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 unique model enzyme for direct measurements of the effects of molecular chaperones and folding catalysts on protein folding and subunit assembly after de novo synthesis of subunits in rabbit reticulocyte lysate. It was observed that luciferase assembly can be separated in time from synthesis of the two subunits and that under these post-translational conditions assembly was inhibited by either ATP depletion or inhibition of peptidylprolyl cis/trans isomerases, 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 first direct evidence for the involvement of peptidylprolyl cis/trans isomerases in protein biogenesis in the eukaryotic cytosol. Here the ATP requirement in luciferase assembly has been characterized. Depletion of either Hsp90 or CCT from reticulocyte lysate did not interfere with luciferase assembly. However, addition of purified Hsc70 stimulated luciferase assembly. While addition of purified Hsp40 did not have any effect on luciferase assembly, the stimulatory effect of Hsc70 was further increased by Hsp40. Thus, after synthesis of the two subunits in reticulocyte lysate assembly of heterodimeric luciferase involves Hsc70 and its co-chaperone Hsp40. Therefore, Hsc70 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 subunits that have to be kept competent for assembly.
Tyedmers
J
J
Medizinische Biochemie und Molekularbiologie, Universität des Saarlandes, Homburg, Germany.
Kruse
M
M
Lerner
M
M
Demand
J
J
Höhfeld
J
J
Solsbacher
J
J
Volkmer
J
J
Zimmermann
R
R
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D018834:Chaperonin 60 / Q000378:metabolism; D016572:Cyclosporine / Q000494:pharmacology; D019281:Dimerization; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D007700:Kinetics; D008156:Luciferases / Q000096:biosynthesis / Q000187:drug effects / Q000378:metabolism*; D019696:Peptidylprolyl Isomerase / Q000378:metabolism; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D012156:Reticulocytes; D016559:Tacrolimus / Q000494:pharmacology
D018834:Chaperonin 60; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D016572:Cyclosporine; D000255:Adenosine Triphosphate; D008156:Luciferases; D019696:Peptidylprolyl Isomerase; D016559:Tacrolimus