The folding of nascent mitochondrial aspartate aminotransferase synthesized in a cell-free extract can be assisted by GroEL and GroES

At 30 degrees C, the precursor to mitochondrial aspartate aminotransferase (pmAspAT) cannot fold after synthesis in rabbit reticulocyte lysate (RRL), a model for studying intracellular protein folding. However, it folds rapid ly once imported into mitochondria. Guanidinium chloride denatured pmAspAT likewise cannot refold at 30 degrees C in a defined in vitro system. Howeve r, it refolds rapidly and in good yield in the presence of the intramitocho ndrial chaperone homologues GroEL and GroES. In this report, we demonstrate that GroEL and GroES can also facilitate the folding of nascent pmAspAT in reticulocyte lysate under conditions where it otherwise would not. When ad ded alone, GroEL arrests the slow folding of nascent pmAspAT and inhibits i mport into mitochondria, These effects are significantly reversed by adding GroES. These observations suggest that added GroEL participates in an equi librium with endogenous chaperones in the cytosol which inhibit folding and promote import competence. Native gel electrophoresis suggests that nascen t pmAspAT exists in RRL as a heterogeneous population of partially folded s pecies, some of which bind to added GroEL more readily than others. The Gro EL-trapped species appear to be among the productive pmAspAT folding interm ediates formed in RRL or they at least appear to equilibrate with these int ermediates, since they become import competent after GroES-stimulated relea se from GroEL, (C) 2000 Academic Press.