PROCESSING AND METHYLATION OF PULG, A PILIN-LIKE COMPONENT OF THE GENERAL SECRETORY PATHWAY OF KLEBSIELLA-OXYTOCA

被引:77
作者
PUGSLEY, AP
机构
[1] Unité de Génétique Moléculaire, Institut Pasteur, Paris, 75724
关键词
D O I
10.1111/j.1365-2958.1993.tb01691.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The signal sequence of the Klebsiella oxytoca pulG gene product, which is required for extracellular secretion of the enzyme pullulanase, is similar in many respects to the corresponding segment of the precursors of type IV (me-Phe) pilins. The significance of this similarity is confirmed by the observation that the pu10 gene product processes prePulG at the consensus type IV prepilin peptidase cleavage site at the amino-terminal end of the PulG signal sequence. Like most type IV pilins, processed PulG was found to have a methylated amino-terminal phenylalanine residue. Site-directed mutagenesis was used to replace amino acids in prePulG that correspond to residues shown by others to be essential for processing, methylation and assembly of type IV pilins. The glycine residue on the amino-terminal side of the prePulG cleavage site is absolutely required for processing and for pullulanase secretion. The glutamate residue at position 11(+5) is also required for pullulanase secretion but not for processing or methylation. This result contrasts with that reported for corresponding variants of Pseudomonas aeruginosa type IV prepilin, which were processed but only inefficiently N-methylated. Cleavage of prePulG and pullulanase secretion were both unaffected by replacement of the phenylalanine residue on the carboxy-terminal side of the cleavage site by leucine, isoleucine or valine, by a conservative substitution within the hydrophobic core of the prePulG signal sequence, or by a glutamine to proline substitution within the processed segment. However, replacement of the same glutamine residue by arginine abolished secretion without affecting either processing or methylation.
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页码:295 / 308
页数:14
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