Identification Of Mitochondria Interacting Protein Of C1QBP

Background and objectives:Protein-protein interaction research is necessary to understand the basic mechanisms of biological processes and functions of proteins. To discover protein-protein inte ractions, Co-immunoprecipitation coupled with mass spectrometry(Co IP-MS) is a hi gh-throughput technique most commonly used. However,utilizing whole cell lysate, t he traditional Co IP may break cellular structure and lead to increase some man-made protein-protein interactions.C1QBP(the complement component 1, q subcomponent binding protein) binding with C1 q is an acidic protein, and it also known as p32/HABP1/ g C1 q R. C1 QBP is a multifunctional protein reported to be related with immune response, metabolism, cancer progression and metastasis, among many others processes. It can be detected in the cytosol, cell surface, and nucleus of different cells, but mostly in the mitochondria. Although C1 QBP is normally referred to as a mitochondrial protein, its functions in the mitochondria are still largely unclear.Mitochondrion is an important subcellular organelle for energy production through respiration. The number and size of mitochondria can vary greatly between different types of tissues and cells. Therefore, it is crucial to optimize the mitochondria isolation method to obtain high isolation efficiency and high purity for the specimen under investigation. Sucrose centrifugation is one of the most widely used methods for mitochondria purification.Materials and methods:1) We developed a modified mitochondria isolation method,and compared the relative amount of proteins in the purified mitochondria with cytoplasmic fractions.2) C1 QBP interacting proteins were isolated from the mitochondria using a optimized Co IP method.3) Western Blotting and confocal microscopy analyses were used to further verify the interactions between C1 QBP and DLAT.4) The PDH enzyme activity was measured in cells with different expression levels of C1 QBP.Results:1) We developed a modified mitochondria isolation method,and the relative amount of proteins in the purified mitochondria was measured to be around 13% as compared to the cytoplasmic fractions.2) DLAT was identified as a potential C1 QBP interacting protein.3) Reciprocal interaction between DLAT and C1 QBP was observed using coimmunoprecipitation and Western Blotting analysis. Confocal immunofluorensence analysis suggested that C1 QBP located in the mitochondria.4) C1 QBP can positively regulate the enzyme activity of PDH in renal cancer cells.Conclusion: In this study, subcellular fractionation was combined with Co IP-MS to characterize protein-protein interactions in the mitochondria. Using this method, DLAT was characterized as a novel mitochondrial C1 QBP binding protein, showing a new mechanism of C1 QBP in mitochondria. This study demonstrates that the subcellular fractionation coupled with Co IP-MS is effective to study the protein-protein interactions in the mitochondria. This method could be easily adapted to study subcellular protein-protein interactions. Furthermore, unlike the traditional Co IP-MS, this method retains the protein localization information, providing more insights regarding the biological implications of the detected protein-protein interactions.