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 rapidly once imported into mitochondria. Guanidinium chloride denatured pmAspAT likewise cannot refold at 30 degrees C in a defined in vitro system. However, it refolds rapidly and in good yield in the presence of the intramitochondrial 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 added alone, GroEL arrests the slow folding of nascent pmAspAT and inhibits import into mitochondria. These effects are significantly reversed by adding GroES. These observations suggest that added GroEL participates in an equilibrium with endogenous chaperones in the cytosol which inhibit folding and promote import competence. Native gel electrophoresis suggests that nascent pmAspAT exists in RRL as a heterogeneous population of partially folded species, some of which bind to added GroEL more readily than others. The GroEL-trapped species appear to be among the productive pmAspAT folding intermediates formed in RRL or they at least appear to equilibrate with these intermediates, since they become import competent after GroES-stimulated release from GroEL.
Mattingly
J R
JR
Division of Molecular Biology and Biochemistry, School of Biological Sciences, University of Missouri-Kansas City 64110, USA.
Yañez
A J
AJ
Martinez-Carrion
M
M
D000818:Animals; D001219:Aspartate Aminotransferases / Q000737:chemistry*; D001692:Biological Transport; D002474:Cell-Free System; D018835:Chaperonin 10 / Q000378:metabolism*; D018834:Chaperonin 60 / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology*; D011485:Protein Binding; D014176:Protein Biosynthesis; D017510:Protein Folding; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D018835:Chaperonin 10; D018834:Chaperonin 60; D001219:Aspartate Aminotransferases