Assembly of heterodimeric luciferase after de novo synthesis of subunits in rabbit reticulocyte lysate involves Hsc70 and Hsp40 at a post-translational stage

Heterodimeric luciferase from Vibrio harveyi had been established as a uniq ue model enzyme for direct measurements of the effects of molecular chapero nes and folding catalysts on protein folding and subunit assembly after de novo synthesis of subunits in rabbit reticulocyte lysate. It was observed t hat luciferase assembly can be separated in time from synthesis of the two subunits and that under these post-translational conditions assembly was in hibited by either ATP depletion or inhibition of peptidylprolyl cis/trans i somerases, that is, by addition of cyclosporin A or FK506. Furthermore, it was observed that the inhibitory effect of FK506 on luciferase assembly can be suppressed by addition of purified cyclophilin, thereby providing the f irst direct evidence for the involvement of peptidylprolyl cis/trans isomer ases in protein biogenesis in the eukaryotic cytosol. Here the ATP requirem ent in luciferase assembly has been characterized. Depletion of either Hsp9 0 or CCT from reticulocyte lysate did not interfere with luciferase assembl y. However, addition of purified Hsc70 stimulated luciferase assembly. Whil e addition of purified Hsp40 did not have any effect on luciferase assembly , the stimulatory effect of Hsc70 was further increased by Hsp40. Thus, aft er synthesis of the two subunits in reticulocyte lysate assembly of heterod imeric luciferase involves Hsc70 and its co-chaperone Hsp40. Therefore, Hsc 70 aids protein biogenesis in the eukaryotic cytosol not only at the levels of nascent polypeptide chains and precursor proteins that have to be kept competent for transport into cell organelles, but also at the level of subu nits that have to be kept competent for assembly.