Folding and assembly of bacterial alkaline phosphatase in vitro and in vivo.
Alkaline phosphatase (PhoA), localized in the periplasmic space of Escherichia coli, is a homodimeric metalloprotein containing two intramolecular disulfide bonds. We attempted to clarify the folding-assembly pathways of this enzyme by allowing in vitro-synthesized PhoA polypeptide to fold into active enzyme and by examining the occurrence of similar pathways in vivo by pulse-chase experiments. PhoA (lacking the signal sequence) that was synthesized in a coupled transcription-translation system was effectively converted into active enzyme when incubated with either oxidized glutathione, periplasmic proteins, or purified DsbA protein in the presence of Zn2+. The first appreciable event in the activation of initially unfolded translation product (species I) was the disulfide bond formation, which was immediately followed by folding into a partially trypsin-resistant monomer (species II), and then by assembly into active dimer (species III). The species II PhoA molecules, but not the species III molecules, were found to be sensitized to trypsin in the presence of a reducing agent, dithiothreitol. Pulse-chase studies showed that PhoA acquires disulfide bonds immediately after the biosynthesis, whereas it acquires resistance to both dithiothreitol and certain endogenous protease over some 2 min at 15 degrees C. These results indicate that PhoA undergoes a series of folding-assembly steps, some of which are of measurable speeds in vivo and mimicable in vitro.
Akiyama
Y
Y
Department of Cell Biology, Kyoto University, Japan.
Ito
K
K
D000469:Alkaline Phosphatase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D002499:Centrifugation, Density Gradient; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D007535:Isomerases / Q000378:metabolism; D007700:Kinetics; D019704:Protein Disulfide-Isomerases; D017510:Protein Folding
D004220:Disulfides; D000469:Alkaline Phosphatase; D007535:Isomerases; D019704:Protein Disulfide-Isomerases; D004229:Dithiothreitol