Luciferase Assembly after Transport into Mammalian Microsomes Involves Molecular Chaperones and Peptidyl-Prolyl Isomerases

Abstract

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...

Characterization of pancreatic ERj3p, ahomolog of yeast DnaJ-like protein Scj1p

“…Where indicated, dog pancreas microsomes and the acceptor peptide NYT (Brunke et al, 1996) were present during translation. Where indicated, dog pancreas microsomes and the acceptor peptide NYT (Brunke et al, 1996) were present during translation.…”

A Scj1p Homolog and Folding Catalysts Present in Dog Pancreas Microsomes

“…Previously, we have presented indirect evidence for the presence of members of the two families of peptidylprolylcis/trans-isomerases in dog pancreas microsomes, cyclophilins and FK506 binding proteins (Klappa et al, 1995;Brunke et al, 1996;Klappa et al, 1996). Here, we employed fractionation of microsomal detergent extracts, prepared under high salt conditions, on cyclosporin Aand ascomycin-affinity resins, followed by gel electro-Folding Catalysts in Dog Pancreas Microsomes 1177 Lumenal proteins which had been extracted from dog pancreas microsomes under low salt conditions were seperated in isoelectric focussing gels (1. dimension).…”

“…Most studies on protein transport into the mammalian endoplasmic reticulum (Blobel and Dobberstein, 1975) as well as many studies on protein folding (Bulleid and Freedman, 1988;Kassenbrock et al, 1988;Brunke et al, 1996;Tyedmers et al, 1996) and on protein degradation (Lyko et al, 1995;Klappa et al, 1996;Xiong et al, 1999) in the mammalian ER are carried out in vitro and employ dog pancreas microsomes. In order to eventually arrive at a full picture of the transport and folding processes we need to know the components (i. e. the ER proteome), their various functions and their respective stoichiometries.…”

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

“…Various laboratories started to use light emitting luciferases in order to address questions related to folding in either the mammalian cytosol (by employing rabbit reticulocyte lysate or wheat germ lysate as a translation and folding system) or in the mammalian endoplasmic reticulum (by employing rabbit reticulocyte lysate as a system for synthesis of precursor proteins and dog pancreas microsomes as a folding system). Specifically, it was asked which molecular chaperones and folding catalysts are involved in folding and assembly of a monomeric or a heterodimeric luciferase [4–10]. Heterodimeric luciferase is a cytosolic enzyme in Vibrio harveyi and catalyzes oxygen‐dependent and FMNH 2 ‐dependent conversion of a long‐chain aldehyde to the corresponding fatty acid.…”

“…In both compartments PPIases appeared not to be involved in the folding of firefly luciferase [8]. In contrast, formation of enzymatically active heterodimeric luciferase was found to involve PPIases in the mammalian cytosol [6] as well as in mammalian microsomes [7]. Furthermore, in both compartments assembly of heterodimeric luciferase was found to depend on ATP hydrolysis, suggesting that molecular chaperones are involved [6,7].…”