Biotinylation in vivo as a sensitive indicator of protein secretion and membrane protein insertion.
Escherichia coli biotin ligase is a cytoplasmic protein which specifically biotinylates the biotin-accepting domains from a variety of organisms. This in vivo biotinylation can be used as a sensitive signal to study protein secretion and membrane protein insertion. When the biotin-accepting domain from the 1.3S subunit of Propionibacterium shermanii transcarboxylase (PSBT) is translationally fused to the periplasmic proteins alkaline phosphatase and maltose-binding protein, there is little or no biotinylation of PSBT in wild-type E. coli. Inhibition of SecA with sodium azide and mutations in SecB, SecD, and SecF, all of which slow down protein secretion, result in biotinylation of PSBT. When PSBT is fused to the E. coli inner membrane protein MalF, it acts as a topological marker: fusions to cytoplasmic domains of MalF are biotinylated, and fusions to periplasmic domains are generally not biotinylated. If SecA is inhibited by sodium azide or if the SecE in the cell is depleted, then the insertion of the MalF second periplasmic domain is slowed down enough that PSBT fusions in this part of the protein become biotinylated. Compared with other protein fusions that have been used to study protein translocation, PSBT fusions have the advantage that they can be used to study the rate of the insertion process.
Jander
G
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Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Cronan
J E
JE
Beckwith
J
J
D000251:Adenosine Triphosphatases / Q000502:physiology; D001426:Bacterial Proteins / Q000378:metabolism* / Q000502:physiology; D001692:Biological Transport; D001710:Biotin / Q000378:metabolism*; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins; D008565:Membrane Proteins / Q000378:metabolism*; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D000069816:SEC Translocation Channels; C065493:SecE protein, E coli; D001710:Biotin; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins