title: The extreme N-terminus of the Caulobacter crescentus surface-layer protein directs export of passenger proteins from the cytoplasm but is not required for secretion of the native protein.
abstract: The paracrystalline surface layer (S-layer) of Caulobacter crescentus is composed of a single protein (RsaA, 1026 amino acids) that associates noncovalently with the lipopolysaccharide of the outer membrane. Like many other extracellular proteins of Gram-negative bacteria, the S-layer protein is not processed during transport to the cell surface. To study the secretion of RsaA, several N-terminal deletions of the protein were made by modifying the 5'-region of the rsaA gene. This analysis showed that portions of the N-terminus totalling the first 775 N-terminal amino acids (75% of the protein) could be removed from RsaA without abolishing secretion of the remainder of the protein. Although the RsaA N-terminus was not required for secretion, an N-terminal domain consisting of either 34 or 52 RsaA-derived amino acids promoted export of the alkaline phosphatase reporter (PhoA) and a cellulase reporter (delta CenA) from the cytoplasm; using the cellulase reporter, the efficiency of hybrid protein export was estimated at 9%. No enzyme activity was detected in the cell-free culture fluids as the result of expressing any gene fusion, indicating that no hybrid protein was completely secreted from the cell. RsaA:PhoA hybrid proteins were also exported from the E. coli cytoplasm, a bacterium not expected to contain the necessary machinery for the secretion of RsaA. Taken together, these data indicate that the secretion pathway of RsaA relies on a C-terminal secretion signal and that once separated from the context of the native protein, the extreme N-terminus of RsaA can act as an inefficient cryptic export signal that is not used during native RsaA secretion.
authors.affiliation: Department of Microbiology and Immunology, University of British Columbia, Vancouver, Canada.
authors.forename: W H
authors.initials: WH
authors.lastname: Bingle
authors.forename: K D
authors.initials: KD
authors.lastname: Le
authors.forename: J
authors.initials: J
authors.lastname: Smit
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D016935:Caulobacter crescentus / Q000235:genetics / Q000378:metabolism*; D002480:Cellulase / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D017930:Genes, Reporter; D007460:Iodoacetamide / Q000494:pharmacology; D008562:Membrane Glycoproteins; D008854:Microscopy, Electron; D008858:Microscopy, Phase-Contrast; D009154:Mutation; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D017384:Sequence Deletion
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C055764:S-layer proteins; D000469:Alkaline Phosphatase; D002480:Cellulase; D004229:Dithiothreitol; D007460:Iodoacetamide