ENZYME ASSEMBLY AFTER DE-NOVO SYNTHESIS IN RABBIT RETICULOCYTE LYSATEINVOLVES MOLECULAR CHAPERONES AND IMMUNOPHILINS

The folding kinetics of two luciferases were studied after synthesis i n reticulocyte lysates to investigate whether molecular chaperones and /or folding catalysts are involved in the folding reactions. Two bacte rial luciferases were used as model proteins: heterodimeric Vibrio har veyi luciferase (LuxAB), and a monomeric luciferase fusion protein (Fa b2). Data indicate that folding of these enzymes to the native state o ccurs in the translation system, and that the extent of folding can be quantified It was found that (i) folding of LuxAB and Faba can clearl y be separated in time from synthesis, (ii) folding of Faba and LuxAB is slow because it involves either transient (Faba) or permanent (LuxA B) interaction of polypeptides, (iii) preservation of the assembly com petent state of LuxA and/or LuxB and folding of Faba depend on ATP-hyd rolysis, (iv) folding of Fab2 and LuxAB is partially sensitive to cycl osporin A (CsA) and FK506, i.e. inhibitors of two distinct peptidylpro lyl cis/trans-isomerases. Thus, bacterial luciferases provide a unique system for direct measurement of the effects of ATP-dependent molecul ar chaperones on protein folding and enzyme assembly in reticulocyte l ysates. Furthermore, these two luciferases provide the first direct ev idence documenting the involvement of peptidylprolyl cis/trans-isomera ses in protein biogenesis in a eukaryotic cytosol.