LUCIFERASE ASSEMBLY AFTER TRANSPORT INTO MAMMALIAN MICROSOMES INVOLVES MOLECULAR CHAPERONES AND PEPTIDYL-PROLYL CIS TRANS-ISOMERASES/

The assembly of a heterodimeric luciferase was studied after de novo s ynthesis of corresponding precursor proteins in reticulocyte lysate an d concomitant transport into dog pancreas microsomes. This cytosolic l uciferase from a prokaryotic organism (Vibrio harveyi) was specificall y used as a model protein to investigate (i) whether the eukaryotic cy tosol and the microsomal lumen have similar folding capabilities and ( ii) whether the requirements of a polypeptide for certain molecular ch aperones and folding catalysts are determined by the polypeptide or th e intracellular compartment, The two luciferase subunits were fused to the preprolactin signal peptide, Data indicate that efficient assembl y of luciferase occurs in the mammalian microsomes, Furthermore, it wa s observed that luciferase assembly can be separated in time from synt hesis and membrane transport, depends on ATP hydrolysis, is partially sensitive to cyclosporin A and FK506, and in the absence of lumenal pr oteins is less efficient as compared with the presence of lumenal prot eins, Thus, heterodimeric luciferase depends on functionally related m olecular chaperones and folding catalysts during its assembly in eithe r the eukaryotic cytosol or the microsomal lumen.