Characterization of a complex involved in lipid transport to mitochondria during plant adaptation to phosphate starvation

Abstract

Phosphate (Pi) starvation is a frequent nutrient stress encountered by plants to which they adapt by exerting different mechanisms. The partial degradation of phospholipids, a common constituent of cellular membranes, is a widespread response observed in plants to increase the intracellular Pi availability. To maintain membrane integrity, the degraded phospholipids are replaced by a non-phosphorous plastid-synthetized lipid, the digalactosyldiacylglycerol (DGDG). This replacement implies a lipid transport by unknown mechanisms from plastids to other organelles, such as mitochondria or plasma membrane. Precursor works in our laboratory have shown that DGDG transport to mitochondria occurs by non-vesicular transfer at plastids-mitochondria membrane contact sites (MCSs), the abundance of which increases during Pi starvation. Recently, we identified the mitochondrial transmembrane lipoprotein (MTL) complex, a huge complex enriched in lipids and mainly composed of proteins located in both mitochondrial membranes. One of its components of the inner membrane, AtMic60, was shown to play a role in intra-mitochondrial DGDG trafficking. Furthermore, the identification of plastidial proteins in the MTL complex suggests it might be located at mitochondria-plastids MCSs to promote lipid transport in response to Pi starvation. The MTL complex is also present in Pi-sufficient conditions and the structure of this huge complex is currently unknown. Thus, deciphering how the MTL structure and composition are modified in response to low Pi will be a key step to understand how this complex could be involved in MCSs formation and lipid transport and to identify new proteins involved in these processes. The goal of this work is to optimize a protocol to purify the MTL complex from Arabidopsis thaliana cell cultures grown in presence or absence of Pi and to analyze its structure and composition. To achieve our goal, we tested a combination of different fractionation methods to separate the MTL complex from other mitochondrial complexes: sucrose gradient, gel filtration and anion exchange chromatography. To date, the best results were obtained by using a combination of anion exchange and size exclusion chromatography. The first imaging attempts by negative stain electron microscopy and cryo-electron microscopy gave encouraging results concerning the homogeneity of our sample. Proteomic analyses of the purified complex are under progress and will help us to decipher whether further purification steps are required or not. To conclude, this preliminary work reveals that the MTL complex is resistant to a combination of chromatography techniques and paves the way for further characterizations of this complex. The new insights gained on the MTL complex will allow us to understand how lipids are transported to mitochondria during adaptation to Pi starvation and will contribute to the understanding of mitochondria MCSs formation in plants.