Balancing redox homeostasis and the metabolic network through metabolite compartmentalisation: SLC25A13, aspartate, and the mitochondrion

The mitochondria are often referred to as the powerhouse of the cell, representing a nexus of the cellular metabolic activity that converts nutrients into useful building blocks for cell repair and growth. Central to this is the ability to get nutrients into and out of the mitochondria so that they are available for use throughout the cell. This is achieved through the nutrient transporters - in the mitochondria, many of these are members of the SLC25 gene family. SLC25A13 is one of two transporters that have been shown to move two amino acids - glutamate and aspartate - into and out of mitochondria. Aspartate is particularly vital for a number of different cellular processes, so its use by cells in different conditions may change. We were therefore surprised when one of our recent experiments showed that when cells are limited for oxygen, the cells suppress this transporter, so they can no longer use it. This suggests that we don't understand how this transporter works - something which is key if we are to better understand how our cells use nutrients when in different conditions. Our proposal here will shed light on all aspects of the SLC25A13 transporter - how it looks, how it is controlled in different cellular environments and what this means for its ability to support cell function and survival.

The malate-aspartate shuttle (MAS) is a fundamental mechanism to maximise ATP synthesis from glucose and balance redox potential between the cytosol and mitochondrion. However, the transporters supporting this function, SLC25A13, A12 and A11 also move important metabolites - particularly aspartate - between these two compartments, which are required for other processes. The interplay between the MAS function and cytosolic requirements for metabolites such as aspartate are poorly understood. This proposal will focus on the determining the structure and cellular function of SLC25A13 under normal and perturbed (hypoxic) conditions. We will investigate the overlapping and distinct functions of SLC25A13 compared to SLC25A12 using stable isotope-enriched nutrients to trace the synthesis and downstream metabolism of aspartate and related metabolites such as malate in knockdown and knockout cell models. We will also determine the first structure of full-length SLC25A13 in normoxia as well as hypoxia, as we have data showing that this is regulated in some which by oxygen tension. We will investigate the mechanism by which this oxygen-mediated regulation occurs, and through re-expression in hypoxia determine the evolutionary drivers for doing so.

In summary, this proposal represents the first in-depth determination of the structure and wider cellular function of SLC25A13, and the mechanisms by which cells respond to changes in their microenvironment by repressing its expression. This fundamental information is an important step towards understanding the rules of life - the interplay between central carbon metabolism, oxygen tension and redox homeostasis.