The F-Box Protein Met30 Is Required for Multiple Steps in the Budding Yeast Cell Cycle

Abstract

The Saccharomyces cerevisiae ubiquitin ligase SCF Met30 is essential for cell cycle progression. To identify and characterize SCF Met30 -dependent cell cycle steps, we used temperature-sensitive met30 mutants in cell cycle synchrony experiments. These experiments revealed a requirement for Met30 during both G 1 /S transition and M phase, while progression through S phase was unaffected by loss of Met30 function. Expression of the G 1 -specific transcripts CLN1, CLN2, and CLB5 was very low in met30 mutants, whereas expression of CLN3 was unaffected. However, overexpression of Cln2 could not overcome the G 1 arrest. Interestingly, overexpression of Clb5 could induce DNA replication in met30 mutants, albeit very inefficiently. Increased levels of Clb5 could not, however, suppress the cell proliferation defect of met30 mutants. Consistent with the DNA replication defects, chromatin immunoprecipitation experiments revealed significantly lower levels of the replication factors Mcm4, Mcm7, and Cdc45 at replication origins in met30 mutants than in wild-type cells. These data suggest that Met30 regulates several aspects of the cell cycle, including G 1 -specific transcription, initiation of DNA replication, and progression through M phase.

Downregulation of Cdc6 and pre-replication complexes in response to methionine stress in breast cancer cells

“…19 By comparison, previous reports using cells from human prostate cancer cell lines and Yoshida sarcoma their methionine stress-sensitive MDAMB468 parental cell line. The proliferation rate of both the MDAMB468 and the isolated R8 clone of resistant MDAMB468 cells in Met+ medium was similar, but only the resistant cells maintained the ability to proliferate in Met-Hcy+ medium ( Fig.…”

“…In the model system, yeast, a regulatory pathway connecting sulfur-containing metabolites, most notably methionine, with cell cycle regulation, has been suggested to respond primarily to S-adenosylmethionine (SAM) levels. [18][19][20] Furthermore, earlier studies showed that reducing SAM synthesis blocks proliferation of leukemic cells. 16,17 Therefore, we hypothesized that methionine dependence is a reflection of limiting SAM availability caused by reduced flux through the methionine metabolic pathway in Met-Hcy+ conditions.…”

“…In budding yeast, methionine stress triggers the SAM-checkpoint and destabilizes pre-replication complexes as measured by loss of Mcm proteins at replication origins. 19 Therefore, we explored the determine if Cdc6 loss is a common response of cancer cells to methionine stress. We prepared lysates from the breast cancer cell lines MCF7 and MDAMB361, and prostate cancer cell lines DU145 and PC-3, grown in Met-Hcy+ medium, and then analyzed Cdc6 levels.…”

Regulation of yeast oscillatory dynamics

“…A major function of this protein is to generate negative feedback by the deactivation of the Met4p through polyubiquitination, resulting in a down-regulation of S-adenosylmethionine, the major methyl donor in the cell, thus providing a strong oscillatory potential. The CMtr complex is conserved in eukaryotes and may constitute a more general redox state checkpoint in the chromosome cycle (30).…”

Regulation of Amino Acid, Nucleotide, and Phosphate Metabolism in Saccharomyces cerevisiae

“…The importance of SCF Met30 control of Met4 activity is clearly demonstrated by the finding that the lethality resulting from the inactivation of MET30, leading to unbridled Met4 activation function, can be bypassed by deletion of the activation domain of Met4 or the deletion of MET32, but not of CBF1, MET28, or MET31 Su et al 2005). Consistent with Met32 having an important role in MET gene expression, a truncated version of Met32 (Met32D145-192) acts as a dominant suppressor of met30 null mutations by interfering with the recruitment of Met4 to both Cbf1 and Met31/32-dependent promoters (Su et al 2008).…”