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The assembly of a heterodimeric luciferase was studied after de novo synthesis of corresponding precursor proteins in reticulocyte lysate and concomitant transport into dog pancreas microsomes. This cytosolic luciferase from a prokaryotic organism (Vibrio harveyi) was specifically used as a model protein to investigate (i) whether the eukaryotic cytosol and the microsomal lumen have similar folding capabilities and (ii) whether the requirements of a polypeptide for certain molecular chaperones and folding catalysts are determined by the polypeptide or the intracellular compartment. The two luciferase subunits were fused to the preprolactin signal peptide. Data indicate that efficient assembly of luciferase occurs in the mammalian microsomes. Furthermore, it was observed that luciferase assembly can be separated in time from synthesis and membrane transport, depends on ATP hydrolysis, is partially sensitive to cyclosporin A and FK506, and in the absence of lumenal proteins is less efficient as compared with the presence of lumenal proteins. Thus, heterodimeric luciferase depends on functionally related molecular chaperones and folding catalysts during its assembly in either the eukaryotic cytosol or the microsomal lumen.In comparison to our knowledge about protein folding after denaturation and subsequent renaturation (Jaenicke, 1987), little is known about protein folding and assembly in the different compartments of the eukaryotic cell, following de novo synthesis of polypeptides. In general, the latter appears to be assisted by various molecular chaperones and folding catalysts (Ellis and van der Vies, 1991;Georgopoulos and Welch, 1993;Hartl et al., 1994;Kunz and Hall, 1993;Schreiber, 1991). However, the intracellular compartments are different from each other with respect to their specific set of molecular chaperones and folding catalysts (Gething and Sambrook, 1992).Recently, we studied the folding and assembly of newly synthesized proteins in the eukaryotic cytosol by employing rabbit reticulocyte lysate as a translation and folding system (Kruse et al., 1995). We asked (i) what are the kinetics of folding and assembly of two model proteins, and (ii) are ATP-dependent molecular chaperones and/or folding catalysts involved in the folding and assembly reactions? In our studies two bacterial luciferases were used as model proteins. The first luciferase was a heterodimeric enzyme (LuxAB) from Vibrio harveyi (Waddle et al., 1987;Escher et al., 1989;Flynn et al., 1993;Ziegler et al., 1993). The second luciferase was a fusion protein (Fab2) that forms a monomeric enzyme comprising LuxA and LuxB (Escher et al., 1989). Both enzymes catalyze the oxygenand FMNH 2 -dependent conversion of a long chain aldehyde to the corresponding fatty acid with concomitant emission of light (490 nm). The genes coding for LuxA, LuxB, and Fab2 were cloned into plasmids that are suitable for in vitro transcription. The plasmids were used to program coupled transcription/ translation in rabbit reticulocyte lysates. The kinetics of foldin...

Folding of polypeptides emerging from the protein translocase in the membrane of mammalian microsomes was analyzed after synthesis of corresponding precursor proteins in a mammalian translation system. Firefly luciferase was used as a model protein; the corresponding hybrid precursor contained the preprolactin signal peptide. The rates and efficiencies of folding of luciferase in microsomes were compared with those of folding of luciferase in the cytosol. Furthermore, folding of luciferase in microsomes was compared with that in proteoliposomes, i.e. in the absence of luminal molecular chaperones and folding catalysts. Folding in microsomes was less efficient compared with folding in the cytosol. Folding in the absence of luminal proteins was more efficient compared with folding in their presence and identical to folding in the cytosol. Thus, firefly luciferase emerging from translocase can efficiently fold to its native conformation without chaperoning by any luminal proteins. There may be molecular chaperones present in the microsomal membrane that can efficiently substitute for the cytosolic chaperone machinery comprising Hsp40, Hsp60, and Hsp70 with respect to folding of firefly luciferase.Little is known about protein folding and subunit assembly in the various compartments of the eukaryotic cell following de novo synthesis of polypeptides. Nevertheless, the latter is generally assumed to be assisted by molecular chaperones and folding catalysts (Ellis and van der

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