Efficient Folding of Firefly Luciferase after Transport into Mammalian Microsomes in the Absence of Luminal Chaperones and Folding Catalysts

Abstract

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

Cotranslational Protein Folding

“…Firefly luciferase (57) and hydroid obelin 2 fold much more efficiently during synthesis than during renaturation under the same conditions. Firefly luciferase also folds efficiently upon translocation into proteoliposomes depleted of chaperones (58). These observations imply a crucial role for vectorial folding of nascent chains.…”

A Scj1p Homolog and Folding Catalysts Present in Dog Pancreas Microsomes

“…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

“…Again, we observed that the PPIases acted rather early in the folding process. We note that we did not detect any role for PPIases in folding of firefly luciferase under our experimental conditions [8] and that the three‐dimensional structure of firefly luciferase did not reveal any prolyl‐containing peptide bonds in the cis configuration [11].…”

“…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.…”

“…Following de novo synthesis in rabbit reticulocyte lysate it was observed that folding of monomeric luciferase depends on the highly organized chaperone machinery, comprising Hsp40, Hsp60 (CCT) and Hsc70, in the mammalian cytosol [4] but not in mammalian microsomes [8]. 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].…”