Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2001 Nov;12(11):3589-600.
doi: 10.1091/mbc.12.11.3589.

Enhanced cell polarity in mutants of the budding yeast cyclin-dependent kinase Cdc28p

S H Ahn  1 , B T Tobe, J N Fitz Gerald, S L Anderson, A Acurio, S J Kron
Affiliations
Free PMC article

Enhanced cell polarity in mutants of the budding yeast cyclin-dependent kinase Cdc28p

S H Ahn et al. Mol Biol Cell. 2001 Nov.
Free PMC article

Abstract

The yeast cyclin-dependent kinase Cdc28p regulates bud morphogenesis and cell cycle progression via the antagonistic activities of Cln and Clb cyclins. Cln G1 cyclins direct polarized growth and bud emergence, whereas Clb G2 cyclins promote isotropic growth of the bud and chromosome segregation. Using colony morphology as a screen to dissect regulation of polarity by Cdc28p, we identified nine point mutations that block the apical-isotropic switch while maintaining other functions. Like a clb2 Delta mutation, each confers tubular bud shape, apically polarized actin distribution, unipolar budding, and delayed anaphase. The mutations are all suppressed by CLB2 overexpression and are synthetically lethal with a CLB2 deletion. However, defects in multiple independent pathways may underlie their common phenotype, because the mutations are scattered throughout the CDC28 sequence, complement each other, and confer diverse biochemical properties. Glu12Gly, a mutation that alters a residue involved in Swe1p inhibition of Cdc28p, was unique in being suppressed by deficiency of SWE1 or CLN1. With wild-type CDC28, filament formation induced by CLN1 overexpression was markedly decreased in a SWE1 deletion. These results suggest that Swe1p, via inhibition of Clb2p/Cdc28p, may mediate much of the effect of Cln1p on filamentous morphogenesis.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Mutations in CDC28 that confer enhanced polar morphogenesis. Photomicrographs are of homozygous mutant cells grown to exponential phase in YPD liquid at 30°C. Bar, 50 μm.
Figure 2
Figure 2
Structural analysis of the cdc28ECP mutants. (A) Cdc28p and human CDK2 sequences aligned by Clustal (Higgins et al., 1996). Boxes delineate consensus kinase subdomains (Hanks et al., 1988) and bars decorate residues altered in cdc28-4, cdc28-1N, and the cdc28ECP mutants. (B) The residues altered in cdc28-4, cdc28-1N, and the cdc28ECP alleles are depicted on a ribbon diagram of Cdc28p modeled after CDK2 in active complex with cyclin A. The PSTAIRE helix is highlighted in red, the inhibitory T-loop in green, and cyclin A in gray. (C) Glu89Lys, Ala55Thr, and Ile59Asn, the mutations in or near PSTAIRE, are shown modeled into the inactive, monomeric form of the enzyme. The view shows the PSTAIRE helix (red) from the perspective of an “approaching” cyclin A. (D) The residues mutated in Leu137Ile, Glu12Gly, and Val21Asp are decorated in a model of Cdc28p with peptide substrate bound. The view shows the ATP as a stick diagram, PSTAIRE in red, the T loop in green, and the substrate peptide HHASPRK (Brown et al., 1999) in gold.
Figure 3
Figure 3
Similar cell cycle kinetics but distinct enzymatic properties of cdc28ECP mutants. Wild-type strain SKY757 and diploids homozygous for the indicated mutations were grown to exponential phase and analyzed as described in MATERIALS AND METHODS. (A) The cdc28ECP mutations confer a G2/M cell cycle shift. Flow cytometry of asynchronous cultures revealed a shift to higher DNA content, and microscopic examination revealed a high percentage of preanaphase (Pre-ana) cells. (B) The cdc28ECP mutations do not alter the expression of key cell cycle regulators. Total RNA was subjected to Northern analysis with the indicated probes. Expression levels were quantitated by phosphorimager analysis and are indicated as the relative ratios of expression of each transcript normalized to the level of ACT1. CLN1 and CLN2 yielded similar results. (C) Western analysis shows that the cdc28ECP alleles do not decrease Cdc28p or Clb2p abundance. (D) The cdc28ECP mutant proteins exhibit diverse enzymatic properties. Whole-cell extracts of the indicated strains were analyzed for p13suc1- and Clb2p-associated histone H1 kinase activity.
Figure 4
Figure 4
Complementation behavior of cdc28ECP mutations. Representative photomicrographs demonstrate cell morphology of homozygous and heteroallelic diploids after 24 h of incubation at 22°C on YPD agar. Polarization indices (+/− to ++++) and G2/G1 ratios (numbers) were determined as described in MATERIALS AND METHODS. Bar, 50 μm.
Figure 5
Figure 5
Modulation of polarized growth by CLB2, CLN1, SWE1. (A) Homozygous diploid shuffle strains of the indicated genotypes expressing the indicated CDC28 alleles were incubated on YPD or YPG agar for 24 h at 22°C. The Phe202Tyr mutant shown is representative of the cdc28ECP alleles other than Glu12Gly. (B) Wild-type or swe1Δ mutant diploid cells carrying integrated GAL-CLN1 or GAL-CLN2 constructs were examined for polarized growth on synthetic medium containing galactose. Bar, 50 μm.
Figure 6
Figure 6
Regulation of yeast cell polarity by Cdc28p. Previous work has established antagonistic functions of Cln and Clb cyclins in morphogenesis. The polarization of cortical actin patches (black dots) and cell wall deposition to the nascent bud site at Start is Cln dependent, whereas the apical-isotropic switch leading to redistribution of actin and swelling growth of the bud in mitosis is Clb dependent. The results of this study suggest that the Cln cyclins may maintain polar growth and prevent the apical-isotropic switch by activating Swe1p, a tyrosine kinase that inhibits Clb-Cdc28p complexes.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Ahn SH, Acurio A, Kron SJ. Regulation of G2/M progression by the STE mitogen-activated protein kinase pathway in budding yeast filamentous growth. Mol Biol Cell. 1999;10:3301–3316. - PMC - PubMed
    1. Amberg DC, Basart E, Botstein D. Defining protein interactions with yeast actin in vivo. Nat Struct Biol. 1995;2:28–35. - PubMed
    1. Amon A, Irniger S, Nasmyth K. Closing the cell cycle circle in yeast: G2 cyclin proteolysis initiated at mitosis persists until the activation of G1 cyclins in the next cycle. Cell. 1994;77:1037–1050. - PubMed
    1. Blacketer MJ, Madaule P, Myers AM. Mutational analysis of morphologic differentiation in Saccharomyces cerevisiae. Genetics. 1995;140:1259–1275. - PMC - PubMed
    1. Brown NR, Noble ME, Endicott JA, Johnson LN. The structural basis for specificity of substrate and recruitment peptides for cyclin-dependent kinases. Nat Cell Biol. 1999;1:438–443. - PubMed

Publication types

MeSH terms

LinkOut - more resources