During oxidative stress, K63-linked polyubiquitin chains accumulate in the cell and modify a variety of proteins including ribosomes. Knowledge of the precise sites of K63 ubiquitination is key to understand its function during the response to stress. To identify the sites of K63 ubiquitin, we developed a new mass-spectrometry based method that quantified >1,100 K63 ubiquitination sites in yeast responding to oxidative stress induced by H2O2. We determined that under stress, K63 ubiquitin modified proteins are involved in several cellular functions including ion transport, protein trafficking, and translation. The most abundant ubiquitination sites localized to the head of the 40S subunit of the ribosome, modified assembled polysomes, and affected the binding of translation factors. The results suggested a new pathway of post-initiation control of translation during oxidative stress and illustrated the importance of high-resolution mapping of noncanonical ubiquitination events.

During oxidative stress, K63-linked polyubiquitin chains accumulate in the cell and modify a variety of proteins including ribosomes. Knowledge of the precise sites of K63 ubiquitin is key to understanding its function during the response to stress. To identify the sites of K63 ubiquitin, we developed a new mass-spectrometry based method that quantified >1,100 K63 ubiquitination sites in yeast responding to oxidative stress induced by H2O2. We determined that under stress, K63 ubiquitin modified proteins involved in several cellular functions including ion transport, protein trafficking, and translation. The most abundant ubiquitination sites localized to the head of the 40S subunit of the ribosome, modified assembled polysomes, and affected the binding of translation factors. The results suggested a new pathway of post-initiation control of translation during oxidative stress and illustrated the importance of high-resolution mapping of noncanonical ubiquitination events.

Local synthesis of proteins near their active site is a critical cellular phenomenon. Recent studies have demonstrated that a subpopulation of cytoplasmic ribosomes is localized to organelles such as the endoplasmic reticulum and mitochondria. Due to the presence of different membranous compartments within eukaryotic cells, proper mRNA localization or targeted translation is required to focus the expensive process of translation to where it is needed most. During stress while global translation has been downregulated translation of stress responsive proteins as well other essential proteins remains active. During ER stress, substantial amounts of remodeling happen near ER localized translation populations. Due to its physical and functional relation with mitochondria it is also essential to regulate the translation process of mitochondrial destined proteins during the ER stress condition.In the current study, a digitonin based extraction method has been successfully established to separate the free cytoplasmic ribosome and mitochondria bound ribosome from HEK293 cells and to perform ribosome profiling in order to capture the actively translating polysome populations of free and mitochondria bound translating ribosome populations. This method can successfully separate the two ribosomal populations and it is enriched with known near‐mitochondria localized ribosome. Previous studies identified a population of mRNAs which are translating near mitochondrial surface in yeast and most of them has been found to be conserved in higher organisms. Interestingly, a set of known near mitochondria localized RNA has been found to involve in active translation in our global riboseq data collected from Hela upon ER stress. It indicates a correlation between the near mitochondrial localized translation and ER stress regulation. Specifically, higher expression of different sub units of oxidative phosphorylation complexes has been found to be enriched upon ER stress. This motivates further detailed study to understand organeller cross talk at translation level during stress. Understanding the near mitochondria localized populations in presence and absence of ER stress in higher organisms would be helpful to understand the complex ER‐mitochondrial cross talk events.Support or Funding InformationNIHR01This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

No abstract