Mitotic Exit Network Controls the Localization of Cdc14 to the Spindle Pole Body in Saccharomyces cerevisiae

AbstractBudding yeast Cdc14 phosphatase plays essential roles in mitotic exit. Cdc14 is sequestered in the nucleolus by its inhibitor Net1/Cfi1 and is only released from the nucleolus during anaphase to inactivate mitotic CDK. It is believed that the mitotic exit network (MEN) is required for the release of Cdc14 from the nucleolus because liberation of Cdc14 by net1/cfi1 mutations bypasses the essential role of the MEN. But how the MEN residing at the spindle pole body (SPB) controls the association of Cdc14 with Net1/Cfi1 in the nucleolus is not yet understood [1, 2]. We found that Cdc14-5GFP was released from the nucleolus in the MEN mutants (tem1, cdc15, dbf2, and nud1), but not in the cdc5 cells during early anaphase. The Cdc14 liberation from the nucleolus was inhibited by the Mad2 checkpoint and by the Bub2 checkpoint in a different manner when microtubule organization was disrupted. We observed Cdc14-5GFP at the SPB in addition to the nucleolus. The SPB localization of Cdc14 was significantly affected by the MEN mutations and the bub2 mutation. We conclude that Cdc14 is released from the nucleolus at the onset of anaphase in a CDC5-dependent manner and that MEN factors possibly regulate Cdc14 release from the SPB

Nutrient-Regulated Antisense and Intragenic RNAs Modulate a Signal Transduction Pathway in Yeast

The budding yeast Saccharomyces cerevisiae alters its gene expression profile in response to a change in nutrient availability. The PHO system is a well-studied case in the transcriptional regulation responding to nutritional changes in which a set of genes (PHO genes) is expressed to activate inorganic phosphate (Pi) metabolism for adaptation to Pi starvation. Pi starvation triggers an inhibition of Pho85 kinase, leading to migration of unphosphorylated Pho4 transcriptional activator into the nucleus and enabling expression of PHO genes. When Pi is sufficient, the Pho85 kinase phosphorylates Pho4, thereby excluding it from the nucleus and resulting in repression (i.e., lack of transcription) of PHO genes. The Pho85 kinase has a role in various cellular functions other than regulation of the PHO system in that Pho85 monitors whether environmental conditions are adequate for cell growth and represses inadequate (untimely) responses in these cellular processes. In contrast, Pho4 appears to activate some genes involved in stress response and is required for G1 arrest caused by DNA damage. These facts suggest the antagonistic function of these two players on a more general scale when yeast cells must cope with stress conditions. To explore general involvement of Pho4 in stress response, we tried to identify Pho4-dependent genes by a genome-wide mapping of Pho4 and Rpo21 binding (Rpo21 being the largest subunit of RNA polymerase II) using a yeast tiling array. In the course of this study, we found Pi- and Pho4-regulated intragenic and antisense RNAs that could modulate the Pi signal transduction pathway. Low-Pi signal is transmitted via certain inositol polyphosphate (IP) species (IP7) that are synthesized by Vip1 IP6 kinase. We have shown that Pho4 activates the transcription of antisense and intragenic RNAs in the KCS1 locus to down-regulate the Kcs1 activity, another IP6 kinase, by producing truncated Kcs1 protein via hybrid formation with the KCS1 mRNA and translation of the intragenic RNA, thereby enabling Vip1 to utilize more IP6 to synthesize IP7 functioning in low-Pi signaling. Because Kcs1 also can phosphorylate these IP7 species to synthesize IP8, reduction in Kcs1 activity can ensure accumulation of the IP7 species, leading to further stimulation of low-Pi signaling (i.e., forming a positive feedback loop). We also report that genes apparently not involved in the PHO system are regulated by Pho4 either dependent upon or independent of the Pi conditions, and many of the latter genes are involved in stress response. In S. cerevisiae, a large-scale cDNA analysis and mapping of RNA polymerase II binding using a high-resolution tiling array have identified a large number of antisense RNA species whose functions are yet to be clarified. Here we have shown that nutrient-regulated antisense and intragenic RNAs as well as direct regulation of structural gene transcription function in the response to nutrient availability. Our findings also imply that Pho4 is present in the nucleus even under high-Pi conditions to activate or repress transcription, which challenges our current understanding of Pho4 regulation

Nob1p is required for biogenesis of the 26S proteasome and degraded upon its maturation in Saccharomyces cerevisiae

Nob1p is a nuclear protein that forms a complex with the 19S regulatory particle of the 26S proteasome and with uncharacterized nuclear protein Pno1p. Overexpression of NOB1 overrode the defects in maturation of the 20S proteasome of ump1Δ cells, and temperature-sensitive nob1 and pno1 mutants exhibited defects in the processing of the β subunits and in the assembly of the 20S and the 26S proteasomes. A defect in either NOB1 or PNO1 caused accumulation of newly formed Pre6p in the cytoplasm, whereas Pre6p of the ump1Δ strain accumulated in the nucleus irrespective of the temperature. Here we present a model proposing that (1) Nob1p serves as a chaperone to join the 20S proteasome with the 19S regulatory particle in the nucleus and facilitates the maturation of the 20S proteasome and degradation of Ump1p, and (2) Nob1p is then internalized into the 26S proteasome and degraded to complete 26S proteasome biogenesis