The extreme C-terminus is required for secretion of both the native polygalacturonase (PehA) and PehA-Bla hybrid proteins in Erwinia carotovora subsp. carotovora.
A set of gene fusions was constructed between the pehA gene encoding the secreted endopolygalacturonase (PehA) and the bla gene coding for a normally periplasmic beta-lactamase (Bla). The resulting hybrid proteins were specifically and actively routed out of the cells via the Out-terminal branch of the general secretory pathway (GSP) in Erwinia carotovora subsp. carotovora (Ecc), provided that no more than the last two amino acids (aa) of the PehA domain were excluded from the fusion. However, both PehA-Bla hybrid proteins and PehA variants lacking at least four aa from the C-terminus of the PehA accumulated in the periplasm. Also, overexpression of the gene fusions prevented extracellular targeting of the hybrid proteins. Site-directed mutagenesis of the codons -4 and -3 (encoding Asn-373 and Val-374, respectively) from the end of the pehA gene and analysis of the protein products suggested that the Val-374 was important both for the structure and secretion of PehA, while the Asn-373 proved to be insignificant. We conclude that: (i) the GSP of Ecc is capable of secreting heterologous proteins; (ii) as the PehA protein can accommodate C-terminal extensions, secretion can occur with no part of the proposed targeting signal lying within the C-terminal extremity of a secreted molecule; and (iii) residues within the C-terminus of PehA play a role in secretion, possibly through stabilization of a structure needed for proper exposition of the proposed targeting motif.
Palomäki
T
T
Department of Biosciences, University of Helsinki, Finland.
Saarilahti
H T
HT
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004274:DNA, Recombinant / Q000235:genetics; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016973:Pectobacterium carotovorum / Q000201:enzymology* / Q000235:genetics*; D011096:Polygalacturonase / Q000235:genetics* / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D001618:beta-Lactamases / Q000235:genetics* / Q000378:metabolism*
D017931:DNA Primers; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; D011096:Polygalacturonase; D001618:beta-Lactamases