Novel tasks of the plant N-end rule degradation pathway

Removal of unwanted proteins from cells is essential in all organisms. It contributes to gene regulation, stress responses and a myriad of other processes in plants. Proteins can be degraded through modification by covalent attachment of the small modifier protein ubiquitin, which marks substrates for destruction by the large protease proteasome. Alternatively, they can be transported into the lytic compartment lysosome via a process called autophagy. The N-end rule pathway of protein degradation was the first degradation route elucidated in molecular detail. In animals and in fungi, it operates via ubiquitin attachment by a ligase that has one binding site for basic amino-terminal residues, and one for hydrophobic amino- terminal residues. Proteins with accessible basic or hydrophobic amino-terminal residues are therefore short-lived. We have been studying the N-end rule pathway of plants. There, the mechanism of degradation is similar to animals for substrates with basic amino termini, but still unknown for proteins with amino-terminal Leu. In unpublished work, we have recently determined components of this new degradation route Leu N-end rule pathway. We want to elucidate how this pathway is integrated into cellular activities. Mutants in the Leu N- end rule pathway, including those previously generated by chemical mutagenesis, shall be used for detailed characterization. We plan to determine the range of amino-termini that turn a protein into a substrate for the Leu N-end rule pathway. To facilitate these studies, we developed interaction assays for systematic tests and screens. These assays shall also serve to screen for proteins binding to any amino terminus of the N-end rule pathway. We hypothesize that some of these proteins may not operate in protein degradation, but may be readers that respond to the abundance of N-end rule substrates.

English summary describing the major results of the grant "Novel tasks of the plant N-end rule degradation pathway" for public relations purposes: Selective proteolysis is essential for cellular housekeeping, and for most regulatory circuits. The process requires recognition of a so-called degradation signal on a protein. The project objective was to elucidate how proteins starting with Leucine as an amino-terminal degradation signal (N-degron) are degraded in plants, and more generally to learn novel facets of N-degron (formerly N-end rule) pathways in plants. We found by pharmacologic, genetic and biochemical data that several different degradation routes exist for Leucine N-degron containing proteins. Both proteasomal and vacuolar turnover participates in the degradation process. This was surprising, because previous work studying how proteins that have amino-terminal Phenylalanine or Arginine as a degradation signal showed that there was only a single major recognition component (and, thus, only one major pathway) each. We could establish improved assays, generate a state-of-the-art model substrate, and set up a functional assay for testing degradation components from Arabidopsis by their functional expression in budding yeast. This latter assay turned out to be particularly useful in light of the existence of several pathways for the Leucine N-degron dependent turnover, because with this assay, individual components could be tested one by one, without background of competing pathways. Moreover, we could, with this assay, also probe minor degradation routes for other N-degrons. We elucidated the molecular identity of two genes involved in Leucine N-degron substrate turnover. These genes had previously been defined as mutant Arabidopsis lines with decreased capacity to degrade a Leu N-degron containing model substrate. Genetic outcrossing, whole genome sequencing, and complementation of the mutant with a wild type copy of the respective gene were among the methods used. We could provide a first answer to the question why plants with decreased capacity to degrade proteins via the ubiquitin-proteasome pathway die. We identified problems with chloroplast biogenesis and regulation as the major cause. This study was supported by transcriptome analysis and identification of suppressor mutations. We could show that turnover of protein complexes that contain the blue light photoreceptor phototropin 2 are particularly important in this respect. Five articles on the topic could be published, one publication was submitted, and a significant fraction of the results from the work will be part of future publications. All in all, the work carried out with FWF support resulted in significant progress, even though this progress was slower than anticipated before start of the grant, due to the unexpected complexity of the investigated degradation pathways.