The replication fork block protein Fob1 functions as a negative regulator of the FEAR network.
BACKGROUND
The protein phosphatase Cdc14 is a key regulator of exit from mitosis in budding yeast. Its activation during anaphase is characterized by dissociation from its inhibitor Cfi1/Net1 in the nucleolus and is controlled by two regulatory networks. The Cdc14 early anaphase release (FEAR) network promotes activation of the phosphatase during early anaphase, whereas the mitotic exit network (MEN) activates Cdc14 during late stages of anaphase.


RESULTS
Here we investigate how the FEAR network component Spo12 regulates Cdc14 activation. We identify the replication fork block protein Fob1 as a Spo12-interacting factor. Inactivation of FOB1 leads to premature release of Cdc14 from the nucleolus in metaphase-arrested cells. Conversely, high levels of FOB1 delay the release of Cdc14 from the nucleolus. Fob1 associates with Cfi1/Net1, and consistent with this observation, we find that the bulk of Cdc14 localizes to the Fob1 binding region within the rDNA repeats. Finally, we show that Spo12 phosphorylation is cell cycle regulated and affects its binding to Fob1.


CONCLUSIONS
Fob1 functions as a negative regulator of the FEAR network. We propose that Fob1 helps to prevent the dissociation of Cdc14 from Cfi1/Net1 prior to anaphase and that Spo12 activation during early anaphase promotes the release of Cdc14 from its inhibitor by antagonizing Fob1 function.
Stegmeier
Frank
F
Center for Cancer Research, Howard Hughes Medical Institute, Massachusetts Institute of Technology, E17-233, 40 Ames St., Cambridge, MA 02139, USA.
Huang
Julie
J
Rahal
Rami
R
Zmolik
Jessica
J
Moazed
Danesh
D
Amon
Angelika
A
D000595:Amino Acid Sequence; D018797:Cell Cycle Proteins / Q000378:metabolism* / Q000502:physiology; D004275:DNA, Ribosomal / Q000235:genetics; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D008938:Mitosis / Q000502:physiology*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011233:Precipitin Tests; D017434:Protein Structure, Tertiary / Q000235:genetics; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism* / Q000502:physiology; D012091:Repetitive Sequences, Nucleic Acid / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism* / Q000502:physiology; D004718:Saccharomycetales; D020798:Two-Hybrid System Techniques
C074860:CDC14 protein, S cerevisiae; D018797:Cell Cycle Proteins; D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; C105579:FOB1 protein, S cerevisiae; D005656:Fungal Proteins; C119300:Net1 protein, S cerevisiae; D009687:Nuclear Proteins; D010761:Phosphorus Radioisotopes; C416648:SPO12 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017027:Protein Tyrosine Phosphatases