Science. 290:19751978.
Detweiler, C.S., and J.J. Li. 1997. Cdc6p establishes and maintains a state of replication competence during G1 phase. J. Cell Sci. 110:753763.
Gietz, R.D., and A. Sugino. 1988. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 74:527534.[CrossRef][Medline]
Gladfelter, A.S., J.R. Pringle, and D.J. Lew. 2001. The septin cortex at the yeast mother-bud neck. Curr. Opin. Microbiol. 4:681689.[CrossRef][Medline]
Guthrie, C., and G.R. Fink. 1991. Guide to Yeast Genetics and Molecular Biology. Methods Enzymol. Vol. 194. Academic Press, San Diego. 933 pp.
Harkins, H.A., N. Pagé, L.R. Schenkman, C. De Virgilio, S. Shaw, H. Bussey, and J.R. Pringle. 2001. Bud8p and Bud9p, proteins that may mark the sites for bipolar budding in yeast. Mol. Biol. Cell. 12:24972518.
Hicks, J.B., J.N. Strathern, and I. Herskowitz. 1977. Interconversion of yeast mating types. III. Action of the homothallism (HO) gene in cells homozygous for the mating type locus. Genetics. 85:395405.
Igual, J.C., A.L. Johnson, and L.H. Johnston. 1996. Coordinated regulation of gene expression by the cell cycle transcription factor SWI4 and the protein kinase C MAP kinase pathway for yeast cell integrity. EMBO J. 15:50015013.[Abstract]
Kang, P.J., A. Sanson, B. Lee, and H.-O. Park. 2001. A GDP/GTP exchange factor involved in linking a spatial landmark to cell polarity. Science. 292:13761378.
Leeds, P., S.W. Peltz, A. Jacobson, and M.R. Culbertson. 1991. The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon. Genes Dev. 5:23032314.[Abstract]
Lillie, S.H., and J.R. Pringle. 1980. Reserve carbohydrate metabolism in Saccharomyces cerevisiae: responses to nutrient limitation. J. Bacteriol. 143:13841394.[Medline]
Liu, H., J. Krizek, and A. Bretscher. 1992. Construction of a GAL1-regulated yeast cDNA expression library and its application to the identification of genes whose overexpression causes lethality in yeast. Genetics. 132:665673.
Long, R.M., W. Gu, X. Meng, G. Gonsalvez, R.H. Singer, and P. Chartrand. 2001. An exclusively nuclear RNA-binding protein affects asymmetric localization of ASH1 mRNA and Ash1p in yeast. J. Cell Biol. 153:307318.
Lord, M., M.C. Yang, M. Mischke, and J. Chant. 2000. Cell cycle programs of gene expression control morphogenetic protein localization. J. Cell Biol. 151:15011511.
Marston, A.L., T. Chen, M.C. Yang, P. Belhumeur, and J. Chant. 2001. A localized GTPase exchange factor, Bud5, determines the orientation of division axes in yeast. Curr. Biol. 11:803807.[CrossRef][Medline]
Maxon, M.E., and I. Herskowitz. 2001. Ash1p is a site-specific DNA-binding protein that actively represses transcription. Proc. Natl. Acad. Sci. USA. 98:14951500.
Menssen, R., A. Neutzner, and W. Seufert. 2001. Asymmetric spindle pole localization of yeast Cdc15 kinase links mitotic exit and cytokinesis. Curr. Biol. 11:345350.[CrossRef][Medline]
Mösch, H.-U., and G.R. Fink. 1997. Dissection of filamentous growth by transposon mutagenesis in Saccharomyces cerevisiae. Genetics. 145:671684.
Oldenburg, K.R., K.T. Vo, S. Michaelis, and C. Paddon. 1997. Recombination-mediated PCR-directed plasmid construction in vivo in yeast. Nucleic Acids Res. 25:451452.
Park, H.-O., A. Sanson, and I. Herskowitz. 1999. Localization of Bud2p, a GTPase-activating protein necessary for programming cell polarity in yeast to the presumptive bud site. Genes Dev. 13:19121917.
Powers, J., and C. Barlowe. 1998. Transport of Axl2p depends on Erv14p, an ER-vesicle protein related to the Drosophila cornichon gene product. J. Cell Biol. 142:12091222.
Pringle, J.R., and L.H. Hartwell. 1981. The Saccharomyces cerevisiae cell cycle. The Molecular Biology of the Yeast Saccharomyces. Life Cycle and Inheritance. Strathern, J.N., E.W. Jones, and J.R. Broach, editors. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 97142.
Pruyne, D., and A. Bretscher. 2000a. Polarization of cell growth in yeast. I. Establishment and maintenance of polarity states. J. Cell Sci. 113:365375.
Pruyne, D., and A. Bretscher. 2000b. Polarization of cell growth in yeast. II. The role of the cortical actin cytoskeleton. J. Cell Sci. 113:571585.
Roemer, T., K. Madden, J. Chang, and M. Snyder. 1996a. Selection of axial growth sites in yeast requires Axl2p, a novel plasma membrane glycoprotein. Genes Dev. 10:777793.[Abstract]
Salmon, E.D., E. Yeh, S. Shaw, B. Skibbens, and K. Bloom. 1998. High-resolution video and digital-enhanced differential interference contrast light microscopy of cell division in budding yeast. Methods Enzymol. 298:317331.[Medline]
Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Sanders, S.L., and I. Herskowitz. 1996. The Bud4 protein of yeast, required for axial budding, is localized to the mother/bud neck in a cell cycle-dependent manner. J. Cell Biol. 134:413427.[Abstract]
Sanders, S.L., M. Gentzsch, W. Tanner, and I. Herskowitz. 1999. O-glycosylation of Axl2p/Bud10p by Pmt4p is required for its stability, localization, and function in daughter cells. J. Cell Biol. 145:11771188.
Sikorski, R.S., and P. Hieter. 1989. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics. 122:1927.
Spellman, P.T., G. Sherlock, M.Q. Zhang, V.R. Iyer, K. Anders, M.B. Eisen, P.O. Brown, D. Botstein, and B. Futcher. 1998. Comprehensive identification of cell cycle-regulated genes of the yeast Saccharomyces cerevisiae by microarray hybridization. Mol. Biol. Cell. 9:32733297.
Taheri, N., T. Köhler, G.H. Braus, and H.-U. Mösch. 2000. Asymmetrically localized Bud8p and Bud9p proteins control yeast cell polarity and development. EMBO J. 19:66866696.
Zahner, J.E., H.A. Harkins, and J.R. Pringle. 1996. Genetic analysis of the bipolar pattern of bud site selection in the yeast Saccharomyces cerevisiae. Mol. Cell. Biol. 16:18571870.[Abstract]
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