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

doi:10.1074/jbc.271.38.23487

Luciferase Assembly after Transport into Mammalian Microsomes Involves Molecular Chaperones and Peptidyl-Prolyl cis/trans-Isomerases*

From the Medizinische Biochemie, Universität des Saarlandes, D-66421 Homburg,
^‡ Institut für Biochemie und Molekulare Zellbiologie, Universität Göttingen, Goßlerstrasse 12d, D-37073 Göttingen, Germany, and the
^§ Plant Molecular Genetics Laboratories, Department of Plant Science, University of Alberta, Edmonton, Alberta T6G 2P5, Canada

^∥ To whom correspondence should be addressed. Tel.: 49/6841/166510; Fax: 49/6841/166288.

↵^¶ Present address: Center for Molecular Biology and Gene Therapy, Loma Linda University, Loma Linda, CA 92350.

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.

Footnotes

↵* This work was supported by Deutsche Forschungsgemeinschaft Grant SFB 236, Fonds der Chemischen Industrie and Human Frontier Science Program Organization grants (to R. Z.), and by National Science and Engineering Research Council and Canadian Bacterial Diseases Network Centers for Excellence grants (to A. A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

PPIase

peptidylprolyl cis/trans-isomerase

RM

rough microsomes

PKRM

puromycin/high salt-treated rough microsomes

Me₂SO

dimethyl sulfoxide

ATPγS

adenosine 5′-O-(thio)triphosphate

FKBPs

FK506 binding proteins

CsA

cyclosporin A.
↵² J. Solsbacher and R. Zimmermann, unpublished results.
- Received February 26, 1996.
- Revision received May 14, 1996.