Secretion of the cell surface lipoprotein pullulanase in Escherichia coli. Cooperation or competition between the specific secretion pathway and the lipoprotein sorting pathway.
The fatty acid-acylated enzyme pullulanase is normally found in either of two locations in Escherichia coli, depending on whether or not the producing strains also express the genes specifically required for the second step in pullulanase secretion. When they are expressed, the enzyme is localized to the cell surface, while in their absence, it is directed to an unidentified location in the cell envelope which, upon lysis, forms vesicles whose density is intermediate between those of outer and cytoplasmic membrane vesicles. In order to test the role of the putative lipoprotein sorting signal, Asp2, in pullulanase sorting and secretion, the structural gene (pulA) was subjected to site-directed mutagenesis. Replacement of the Asp2 residue by Asn, Glu, or Ser caused the enzyme to fractionate with outer membrane-derived vesicles rather than with intermediate density vesicles from E. coli cells devoid of pullulanase secretion genes. A pronounced secretion defect was observed in a two-step secretion assay in which the first (sec gene-dependent) and second (pul gene-dependent) secretion steps were uncoupled. We propose that the Asp residue increases the efficiency of pullulanase secretion by allowing the enzyme to be initially sorted to a region of the cell envelope wherein most of the pullulase-specific secretion factors are located.
Pugsley
A P
AP
Unité de Génétique Moléculaire (Centre National de la Recherche Scientifique UA1149), Institut Pasteur, Paris, France.
Kornacker
M G
MG
D001483:Base Sequence; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000201:enzymology; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism*; D008074:Lipoproteins / Q000378:metabolism*; D009841:Oligonucleotides / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D008074:Lipoproteins; D009841:Oligonucleotides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D006026:Glycoside Hydrolases; C100181:pullulanase