Transcriptional regulation of meiosis in budding yeast

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the established model, however, the ultimate proof would be the demonstration that Ime1 is present on the complex formed on URS1, and/or that it associates with the transcription machinery. Both models assume that since Ime1 functions through Ume6, deletions of either one of these genes would have a similar phenotype in meiotic cultures. However, ime1∆ diploids arrest in G1, whereas ume6∆ cells mainly arrest at G2. We assume that the expression of EMG in vegetative cultures promotes the entry and progression through some meiotic events. Ime1 has at least two functions, it supplies a transcriptional activation domain, but it is also required to relieve repression of Sin3/Rpd3 (Washburn and Esposito, 2001). Diploid cells deleted for IME1 and expressing Gal4(ad)-Ume6 from the ADH1 promoter are sporulation deficient (Washburn and Esposito, 2001), but the Gal4(ad)-Ume6(M530T) mutant protein promotes low levels of sporulation (10%). Since the latter protein is defective in association with Sin3, it was concluded that Ime1 is required to relieve repression mediated by the Sin3/Rpd3 complex (Washburn and Esposito, 2001). The interaction between Ime1 and Ume6 is regulated by nutrients, Rim11 and Rim15. Since two-hybrid assays were used to determine this interaction, the regulated expression of the reporter genes might result from regulation at any of the following levels: i. Regulated physical association between Ime1 and Ume6, or ii. Regulated transcriptional activation by the Ume6/Ime1 complex. The latter model assumes that Ime1 and Ume6 physically associate under all growth conditions, but in glucose growth media the resulting complex does not function as a transcriptional activator. This hypothesis is supported by the following results. i. In vegetative growth media association of Ume6 with the Sin3/Rpd3 complex represses transcription (Kadosh and Struhl, 1997). ii. A point mutation in Ume6 that prevents its association with Sin3 promote transcriptional activation when the mutant protein is fused to Gal4(ad) (Washburn and Esposito, 2001). Direct demonstration of regulated (or not) physical association between Ime1 and Ume6 is required to distinguish between these models. In accord with the role of Ime1 as a transcriptional activator, the protein is localized to the nucleus (Colomina et al., 1999; Rubin-Bejerano et al., 1996; Smith et al., 1993). Localization of Ime1 is independent of Rim11 (Rubin-Bejerano et al., 1996), but as described above, it is inhibited by the Cln/Cdc28 function (Colomina et al., 1999). Fig. 10 schematically illustrates how nutrients control the availability and activity of Ime1 25
3. Proteins required for the association of Ime1 with Ume6. 3.1. The function of Rim11 and its homologs Mck1 and Mrk1. Rim11 is absolutely required for the interaction between Ime1 and Ume6 (Colomina et al., 1999; Rubin-Bejerano et al., 1996), the expression of EMG, and spore formation (Bowdish et al., 1994; Rubin-Bejerano et al., 1996). Rim11 associates with Ime1(id) under all growth conditions (Bowdish et al., 1994; Rubin-Bejerano et al., 1996), and in vitro it phosphorylates both Ime1 (Bowdish et al., 1994) and Ume6 (Malathi et al., 1997). The Rim11 homologs Mck1 and Mrk1 have only minor effects on these processes (Neigeborn and Mitchell, 1991; Rabitsch et al., 2001). The transcription of RIM11 is constitutive in vegetative and meiotic conditions; however, in ime1∆ cells its expression in meiotic cultures is reduced, probably reflecting the use of the URS1 like site present in its promoter (Bowdish et al., 1994). The level of Rim11 protein is slightly reduced in glucose growth media in comparison to acetate (Bowdish et al., 1994). Rim11 functions as a kinase that shows autophosphorylation (Bowdish et al., 1994). Rim11 is phosphorylated on tyrosine 199 (Zhan et al., 2000). Tyrosine to phenylalanine mutation in this residue results in impaired expression of ime2-lacZ in vivo, and in a defect in phosphorylation Ume6 in vitro (Zhan et al., 2000). This mutation does not impair autophosphorylation (Zhan et al., 2000), suggesting different requirement for phosphorylation of exogenous substrates (see section IIID1). Rim11 associates with the C-terminal domain of Ime1, amino acids 270-360 (Malathi et al., 1999; Rubin-Bejerano et al., 1996). In vitro, Rim11 phosphorylates the C-terminal 20 amino acids residues in this region of Ime1 (Malathi et al., 1999). Simultaneous mutations of 8 serine threonine and tyrosine of these residues have the following effects: i. In vitro phosphorylation of Ime1 by Rim11 is absent, ii. Ime1 does not interact with Ume6, iii. IME2 is not expressed, and iv. Asci are not formed (Malathi et al., 1999). Ime1 is detected by antibodies directed against phosphotyrosines, and tyrosine to phenylalanine mutation in this domain results in impaired expression of ime2-lacZ in vivo, suggesting that Rim11 phosphorylates these tyrosine resides in Ime1. Rim11 controls meiosis by regulating not only Ime1, but also Ume6. Using the two-hybrid method, coIP, and in vitro kinase assay, Malathi et al show that Rim11 physically associates with Ume6, it phosphorylates Ume6(1-232), and the association between Rim11 and Ume6 is independent of Ime1 (Malathi et al., 1997). In addition, the two-hybrid assay reveals interaction between Ume6 and Mck1 (Xiao and Mitchell, 2000). In vivo, Ume6 is a phosphoprotein, whose level of phosphorylation is increased in vegetative growth media with acetate as the sole carbon source in comparison to glucose (Xiao and Mitchell, 2000). Moreover, hyper phosphorylation is 26
Page 1 and 2: Transcriptional regulation of meios
Page 3 and 4: 3. Proteins required for the associ
Page 5 and 6: I. Introduction The budding yeast S
Page 7 and 8: et al., 2000). On the other hand, i
Page 9 and 10: the UCS4 element upstream of the CY
Page 11 and 12: egulator of Cyr1, CDC25 encodes the
Page 13 and 14: 1998). Furthermore, unlike IREu, IR
Page 15 and 16: In the absence of glucose Sok2 does
Page 17 and 18: autophosphorylation, including on a
Page 19 and 20: hours in SPM, followed by a decline
Page 21 and 22: 4. The SIN3, UME6, and RPD3 genes.
Page 23 and 24: 1993). Nevertheless, when Ime1 is t
Page 25: However, direct demonstration of ph
Page 29 and 30: heterologous promoter does not supp
Page 31 and 32: expressed only in the absence of gl
Page 33 and 34: structure of hCDK2 reveals that cyc
Page 35 and 36: terminal non-essential domain (Komi
Page 37 and 38: is dependent on Ime2, but in cells
Page 39 and 40: VI. Transcriptional regulation of l
Page 41 and 42: The transcriptional activator that
Page 43 and 44: Functional interaction between Ime1
Page 45 and 46: Bowdish, K. S., and Mitchell, A. P.
Page 47 and 48: Foiani, M., Nadjar-Boger, E., Capon
Page 49 and 50: Jeffrey, P. D., Russo, A. A., Polya
Page 51 and 52: Lindgren, A., Bungard, D., Pierce,
Page 53 and 54: Pazin, M. J., and Kadonaga, J. T. (
Page 55 and 56: Shenhar, G. (2001). TRANSCRIPTIONAL
Page 57 and 58: Szent-Gyorgyi, C. (1995). A biparti
Page 59 and 60: Yoshida, M., Kawaguchi, H., Sakata,
Page 61 and 62: Fig. 4. The function of positive an
Page 63 and 64: Fig. 9. A schematic structure of IM
Page 65 and 66: activation by Ime2 is due to phosph
Page 67 and 68: MCK1 Protein kinase required for ef
Page 69: UME3 A cyclin C homolog that associ

Transcriptional regulation of meiosis in budding yeast

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