Regulation of PHO4 nuclear localization by the PHO80-PH085 cyclin-CDK complex

Science

Volumn 271, Issue 5246, 1996, Pages 209-212

  O'Neill, Elizabeth M ^a   Kaffman, Arie ^a   Jolly, Emmitt R ^a   O'Shea, Erin K ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

CYCLIN DEPENDENT KINASE; PHOSPHATE; TRANSCRIPTION FACTOR;

ARTICLE; CELL NUCLEUS; CELLULAR DISTRIBUTION; CONTROLLED STUDY; CYTOPLASM; NONHUMAN; PRIORITY JOURNAL; PROTEIN PHOSPHORYLATION; TRANSCRIPTION TERMINATION; YEAST;

AMINO ACID SEQUENCE; CELL NUCLEUS; CULTURE MEDIA; CYCLIN-DEPENDENT KINASES; CYCLINS; CYTOPLASM; DIPEPTIDES; DNA-BINDING PROTEINS; FUNGAL PROTEINS; GENE EXPRESSION REGULATION, FUNGAL; MEMBRANE TRANSPORT PROTEINS; MOLECULAR SEQUENCE DATA; MUTATION; PHOSPHATE TRANSPORT PROTEINS; PHOSPHATES; PHOSPHORYLATION; REPRESSOR PROTEINS; SACCHAROMYCES CEREVISIAE; SACCHAROMYCES CEREVISIAE PROTEINS; TRANSCRIPTION FACTORS;

EID: 0030059223     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.271.5246.209     Document Type: Article

References (34)

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8. SA4 (Fig. 2C). Loss of these two peptides is probably a result of incomplete trypsin digestion, because the seven trypsin cleavage sites COOH-terminal to Ser-Pro dipeptide 4 are all in amino acid contexts that are not efficiently cleaved by trypsin (16)
9. SA1 was uninformative because of anomalous migration of the phosphopeptide containing Ser-Pro dipeptide 1 or insolubility of this particular peptide in the buffer used to prepare the samples (16).
10. Sennes 100, 114, 128, 152, and 223 of PHO4 are phosphorylated by PHO80-PHO85. Four of the five senne residues phosphorylated by PHO80-PHO85 are found in a similar amino acid context, conforming to the consensus Ser-Pro-X-lle/Leu. The fifth site has a threonine instead of a hydrophobic amino acid at the third residue following the phosphoacceptor
11. E. K O'Shea, D. A Jeffery, D. King, unpublished data.
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17. 600 of cells Spheroplasts were placed on glass microscope slides that were first treated with poly-L-lysine (1 mg/ml) for 5 min, washed twice with water, and air-dried Attached spheroplasts were treated with blocking buffer (PBS + 0.1% Triton X-100, 1% bovine serum albumin) for 30 min at room temperature and incubated with preadsorbed primary antibody (diluted 1.200 in blocking buffer) overnight at 4°C. Spheroplasts were then washed three times with PBS, incubated with preadsorbed secondary antibody (diluted 1:200 in blocking buffer) for 1 hour at room temperature, washed three times with PBS, and mounted in Fluoromount G (Southern Biotechnology Associates, Inc.) + 4′,6′ diamidino-2-phenylindole dihydrochlonde (DAPI; 22.5 ng/ml). Primary antibody was polyclonal rabbit antiserum directed against PHO4 protein (2). Secondary antibody was rhodamine-conjugated goat antibody to rabbit immunoglobulin G (Boehringer Mannheim). Both secondary and primary antibodies were preadsorbed as described (30) with spheroplasts of either a wild-type strain or a strain deleted for the PHO4 gene, respectively.
18. The wild-type strain used in this study was K699 MATa ade2-1 trpl-1 can1-100 leu2-3,112 his3-11, 15 ura3 (30). PHO4, PHO8O, PHO81. and PHO85 were each deleted in this strain by standard gene replacement techniques [R. Rothstein, Methods Enzymol. 194, 281 (1991)] to generate strains EY0130 (pho4△::TRP1), EY0134 (pho80△::HIS3), EY0150 (pho81△::TRP1), and EY0140 (pho85△::LEU2), respectively
19. 600 of 0 5 to 1.5 before analysis.
20. ST6 (26) were used to express the mutant versions of PHO4 in Escherichia coli. Phosphoamino acid analysis was performed as described (16)
21. Abbreviations for the amino acid residues are as follows: A, Ala, C, Cys; D, Asp, E, Glu; F, Phe; G, Gly; H, His; I, lie; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S. Ser, T, Thr; V, Val; W, Trp; and Y, Tyr.
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24. SA12346 (26) were used to express the mutant versions of PH04 in E coli
25. SA12346.
26. SA12346 under the control of the PHO4 promoter.
27. Each strain was patched onto a plate containing standard SC medium (31) and allowed to grow over-night at 30°C. This plate was then replica-plated, both back onto standard SC medium and onto phosphate-depleted medium (32) These two plates were allowed to grow at 3o°C for 12 hours and then were overlaid with agar contaning a chromagenic phosphatase substrate (34).
28. SA12346 = 22 ± 5.6 For comparison, the acid phosphatase activity of a pho80△ strain is 51.3 ± 10.2 in high phosphate medium and 59.9 ± 14.8 in low phosphate medium.
29. K. Nasmyth, G. Adolf, D. Lydall, A. Seddon, Cell 62, 631 (1990).
30. F. Sherman, Methods Enzymol 194, 3 (1991).
31. K. F. O'Connell and R. E. Baker, Genetics 132, 63 (1992).
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34. SA1-6 mutant, S. Sanders and N. Valtz for advice regarding immunofluorescence, Y. Oshima for providing plasmids; and I Herskowitz, M. Lenburg, M. Peter, M. Maxon, D. Jeffery, R. Tjian, and K. Schneider for comments on the manuscript. E.M.O was supported by a National Institutes of Health (NIH) postdoctoral training grant awarded to the Department of Biochemistry and Biophysics at the University of California, San Francisco (UCSF) and by the Jane Coffin Childs Memorial Fund for Medical Research Support for this work was provided to EKO by the NIH, the Lucille P Markey Foundation, the David and Lucile Packard Foundation, and the UCSF Simon Fund.