The role of the lipoprotein sorting signal (aspartate +2) in pullulanase secretion.
The analyses of hybrid proteins and of deletion and insertion mutations reveal that the only amino acid at the amino-proximal end of the cell surface lipoprotein pullulanase that is specifically required for its extracellular secretion is an aspartate at position +2, immediately after the fatty acylated amino-terminal cysteine. To see whether the requirement for this amino acid is related to its proposed role as a cytoplasmic membrane lipoprotein sorting signal, we used sucrose gradient floatation analysis to determine the subcellular location of pullulanase variants (with or without the aspartate residue) that accumulated in cells lacking the pullulanase-specific secretion genes. A non-secretable pullulanase variant with a serine at position +2 cofractionated mainly with the major peak of outer membrane porin. In contrast, most (55%) of a pullulanase variant with an aspartate at position +2 cofractionated with slightly lighter fractions that contained small proportions of both outer membrane porin and the cytoplasmic membrane marker NADH oxidase. Only 5% of this pullulanase variant cofractionated with the major NADH oxidase peak, while the rest (c. 40%) remained at the bottom of the gradient in fractions totally devoid of porin and NADH oxidase. When analysed by sedimentation through sucrose gradients, however, a large proportion of this variant was recovered from fractions near the top of the gradient that also contained the major NADH oxidase peak. When this peak fraction was applied to a floatation gradient the pullulanase activity remained at the bottom while the NADH oxidase floated to the top.(ABSTRACT TRUNCATED AT 250 WORDS)
Poquet
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I
Unité de Génétique Moléculaire (CNRS-URA1149), Institut Pasteur, Paris, France.
Kornacker
M G
MG
Pugsley
A P
AP
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D014644:Genetic Variation; D006026:Glycoside Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D008074:Lipoproteins / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016254:Mutagenesis, Insertional; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals / Q000378:metabolism*; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid
D008074:Lipoproteins; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D001224:Aspartic Acid; D006026:Glycoside Hydrolases; C100181:pullulanase