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

• Published in: Canadian journal of microbiology Vol. 42; no. 7; p. 672
• Main Authors: Bingle, W H, Le, K D, Smit, J
• Format: Journal Article
• Language: English
• Published: Canada 01-07-1996
• Subjects: Alkaline Phosphatase - genetics; Alkaline Phosphatase - metabolism; Bacterial Outer Membrane Proteins - chemistry; Bacterial Outer Membrane Proteins - genetics; Bacterial Outer Membrane Proteins - metabolism; Bacterial Proteins - metabolism; Caulobacter crescentus - genetics; Caulobacter crescentus - metabolism; Cellulase - genetics; Cellulase - metabolism; Cloning, Molecular; Cytoplasm - metabolism; Dithiothreitol - pharmacology; Genes, Reporter; Iodoacetamide - pharmacology; Membrane Glycoproteins; Microscopy, Electron; Microscopy, Phase-Contrast; Mutation; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - pharmacology; Sequence Deletion
• ISSN: 0008-4166
• DOI: 10.1139/m96-092

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.