Molecular characterization of PulE, a protein required for pullulanase secretion.
pulE, one of 14 genes specifically required for pullulanase secretion in Klebsiella oxytoca, codes for a putative nucleotide-binding protein. Subcellular fractionation indicated that the majority of PulE in Escherichia coli cells expressing all 14 secretion genes is mainly associated with the cytoplasmic membrane through both hydrophobic and non-hydrophobic interactions. Mutational analysis revealed that one of the two regions of PulE that are conserved in many nucleotide-binding proteins (Walker box A) is essential for pullulanase secretion. Likewise, mutations that removed aspartate residues from each of two regions immediately downstream from the Walker box A also reduced secretion. These aspartate-rich regions are highly conserved in all 16 known PulE homologues but not in any other nucleotide-binding proteins. Altogether, these results indicate that PulE might belong to a new family of nucleotide-binding proteins. The protein could not be cross-linked to the photoactivatable ATP analogue azido-ATP, however. Most pulE point or deletion mutations which prevented pullulanase secretion exhibited transdominance when expressed at high levels in cells producing wild-type PulE protein. Evidence presented suggests that PulE might be a homodimer.
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Unité de Génétique Moléculaire, CNRS URA 1149, Institut Pasteur, Paris, France.
Pugsley
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AP
D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000235:genetics; D016384:Consensus Sequence; D029968:Escherichia coli Proteins; D006026:Glycoside Hydrolases / Q000378:metabolism; D007709:Klebsiella / Q000737:chemistry / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C072863:PulE protein, E coli; D000255:Adenosine Triphosphate; D006026:Glycoside Hydrolases; C100181:pullulanase; D000251:Adenosine Triphosphatases