Abstract

The mitochondrial matrix constitutes a biochemical reaction environment with a highly complex structure. During normal and pathological conditions, this mitochondrial compartment displays dynamic changes in its (ultra)structure and physicochemical properties, which affect diffusion-limited reactions and molecular target finding. However, there is little quantitative information on how the viscosity of the mitochondrial matrix solvent (ηsolvent) impacts on solute diffusion in living cells. This precludes a proper understanding of how mitochondrial structural and functional dynamics affect mitochondrial, and thereby cellular, functioning. It was previously demonstrated that matrix-protruding folds (cristae) in the mitochondrial inner membrane substantially hinder the free diffusion of fluorescent proteins (FPs). Using HeLa cell lines expressing matrix-targeted FP-concatemers of increasing MW (AcGFP1, AcGFP12, AcGFP13, AcGFP14) we here provide evidence that: (i) ηsolvent equals 2.69-3.32 cP, (ii) all FPs assume a molecular conformation of maximal size (“extended”), (iii) the mitochondrial matrix fluid modulates FP diffusion in a MW-dependent manner via viscosity-dependent and -independent mechanisms. Treatment with chloramphenicol (CAP), a mitochondrial protein synthesis inhibitor that induces the mitochondrial unfolded protein response (UPRmito), 2-fold reduced the number of cristae and 24-fold increased ηsolvent (64.7-80.0 cP). Under these conditions AcGFP14 assumed its minimal size (“compact”), compatible with macromolecular crowding. These findings support a mechanism in which (combined) changes in mitochondrial nanostructure and matrix viscosity modulate mitochondrial bioreactions by altering the diffusion and molecular conformation of matrix solutes in a MW-dependent manner.