Whi5 hypo- and hyper-phosphorylation dynamics control cell-cycle entry and progression.

Progression through the cell cycle depends on the phosphorylation of key substrates by cyclin-dependent kinases. In budding yeast, these substrates include the transcriptional inhibitor Whi5 that regulates G1/S transition. In early G1 phase, Whi5 is hypo-phosphorylated and inhibits the Swi4/Swi6 (SBF) complex that promotes transcription of the cyclins CLN1 and CLN2. In late G1, Whi5 is rapidly hyper-phosphorylated by Cln1 and Cln2 in complex with the cyclin-dependent kinase Cdk1. This hyper-phosphorylation inactivates Whi5 and excludes it from the nucleus. Here, we set out to determine the molecular mechanisms responsible for Whi5's multi-site phosphorylation and how they regulate the cell cycle. To do this, we first identified the 19 Whi5 sites that are appreciably phosphorylated and then determined which of these sites are responsible for G1 hypo-phosphorylation. Mutation of 7 sites removed G1 hypo-phosphorylation, increased cell size, and delayed the G1/S transition. Moreover, the rapidity of Whi5 hyper-phosphorylation in late G1 depends on "priming" sites that dock the Cks1 subunit of Cln1,2-Cdk1 complexes. Hyper-phosphorylation is crucial for Whi5 nuclear export, normal cell size, full expression of SBF target genes, and timely progression through both the G1/S transition and S/G2/M phases. Thus, our work shows how Whi5 phosphorylation regulates the G1/S transition and how it is required for timely progression through S/G2/M phases and not only G1 as previously thought.

Whi5 hypo- and hyper-phosphorylation dynamics control cell cycle entry and progression.

Progression through the cell cycle depends on the phosphorylation of key substrates by cyclin-dependent kinases. In budding yeast, these substrates include the transcriptional inhibitor Whi5 that regulates the G1/S transition. In early G1 phase, Whi5 is hypo-phosphorylated and inhibits the SBF complex that promotes transcription of the cyclins CLN1 and CLN2 . In late-G1, Whi5 is rapidly hyper-phosphorylated by Cln1,2 in complex with the cyclin-dependent kinase Cdk1. This hyper-phosphorylation inactivates Whi5 and excludes it from the nucleus. Here, we set out to determine the molecular mechanisms responsible for Whi5's multi-site phosphorylation and how they regulate the cell cycle. To do this, we first identified the 19 Whi5 sites that are appreciably phosphorylated and then determined which of these sites are responsible for G1 hypo-phosphorylation. Mutation of 7 sites removed G1 hypo-phosphorylation, increased cell size, and delayed the G1/S transition. Moreover, the rapidity of Whi5 hyper-phosphorylation in late G1 depends on 'priming' sites that dock the Cks1 subunit of Cln1,2-Cdk1 complexes. Hyper-phosphorylation is crucial for Whi5 nuclear export, normal cell size, full expression of SBF target genes, and timely progression through both the G1/S transition and S/G2/M phases. Thus, our work shows how Whi5 phosphorylation regulates the G1/S transition and how it is required for timely progression through S/G2/M phases and not only G1 as previously thought.

G1 cyclin-Cdk promotes cell cycle entry through localized phosphorylation of RNA polymerase II.

Cell division is thought to be initiated by cyclin-dependent kinases (Cdks) inactivating key transcriptional inhibitors. In budding yeast, the G1 cyclin Cln3-Cdk1 complex is thought to directly phosphorylate the Whi5 protein, thereby releasing the transcription factor SBF and committing cells to division. We report that Whi5 is a poor substrate of Cln3-Cdk1, which instead phosphorylates the RNA polymerase II subunit Rpb1's C-terminal domain on S5 of its heptapeptide repeats. Cln3-Cdk1 binds SBF-regulated promoters and Cln3's function can be performed by the canonical S5 kinase Ccl1-Kin28 when synthetically recruited to SBF. Thus, we propose that Cln3-Cdk1 triggers cell division by phosphorylating Rpb1 at SBF-regulated promoters to promote transcription. Our findings blur the distinction between cell cycle and transcriptional Cdks to highlight the ancient relationship between these two processes.

Punctuated evolution and transitional hybrid network in an ancestral cell cycle of fungi.

Although cell cycle control is an ancient, conserved, and essential process, some core animal and fungal cell cycle regulators share no more sequence identity than non-homologous proteins. Here, we show that evolution along the fungal lineage was punctuated by the early acquisition and entrainment of the SBF transcription factor through horizontal gene transfer. Cell cycle evolution in the fungal ancestor then proceeded through a hybrid network containing both SBF and its ancestral animal counterpart E2F, which is still maintained in many basal fungi. We hypothesize that a virally-derived SBF may have initially hijacked cell cycle control by activating transcription via the cis-regulatory elements targeted by the ancestral cell cycle regulator E2F, much like extant viral oncogenes. Consistent with this hypothesis, we show that SBF can regulate promoters with E2F binding sites in budding yeast.

The putative Poc complex controls two distinct Pseudomonas aeruginosa polar motility mechanisms.

Each Pseudomonas aeruginosa cell localizes two types of motility structures, a single flagellum and one or two clusters of type IV pili, to the cell poles. Previous studies suggested that these motility structures arrive at the pole through distinct mechanisms. Here we performed a swimming motility screen to identify polar flagellum localization factors and discovered three genes homologous to the TonB/ExbB/ExbD complex that have defects in both flagella-mediated swimming and pilus-mediated twitching motility. We found that deletion of tonB3, PA2983 or PA2982 led to non-polar localization of the flagellum and FlhF, which was thought to sit at the top of the flagellar localization hierarchy. Surprisingly, these mutants also exhibited pronounced changes in pilus formation or localization, indicating that these proteins may co-ordinate both the pilus and flagellum motility systems. Thus, we have renamed PA2983 and PA2982, pocA and pocB, respectively, for polar organelle co-ordinator to reflect this function. Our results suggest that TonB3, PocA and PocB may form a membrane-associated complex, which we term the Poc complex. These proteins do not exhibit polar localization themselves, but are required for increased expression of pilus genes upon surface association, indicating that they regulate motility structures through either localization or transcriptional mechanisms.

Cell size control in yeast.

Cell size is an important adaptive trait that influences nearly all aspects of cellular physiology. Despite extensive characterization of the cell-cycle regulatory network, the molecular mechanisms coupling cell growth to division, and thereby controlling cell size, have remained elusive. Recent work in yeast has reinvigorated the size control field and suggested provocative mechanisms for the distinct functions of setting and sensing cell size. Further examination of size-sensing models based on spatial gradients and molecular titration, coupled with elucidation of the pathways responsible for nutrient-modulated target size, may reveal the fundamental principles of eukaryotic cell size control.