Quantitative Proteomics Reveals UGA-Independent Misincorporation of Selenocysteine throughout the Escherichia coli Proteome

Selenocysteine is cotranslationally inserted into polypeptide chains by recoding the stop codon UGA. However, selenocysteine has also been found to be misincorporated into a small number of proteins displacing cysteines in previous studies, but such misincorporation has not yet been examined at the proteome level thoroughly. We performed label-free quantitative proteomics analysis on Escherichia coli grown in a high-selenium medium to obtain a fuller picture of selenocysteine misincorporation in its proteome. We found 139 misincorporation sites, including 54 recurred in all biological replicates, suggesting that some cysteine sites are more prone to be misincorporated than others. However, sequence and evolutionary conservation analysis showed no clear pattern among these misincorporation sites. We hypothesize that misincorporations occur randomly throughout the proteome, but the degradation rate of such misincorporated proteins varies depending on the impact of the misincorporation on protein function and stability, leading to the differential detectability of misincorporated sites by proteomics. Our hypothesis is further supported by two observations: (1) cells cultured with severely limited sulfur still retained a substantial proportion of normal cysteine counterparts of all of the found misincorporated proteins and (2) proteins involved in protein folding and proteolysis were highly upregulated in high-selenium culture.

Automated summary

Selenocysteine can be inserted into polypeptide chains in place of cysteine, but the misincorporation of selenocysteine in the proteome has not been extensively studied. Through proteomics analysis of E. coli grown in a high-selenium medium, 139 misincorporation sites were identified, indicating a random distribution throughout the proteome. The differential detectability of misincorporated sites may be due to the impact of misincorporation on protein function and stability.