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Host function of MAK16: G1 arrest by a mak16 mutant of Saccharomyces cerevisiae
1Section on Genetics of Simple Eukaryotes, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD 20892.
Abstract:
The MAK16 gene was first defined as a gene whose mutation resulted in loss of M1 double-stranded RNA virus-like particles. The mak16-1 mutation also produces temperature-sensitive cell growth. We report here that mak16-1 cells arrest at the nonpermissive temperature in G1 phase, such that they are mating competent. We sequenced the MAK16 gene and found an open reading frame of 306 amino acids encoding a predicted protein of Mr 35,694. Two typical nuclear localization signal sequences were found. MAK16-LacZ fusion proteins that include one of these putative signals entered the nucleus, while unfused beta-galactosidase did not, as judged by subcellular fractionation experiments. In the C-terminal third of the MAK16 open reading frame is an acidic region in which 25 of 41 residues are either glutamate or aspartate. This region contains potential phosphorylation sites for "casein kinases," protein kinases specific for serine or threonine residues in an acidic environment.
Insights
The MAK16 gene mutation affects cell growth and causes cells to arrest in G1 phase, impacting their ability to mate. This study characterizes the MAK16 protein
Area of Science:
- Molecular biology
- Cell biology
- Virology
Background:
- The MAK16 gene was initially identified by its role in the formation of M1 double-stranded RNA virus-like particles.
- Mutations in MAK16 lead to temperature-sensitive cell growth defects.
Purpose of the Study:
- To investigate the cell cycle effects of the mak16-1 mutation.
- To characterize the MAK16 protein, including its localization and potential regulatory features.
Main Methods:
- Cell cycle analysis of mak16-1 mutant cells at permissive and non-permissive temperatures.
- Gene sequencing to determine the MAK16 open reading frame.
- Subcellular localization studies using MAK16-LacZ fusion proteins and beta-galactosidase as a control.
- Bioinformatic analysis to identify protein domains and post-translational modification sites.
Main Results:
- mak16-1 cells arrest in the G1 phase of the cell cycle at the non-permissive temperature, while retaining mating competence.
- The MAK16 gene encodes a predicted protein of 306 amino acids with a molecular weight of 35,694 Da.
- MAK16 protein contains two nuclear localization signals, and fusion proteins demonstrated nuclear import.
- An acidic region rich in glutamate and aspartate was identified in the C-terminal third, suggesting potential phosphorylation by casein kinases.
Conclusions:
- The MAK16 gene product is a nuclear protein involved in cell cycle progression, specifically regulating the G1 phase.
- The identified structural features, including nuclear localization signals and potential phosphorylation sites, provide insights into MAK16 function and regulation.