Secretory S complex of Bacillus subtilis forms a large, organized structure when released from ribosomes.

Abstract

The S complex of Bacillus subtilis, a set of four proteins that appears to be involved in protein secretion, is shown to be attached to 70S ribosomes: antibody to its 64-kDa component can aggregate these ribosomes, and the complex can be chemically crosslinked to ribosomal proteins. Low Mg2+ or prolonged high-speed centrifugation in a sucrose gradient releases the S complex from the ribosomes, and it is recovered as an aggregate with an S value of 76. Electron microscopy shows that these aggregates have a regular structure, somewhat resembling clathrin cages, with a diameter of about 45 nm. If these aggregates are physiological, their function would differ significantly from that of the signal recognition particle of eukaryotes.A 64-kDa protein of Bacillus subtilis, present in the membrane fraction complexed with ribosomes (complexed membrane, CM), appears to be involved in protein secretion since the ribosomes protect it from interaction with protease or antibody (1); it is also present in the cytosol (2, 3). In addition, this protein is present in a complex of four proteins (the S complex), of 64, 60, 41, and 36 kDa, found in large amounts in the membrane-free monosomes (but not in the membrane-polysome complexes or in the cytosol) (3). These findings suggest that the S complex functions in the initiation step in protein secretion, like the signal recognition particle of eukaryote cells (4), but it differs significantly: it lacks an RNA molecule (at least as isolated), and it failed to cause translational arrest at the signal sequence of a secretory protein (3). The distribution of the 64-kDa protein further suggests that in the initiation cycle the other proteins of the S complex may be released after contact with the membrane, while the 64-kDa protein remains attached and then is released at a later stage. This paper shows that the S complex separates from the ribosomes when they are dissociated into subunits by low Mg2+ or by high-speed centrifugation. However, the complex then sediments at about 76 S, rather than at the expected low values. Since the S complex in the monosome fraction can be chemically crosslinked to certain ribosomal proteins and anti-64-kDa IgG can aggregate the monosomes (as shown by electron microscopy), the 76S particle is evidently an aggregate of S complex, formed after release from attachment to the ribosomes. Moreover, after release, but not in the monosome fraction, the S complex is seen in the electron microscope as a specific, organized structure, larger than the ribosome. It is intriguing that this structure somewhat resembles the clathrin cages of eukaryotic cells, which have been implicated in membrane traffic (5 buffer. To decrease contamination with 50S subunits and disomes the preparation was passed through a second 10-30% sucrose gradient and the 70S region was recovered. In another method for preparing monosomes associated with S complex, the initial supernate from the lysate, after the bulk ofthe membrane/ribosomes had been removed by a 1-hr centrifugation (3),...

Purification and characterization of the flagellar hook–basal body complex of Bacillus subtilis

“…The amino acid sequencing revealed that the 65 kDa protein was pyruvate dehydrogenase C (PdhC; data not shown), which has been known to be a component of the membrane-bound S complex (Hemila et al, 1990). By electron microscopy, among the HBB complexes we observed many small particles of c. 45 nm in diameter, as described previously (Caulfield et al, 1985). Thus, the S complex seemed to be co-isolated with the HBB complex.…”

Nonclassical Protein Secretion by Bacillus subtilis in the Stationary Phase Is Not Due to Cell Lysis

“…A recent report introduced a new prediction algorithm for the identification of nonclassical secretory proteins; the new algorithm is based on biological and chemical properties, such as threonine contents, trans-membrane helices, and protein disorder in the structure (6). It is noted that enolase, pyruvate dehydrogenase (S-complex) (10,11,17), and GroEL are predicted to be nonsecreted proteins using this prediction algorithm, while in our study, they are identified as abundant, secreted proteins.…”

Secretory S complex of Bacillus subtilis: sequence analysis and identity to pyruvate dehydrogenase

“…The B. stearothermophilus PDH complex sediments at 75S, and in electron microscopy these complexes have a diameter of 40 nm (29). Similarly, the S complex, released from the ribosomes by a low concentration of Mge' ions, can be recovered as particles sedimenting at 76S and having a diameter of 45 nm in electron microscopy (14). Furth'ermore, the PDH complex from B. stearothermophilus shows a striking resemblance to the mammalian (mitochondrial) PDH in terms of morphology, subunit composition, and molecular weight, sharply contrasting with the E. coli PDH, which consists of only three subunits (29).…”