Studies of the expression of human poly(ADP-ribose) polymerase-1 in Saccharomyces cerevisiae and identification of PARP-1 substrates by yeast proteome microarray screening.
Poly(ADP-ribosyl)ation of various nuclear proteins catalyzed by a family of NAD(+)-dependent enzymes, poly(ADP-ribose) polymerases (PARPs), is an important posttranslational modification reaction. PARP activity has been demonstrated in all types of eukaryotic cells with the exception of yeast, in which the expression of human PARP-1 was shown to lead to retarded cell growth. We investigated the yeast growth inhibition caused by human PARP-1 expression in Saccharomyces cerevisiae. Flow cytometry analysis reveals that PARP-1-expressing yeast cells accumulate in the G(2)/M stage of the cell cycle. Confocal microscopy analysis shows that human PARP-1 is distributed throughout the nucleus of yeast cells but is enriched in the nucleolus. Utilizing yeast proteome microarray screening, we identified 33 putative PARP-1 substrates, six of which are known to be involved in ribosome biogenesis. The poly(ADP-ribosyl)ation of three of these yeast proteins, together with two human homologues, was confirmed by an in vitro PARP-1 assay. Finally, a polysome profile analysis using sucrose gradient ultracentrifugation demonstrated that the ribosome levels in yeast cells expressing PARP-1 are lower than those in control yeast cells. Overall, our data suggest that human PARP-1 may affect ribosome biogenesis by modifying certain nucleolar proteins in yeast. The artificial PARP-1 pathway in yeast may be used as a simple platform to identify substrates and verify function of this important enzyme.
Tao
Zhihua
Z
Institute for Cellular andMolecular Biology, University of Texas at Austin, Austin, Texas 78712, USA.
Gao
Peng
P
Liu
Hung-Wen
HW
D016195:G2 Phase / Q000235:genetics; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis / Q000235:genetics; D009687:Nuclear Proteins / Q000096:biosynthesis / Q000235:genetics; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D040081:Protein Array Analysis / Q000379:methods; D020543:Proteome / Q000032:analysis* / Q000235:genetics; D040901:Proteomics / Q000379:methods; D012270:Ribosomes / Q000201:enzymology / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics
D009687:Nuclear Proteins; D020543:Proteome; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases