Stress-dependent macromolecular crowding in the mitochondrial matrix.

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.

Sequencing of glycosaminoglycans with potential to interrogate sequence-specific interactions.

Technologies to sequence nucleic acids/proteins are widely available, but straightforward methodologies to sequence complex polysaccharides are lacking. We here put forward a strategy to sequence glycosaminoglycans, long linear polysaccharides involved in many biochemical processes. The method is based on the covalent immobilization and (immuno)chemical characterization of only those size-separated saccharides that harbor the original reducing end of the full-length chain. Using this methodology, the saccharide sequence of the chondroitin sulfate chain of the proteoglycan bikunin was determined. The method can be performed in any standard biochemical lab and opens studies to the interaction of complex saccharide sequences with other biomolecules.