Substrates Of N-end Rule Pathway Research Articles

To Investigate the Relationship between Substrates of the N-end Rule Pathway and Genes Regulated by EBP Binding Sites (GCCGCC) in Arabidopsis thaliana

The N-end rule pathway is a highly conserved mechanism present in all eukaryotes. It targets specific proteins based on the recognition of the substrate's N-terminal amino acid by E3-ligase for degradation via ubiquitin-proteasome pathway in the cell. In A.thaliana, the Group VII ERFs transcription factors are substrates of N-end rule pathway that begin with ‘Met-Cys. In presence of oxygen and nitric oxide, cysteine is oxidized and arginylated and recognized by the E3-ligase PROTEOLYSIS 6 (PRT6). Previous studies on prt6 mutant endosperm containing promABI5::GUS showed that the ABI5 5’UTR region that contains 2 EBP boxes (GCCGCC) mediate regulation via Group VII ERFs. Whereas, expression was inhibited when two EBP boxes were absent. Hence, suggesting that genes containing EBP boxes as similar as ABI5 might also be regulated through the N-end rule pathway.In this project fourteen genes containing two copies of GCCGCC in the 5’UTR/promoter were identified as potential candidate genes to be regulated by Group VII ERFs, similarly to ABI5. Successful amplification of the promoter region of five of these genes [AT4G01026 (PYL7), AT1G14810, AT3G54510 (ERD4), At3G13440 and AT5G44420 (PDF1.2)] was followed by cloning into a GUS-reporter plasmid and transformation into Arabidopsis wild type (Col-0) and prt6-1 plants. Leaves and flowers of transgenic plants were analysed by GUS staining to reveal promoter activity. This analysis has shown that these promoters enhanced GUS expression in prt6 in comparison to Col-0, suggesting that genes are regulated by the Group VII ERFs.Analysis of in-vivo binding of ERFs to selected promoters was done by Chromatin Immuno- precipitation (ChIP). To confirm the hypothesis that promoters carrying GCCGCC binding sites are bound in-vivo by Group VII ERFs. Future work involves analysis of mutant combinations of prt6 with genes such as PYL7 and ERD4 for ABA assay.

PRT1 of Arabidopsis is a ubiquitin protein ligase of the plant N-end rule pathway with specificity for aromatic amino-terminal residues.

The gene PRT1 of Arabidopsis, encoding a 45-kD protein with two RING finger domains, is essential for the degradation of F-dihydrofolate reductase, a model substrate of the N-end rule pathway of protein degradation. We have determined the function of PRT1 by expression in yeast (Saccharomyces cerevisiae). PRT1 can act as a ubiquitin protein ligase in the heterologous host. The identified substrates of PRT1 have an aromatic residue at their amino-terminus, indicating that PRT1 mediates degradation of N-end rule substrates with aromatic termini but not of those with aliphatic or basic amino-termini. Expression of model substrates in mutant and wild-type plants confirmed this substrate specificity. A ligase activity exclusively devoted to aromatic amino-termini of the N-end rule pathway is apparently unique to plants. The results presented also imply that other known substrates of the plant N-end rule pathway are ubiquitylated by one or more different ubiquitin protein ligases.