Abstract
The Spo12 protein plays a regulatory role in two of the most fundamental processes of biology, mitosis and meiosis, and yet its biochemical function remains elusive. In this study we concentrate on the genetic and biochemical analysis of its mitotic function. Since high-copy SPO12 is able to suppress a wide variety of mitotic exit mutants, all of which arrest with high Clb-Cdc28 activity, we speculated whether SPO12 is able to facilitate exit from mitosis when overexpressed by antagonizing mitotic kinase activity. We show, however, that Spo12 is not a potent regulator of Clb-Cdc28 activity and can function independently of either the cyclin-dependent kinase inhibitor (CDKi), Sic1, or the anaphase-promoting complex (APC) regulator, Hct1. Spo12 protein level is regulated by the APC and the protein is degraded in G1 by an Hct1-dependent mechanism. We also demonstrate that in addition to localizing to the nucleus Spo12 is a nucleolar protein. We propose a model where overexpression of Spo12 may lead to the delocalization of a small amount of Cdc14 from the nucleolus, resulting in a sufficient lowering of mitotic kinase levels to facilitate mitotic exit. Finally, site-directed mutagenesis of highly conserved residues in the Spo12 protein sequence abolishes both its mitotic suppressor activity as well as its meiotic function. This result is the first indication that Spo12 may carry out the same biochemical function in mitosis as it does in meiosis.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Amino Acid Sequence
-
Anaphase
-
CDC28 Protein Kinase, S cerevisiae / metabolism
-
Cdh1 Proteins
-
Cell Cycle
-
Cell Cycle Proteins / metabolism
-
Cell Nucleolus / metabolism
-
Cell Nucleus / metabolism
-
Cyclin B / metabolism
-
Cyclin-Dependent Kinase Inhibitor Proteins
-
Cyclin-Dependent Kinases / antagonists & inhibitors
-
Diploidy
-
Fluorescent Antibody Technique, Indirect
-
Fungal Proteins / genetics*
-
Fungal Proteins / metabolism
-
Fungal Proteins / physiology*
-
G1 Phase
-
Galactose / pharmacology
-
Genotype
-
Glucose / pharmacology
-
Meiosis
-
Microscopy, Fluorescence
-
Mitosis*
-
Models, Biological
-
Molecular Sequence Data
-
Mutagenesis, Site-Directed
-
Mutation
-
Nuclear Proteins
-
Phenotype
-
Plasmids / metabolism
-
Protein Kinases / metabolism
-
Protein Serine-Threonine Kinases
-
Protein Tyrosine Phosphatases*
-
Saccharomyces cerevisiae / genetics*
-
Saccharomyces cerevisiae / physiology*
-
Saccharomyces cerevisiae Proteins*
-
Sequence Homology, Amino Acid
-
Temperature
-
Time Factors
Substances
-
CDC14 protein, S cerevisiae
-
CDH1 protein, S cerevisiae
-
CLB2 protein, S cerevisiae
-
Cdh1 Proteins
-
Cell Cycle Proteins
-
Cyclin B
-
Cyclin-Dependent Kinase Inhibitor Proteins
-
Fungal Proteins
-
Nuclear Proteins
-
SIC1 protein, S cerevisiae
-
SPO12 protein, S cerevisiae
-
Saccharomyces cerevisiae Proteins
-
Protein Kinases
-
DBF2 protein, S cerevisiae
-
DBF20 protein, S cerevisiae
-
Protein Serine-Threonine Kinases
-
CDC28 Protein Kinase, S cerevisiae
-
Cyclin-Dependent Kinases
-
Protein Tyrosine Phosphatases
-
Glucose
-
Galactose