Affinity purification, overexpression, and characterization of chaperonin 10 homologues synthesized with and without N-terminal acetylation.
Utilizing the ability of bacterial chaperonin 60 (GroEL) to functionally interact with chaperonin 10 (Cpn10) homologues in an ATP-dependent fashion, we have purified substantial amounts of mammalian, chloroplast, and thermophilic Cpn10 homologues from their natural host. In addition, large amounts of recombinant rat Cpn10 were produced in Escherichia coli and found to be identical to its authentic counterpart except for the lack of N-terminal acetylation. By comparing these two forms of Cpn10, it was found that acetylation does not influence the oligomeric structure of Cpn10 and is not essential for chaperone activity or mitochondrial import in vitro. In contrast, N-terminal acetylation proved crucial in the protection of Cpn10 against degradation by N-ethylmaleimide-sensitive proteases derived from organellar preparations of rat liver. The availability of large amounts of both affinity-purified and recombinant Cpn10 will facilitate not only further characterization of the eukaryotic folding machinery but also further scrutiny of the reported function of Cpn10 as early pregnancy factor.
Ryan
M T
MT
School of Biochemistry, La Trobe University, Bundoora, Victoria, Australia.
Naylor
D J
DJ
Hoogenraad
N J
NJ
Høj
P B
PB
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D018835:Chaperonin 10 / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D018834:Chaperonin 60 / Q000302:isolation & purification; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents; D017931:DNA Primers; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli; D005033:Ethylmaleimide / Q000378:metabolism; D005976:Glutaral; D046911:Macromolecular Substances; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000302:isolation & purification; D016133:Polymerase Chain Reaction; D017510:Protein Folding; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013552:Swine
D018835:Chaperonin 10; D018834:Chaperonin 60; D003432:Cross-Linking Reagents; D017931:DNA Primers; D046911:Macromolecular Substances; D010455:Peptides; D011994:Recombinant Proteins; D008291:Malate Dehydrogenase; D010450:Endopeptidases; D005033:Ethylmaleimide; D005976:Glutaral