Essential role of a sodium dodecyl sulfate-resistant protein IV multimer in assembly-export of filamentous phage.
Filamentous phage f1 encodes protein IV (pIV), a protein essential for phage morphogenesis that localizes to the outer membrane of Escherichia coli, where it is found as a multimer of 10 to 12 subunits. Introduction of internal His or Strep affinity tags at different sites in pIV interfered with its function to a variable extent. A spontaneous second-site suppressor mutation in gene IV allowed several different insertion mutants to function. The identical mutation was also isolated as a suppressor of a multimerization-defective missense mutation. A high-molecular-mass pIV species is the predominant form of pIV present in cells. This species is stable in 4% sodium dodecyl sulfate at temperatures up to 65 degrees C and is largely preserved at 100 degrees C in Laemmli protein sample buffer containing 4% sodium dodecyl sulfate. The suppressor mutation makes the high-molecular-mass form of wild-type pIV extremely resistant to dissociation, and it stabilizes the high-molecular-mass form of several mutant pIV proteins to extents that correlate with their level of function. Mixed multimers of pIV(f1) and pIV(Ike) also remain associated during heating in sodium dodecyl sulfate-containing buffers. Thus, sodium dodecyl sulfate- and heat-resistant high-molecular-mass pIV is derived from pIV multimer and reflects the physiologically relevant form of the protein essential for assembly-export.
Linderoth
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Laboratory of Genetics, The Rockefeller University, New York, New York 10021-6399, USA.
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Russel
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D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western / Q000379:methods; D002213:Capsid / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000502:physiology*; D003090:Coliphages / Q000737:chemistry / Q000502:physiology*; D004591:Electrophoresis, Polyacrylamide Gel; D005990:Glycerol / Q000494:pharmacology; D006361:Heating; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013329:Structure-Activity Relationship; D014508:Urea / Q000494:pharmacology; D019065:Virus Assembly / Q000502:physiology*
D009838:Oligodeoxyribonucleotides; D012967:Sodium Dodecyl Sulfate; D014508:Urea; D005990:Glycerol