An Enigma of N-termini dependent protein degradation.

The N-Degron rules that target proteins for degradation via their N-terminal sequences has expanded to encompass most N-terminal sequences. These destabilizing N-termini include many sequence combinations with the initiator methionine intact, including the recently reported N-terminal methionine followed by a basic residue. Despite the diverse sequences reported for N-Degron recognition and degradation system wide proteomic analysis has currently not observed these rules with endogenous proteins. Here we report on these apparent inconsistencies by validating the degradation of reporter proteins with N-terminal MK- and MR-sequences and also investigating global endogenous protein turnover by proteomics. In addition to verifying the reported degradation of proteins with MK- and MR- N-termini we have also identified an additional sequence dependency where an acidic residue following the basic residue inhibits protein degradation. Global protein degradation analysis using a metabolic labelling approach with azidohomoalanine failed to observe trends in cytoplasmic protein stability that correlates to the N-terminal sequence. Together we have been able to reproduce the apparent contradictory results reported using different methodologies. This included the use of different recombinant protein reporters to investigate MK- and MR- N-termini dependent protein degradation and an alternative proteomic method to quantify global protein degradation. This highlights the need into further investigations to complete our understanding of the mechanisms and roles of N-Degron pathways.