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 Esc herichia coli cells expressing all 14 secretion genes is mainly associ ated with the cytoplasmic membrane through both hydrophobic and non-hy drophobic interactions. Mutational analysis revealed that one of the t wo regions of PulE that are conserved in many nucleotide-binding prote ins (Walker box A) is essential for pullulanase secretion. Likewise, m utations that removed aspartate residues from each of two regions imme diately downstream from the Walker box A also reduced secretion. These aspartate-rich regions are highly conserved in all 16 known PulE homo logues but not in any other nucleotide-binding proteins. Altogether, t hese results indicate that PulE might belong to a new family of nucleo tide-binding proteins. The protein could not be cross-linked to the ph otoactivatable ATP analogue azido-ATP, however. Most pulE point or del etion mutations which prevented pullulanase secretion exhibited transd ominance when expressed at high levels in cells producing wild-type Pu lE protein. Evidence presented suggests that PulE might be a homodimer .