Links between the Ubiquitin-Proteasome System and Cell Death Pathways in Arabidopsis thaliana

The dominant pathway for protein degradation in eukaryotes is the ubiquitin-proteasome system. In an enzymatic cascade with three different enzymes (E1, E2, E3), the small protein ubiquitin is covalently linked to a target protein, and substrates carrying a multi-ubiquitin chain consisting of at least four ubiquitin moieties are rapidly degraded by the 26S proteasome. Programmed cell death (PCD) is the genetically controlled suicide of cells that occurs in all eukaryotes. Examples for PCD in plants are leaf senescence, the hypersensitive response (HR) after pathogen attack and processes during development such as the formation of tracheary elements. Several proteins involved in PCD in animals are regulated by the ubiquitin-proteasome pathway. In this work, the ubiquitin-proteasome system was perturbed in the model plant Arabidopsis by expression of UbK48R, a variant ubiquitin that was able to induce cell death in plants. Physiological characterization of the UbK48R-induced cell death could not precisely pinpoint the type of PCD observed. However, DNA degradation at a very late time point during PCD indicated that the UbK48R-induced PCD was related to senescence. It could also be shown that exposure to additional, normally harmless stress factors is able to accelerate the cell death process, and the isolation and characterization of suppressor of UbK48R-induced cell death (sud) mutants indicated changes in distinct, rather specialized pathways. One dominant mutant (sud1) resulted in higher resistance to the proteasome inhibitor MG132, and the recessive sud2 mutation could be positioned in a first-pass mapping to chromosome III between 10.7 Mbp and 18.9 Mbp. A second line of research dealt with the N-end rule pathway, a distinct ubiquitin-dependent protein degradation pathway that connects the fate of a protein with the nature of its amino-terminal residue. This pathway is also involved in leaf senescence. By using model substrates with defined amino acids at the amino terminus, it was shown that the Arabidopsis gene At5g02310 (now PRT6) encodes a ligase of the N-end rule pathway that recognizes and degrades proteins carrying Arg as amino-terminal residue. Model substrates with Leu as amino-terminal residue were, although also short-lived, not degraded by PRT6 or the second known ligase PRT1. Therefore, the existence of at least one more N-end rule ligase in plants was postulated