Dependence of the folding and import of the precursor to mitochondrial aspartate aminotransferase on the nature of the cell-free translation system.

The precursor to mitochondrial aspartate amino-transferase (pmAspAT), when newly synthesized in vitro using either rabbit reticulocyte lysate (RRL) or wheat germ extract (WGE), is highly susceptible to proteolysis. Treatment of these translation products with trypsin generates a characteristic pattern of proteolytic fragments which differs between WGE and RRL. pmAspAT synthesized in RRL acquires over time the trypsin resistance characteristic of purified recombinant pmAspAT in a process that reflects folding of the nascent protein in a cytosolic-like environment and results in the loss of its ability to be imported into mitochondria (Mattingly, J.R., Jr., Youssef, J., Iriarte, A., and Martinez-Carrion, M. (1993) J. Biol. Chem. 268, 3925-3937). Folding in RRL is temperature-dependent. By contrast, pmAspAT synthesized in WGE does not acquire trypsin resistance at any temperature and is unable to be imported into isolated mitochondria. Yet, addition of RRL to the protein synthesized in WGE restores both the folding and import processes. This effect is temperature and N-ethylmaleimide-sensitive and requires ATP. We have also detected that newly synthesized pmAspAT, in both the mammalian (RRL) and plant (WGE) environments, associates at an early stage, possibly cotranslationally, with cytosolic hsp70. This complex is transient and short-lived in RRL but is stable for protein synthesized in WGE. The mature form of the protein is also found associated with hsp70 early on during its synthesis. Yet, in RRL, it dissociates from the complex more easily than its precursor form. This suggests that the interaction between the precursor and hsp70 is at least partially dependent on the signal peptide. The dissociation of the precursor/hsp70 complex seems to precede ATP-requiring subsequent folding steps. Furthermore, release of precursor from this complex, either to be imported into mitochondria or to resume folding when in the absence of mitochondria, appears to be influenced by other factor(s) present in RRL but absent or inhibited in WGE.