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Yeast homolog of human SAG/ROC2/Rbx2/Hrt2 is essential for cell growth, but not for germination: chip profiling implicates its role in cell cycle regulation

Oncogene volume 19, pages 2855–2866 (2000)Cite this article

Abstract

In an attempt to understand the signaling pathway mediating redox-induced apoptosis, we cloned SAG, an evolutionarily conserved zinc RING finger gene that, when overexpressed, protects cells from apoptosis induced by redox agents. Here we report functional characterization of SAG by the use of yeast genetics approach. Targeted disruption of ySAG, yeast homolog of human SAG, and subsequent tetrad analysis revealed that ySAG is required for yeast viability. Complementation experiment showed that the lethal phenotype induced by the ySAG deletion is fully rescued by wildtype SAG, but not by several hSAG mutants. Complementation experiment has also confirmed that ySAG is essential for normal vegetative growth, rather than being required for sporulation. Furthermore, cell death induced by SAG deletion was accompanied by cell enlargement and abnormal cell cycle profiling with an increased DNA content. Importantly, SAG was found to be the second family member of Rbx (RING box protein) or ROC (Regulator of cullins) or Hrt that is a component of SCF E3 ubiquitin ligase. Indeed, like ROC1/Rbx1/Hrt1, SAG binds to Cul1 and SAG-Cul1 complex has ubiquitin ligase activity to promote poly-ubiquitination of E2/Cdc34. This ligase activity is required for complementation of death phenotype induced by ySAG disruption. Finally, chip profiling of the entire yeast genome revealed induction of several G1/S as well as G2/M checkpoint control genes upon SAG withdrawal. Thus, SAG appears to control cell cycle progression in yeast by promoting ubiquitination and degradation of cell cycle regulatory proteins.

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Abbreviations

Cul1:

cullin1

5-FOA:

5-fluoroorotic acid

GSS:

glutathione synthetase

HA:

hemagglutinin A epitope

OP:

1,10-phenanthroline

Rbx:

RING box protein

ROC:

regulator of cullins

ROS:

reactive oxygen species

SAG:

sensitive to apoptosis gene

SCF:

Skp/cullin/F-box

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Acknowledgements

We would like to thank Drs Dennis Thiele and Kunliang Guan at the University of Michigan for the instructions of yeast dissection (D Thiele), for providing yeast strains and expression vectors, p414 and p426 (K Guan). We thank Dr Peter Philippsen at University Basel for plasmid pFA6-KanMX4 used in yeast knockout experiment. We also thank Dr Zhen-Qiang Pan and Augus Chen at the Mount Sinai School of Medicine for their help in setting up ubiquitin ligase assay and for providing assay reagents, Dr Yue Xiong at University of North Carolina at Chapel Hill for providing ROC1 and Cul1 plasmids and Dr Hui Zhang at Yale University for Cul1 antibody. Our acknowledgements are extended to Drs Steve Hunt and Joe Menetsky at Parke-Davis, Edith Gralla at University of California at Los Angeles for stimulating discussion, and Dr Ken Hu and Jobi Wong at Parke-Davis for yeast promoter analysis and for help in graphic preparation, respectively.

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Authors and Affiliations

  1. Department of Molecular Biology, Division of Warner-Lambert Company, Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan, USA

    Manju Swaroop, Yixin Wang, Hangjun Duan, Tim Jatkoe, Steven J Madore & Yi Sun

  2. Department of Infectious Disease, Division of Warner-Lambert Company, Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan, USA

    Paul Miller

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Swaroop, M., Wang, Y., Miller, P. et al. Yeast homolog of human SAG/ROC2/Rbx2/Hrt2 is essential for cell growth, but not for germination: chip profiling implicates its role in cell cycle regulation. Oncogene 19, 2855–2866 (2000). https://doi.org/10.1038/sj.onc.1203635

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