Localization of the type I restriction-modification enzyme EcoKI in the bacterial cell.
To localise the type I restriction-modification (R-M) enzyme EcoKI within the bacterial cell, the Hsd subunits present in subcellular fractions were analysed using immunoblotting techniques. The endonuclease (ENase) as well as the methylase (MTase) were found to be associated with the cytoplasmic membrane. HsdR and HsdM subunits produced individually were soluble, cytoplasmic polypeptides and only became membrane-associated when coproduced with the insoluble HsdS subunit. The release of enzyme from the membrane fraction following benzonase treatment indicated a role for DNA in this interaction. Trypsinization of spheroplasts revealed that the HsdR subunit in the assembled ENase was accessible to protease, while HsdM and HsdS, in both ENase and MTase complexes, were fully protected against digestion. We postulate that the R-M enzyme EcoKI is associated with the cytoplasmic membrane in a manner that allows access of HsdR to the periplasmic space, while the MTase components are localised on the inner side of the plasma membrane.
Holubová
I
I
Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, Prague 4, 14220, Czech Republic.
Vejsadová
S
S
Weiserová
M
M
Firman
K
K
D001426:Bacterial Proteins / Q000302:isolation & purification; D002451:Cell Compartmentation; D003593:Cytoplasm / Q000201:enzymology; D004262:DNA Restriction Enzymes / Q000302:isolation & purification*; D015280:DNA Restriction-Modification Enzymes / Q000302:isolation & purification; D015253:Deoxyribonucleases, Type I Site-Specific; D004720:Endonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000302:isolation & purification; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000302:isolation & purification*
D001426:Bacterial Proteins; D015280:DNA Restriction-Modification Enzymes; D029968:Escherichia coli Proteins; C113046:HSDS protein, Bacteria; D008565:Membrane Proteins; C093244:DNA modification methylase EcoKI; C109829:HsdM protein, Bacteria; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D004720:Endonucleases; D004262:DNA Restriction Enzymes; C089629:endodeoxyribonuclease EcoKI; D015253:Deoxyribonucleases, Type I Site-Specific; C109830:HsdR protein, E coli