Mechanisms Coordinating Ribosomal Protein Gene Transcription in Response to Stress

While expression of ribosomal protein genes (RPGs) in the budding yeast Saccharomyces cerevisiae has been extensively studied, a longstanding enigma persists regarding their co-regulation under fluctuating nutrient and stress conditions. Most (<90%) of RPG promoters display one of two distinct arrangements of a core set of transcription factors (TFs; Rap1, Fhl1 and Ifh1) and are further differentiated by the presence or absence of the HMGB box protein Hmo1. However, a third group of promoters appears not to bind any of these proteins, raising the question of how the whole suite of genes is co-regulated. We demonstrate that all RPGs are regulated by two distinct, but complementary mechanisms driven by the TFs Ifh1 and Sfp1, both of which are required for maximal expression in optimal conditions and coordinated down-regulation upon stress. At the majority of RPG promoters Ifh1-dependent regulation predominates, whereas Sfp1 plays the major role at all other genes. We also uncovered an unexpected, protein homeostasis-dependent binding property of Hmo1 at a large subset of RPG promoters. Finally, we show that the Ifh1 paralog Crf1, previously described as a transcriptional repressor, can act as a constitutive RPG activator in the W303 strain background when overexpressed. Our study thus provides a more complete picture of RPG regulation and may serve as a paradigm for unravelling RPG regulation in multicellular eukaryotes.

Off-Target Effects of the Septin Drug Forchlorfenuron on Nonplant Eukaryotes

ABSTRACTThe septins are a family of GTP-binding proteins that form cytoskeletal filaments. Septins are highly conserved and evolutionarily ancient but are absent from land plants. The synthetic plant cytokinin forchlorfenuron (FCF) was shown previously to inhibit budding yeast cell division and induce ectopic septin structures (M. Iwase, S. Okada, T. Oguchi, and A. Toh-e, Genes Genet. Syst. 79:199–206, 2004,http://dx.doi.org/10.1266/ggs.79.199). Subsequent studies in a wide range of eukaryotes have concluded that FCF exclusively inhibits septin function, yet the mechanism of FCF action in nonplant cells remains poorly understood. Here, we report that the cellular effects of FCF are far more complex than previously described. The reported growth arrest of budding yeast cells treated with 1 mM FCF partly reflects sensitization caused by abud4mutation present in the W303 strain background. In wild-type (BUD4+) budding yeast, growth was inhibited at FCF concentrations that had no detectable effect on septin structure or function. Moreover, FCF severely inhibited the proliferation of fission yeast cells, in which septin function is nonessential. FCF induced fragmentation of budding yeast mitochondrial reticula and the loss of mitochondrial membrane potential. Mitochondria also fragmented in cultured mammalian cells treated with concentrations of FCF that previously were assumed to target septins only. Finally, FCF potently inhibited ciliation and motility and induced mitochondrial disorganization inTetrahymena thermophilawithout apparent alterations in septin structure. None of these effects was consistent with the inhibition of septin function. Our findings point to nonseptin targets as major concerns when using FCF.