Stress‐dependent macromolecular crowding in the mitochondrial matrix

Abstract

Macromolecules of various sizes induce crowding of the cellular environment. This crowding impacts on biochemical reactions by increasing solvent viscosity, decreasing the water‐accessible volume and altering protein shape, function, and interactions. Although mitochondria represent highly protein‐rich organelles, most of these proteins are somehow immobilized. Therefore, whether the mitochondrial matrix solvent exhibits macromolecular crowding is still unclear. Here, we demonstrate that fluorescent protein fusion peptides (AcGFP1 concatemers) in the mitochondrial matrix of HeLa cells display an elongated molecular structure and that their diffusion constant decreases with increasing molecular weight in a manner typical of macromolecular crowding. Chloramphenicol (CAP) treatment impaired mitochondrial function and reduced the number of cristae without triggering mitochondrial orthodox‐to‐condensed transition or a mitochondrial unfolded protein response. CAP‐treated cells displayed progressive concatemer immobilization with increasing molecular weight and an eightfold matrix viscosity increase, compatible with increased macromolecular crowding. These results establish that the matrix solvent exhibits macromolecular crowding in functional and dysfunctional mitochondria. Therefore, changes in matrix crowding likely affect matrix biochemical reactions in a manner depending on the molecular weight of the involved crowders and reactants. image It is still unclear whether presence of macromolecules in the highly protein‐rich mitochondrial matrix fluid affects solute diffusion and biochemical reactions remains unclear. This study identifies macromolecular crowding as a stress‐dependent determinant of matrix solute mobility and solvent viscosity, suggesting additional regulatory layers of mitochondrial biology. Mitochondrial matrix‐targeted fusion peptides of increasing molecular weight show a decreasing solvent‐dependent diffusion constant indicative of macromolecular crowding. Mitochondrial impairment by chloramphenicol decreases the number of cristae and increases matrix solvent crowding and viscosity. The mitochondrial matrix solvent exhibits macromolecular crowding, suggesting that the kinetics of matrix biochemical reactions depend on the molecular weight of the involved crowders and reactants.