The fate of extracellular proteins tagged by the SsrA system of Bacillus subtilis.
In bacteria, SsrA, a highly conserved RNA molecule, functions in a mechanism meant to rescue stalled ribosomes. In this process, a peptide tag encoded by SsrA is cotranslationally added to truncated polypeptides, thereby targeting these molecules for proteolytic degradation, at least when they stay inside the cell. This study examined the fate of two extracellular proteins that were tagged by the SsrA system of Bacillus subtilis. Gene constructs encoding human interleukin-3 (hIL-3) fused to a signal peptide and B. subtilis alpha-amylase, both lacking an in-frame stop codon, were used as models to achieve ribosome stalling and activation of the SsrA system. Introduction of these gene constructs into B. subtilis led to tagging of the gene products by SsrA RNA. The tagged protein products bound to antibodies that were raised against the proteolysis tag encoded by B. subtilis SsrA [(A)GKTNSFNQNVALAA]. The apolar C-terminal SsrA-tag does not function as a specific signal for proteolytic degradation of SsrA-tagged amylase directly after trans-translation or during the secretion process. Also, SsrA-tagged amylase appeared to be very stable once outside the cell. In contrast, hIL-3 molecules tagged with the native, apolar SsrA-tag were considerably less stable than hIL-3 molecules that received a negatively charged control tag. Not one specific protease, but several non-specific proteases seem to play a role in the rapid degradation of SsrA-tagged hIL-3. The polarity of the C-terminus of heterologous hIL-3 protein proved to be an important determinant for protein stability when produced by B. subtilis. As observed previously in Escherichia coli and B. subtilis, SsrA tagging also occurs frequently in normally growing Gram-positive bacilli and it appears that intracellular proteins are the predominant natural substrates of SsrA.
Kolkman
Marc A B
MAB
Genencor International BV, Archimedesweg 30, 2333 CN Leiden, The Netherlands.
Ferrari
Eugenio
E
Genencor International, Inc., 925 Page Mill Road, Palo Alto, CA 94304, USA.
D001412:Bacillus subtilis / Q000235:genetics*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D012329:RNA, Bacterial / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000516:alpha-Amylases / Q000235:genetics
D004269:DNA, Bacterial; D009838:Oligodeoxyribonucleotides; D012329:RNA, Bacterial; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C096222:tmRNA; D000516:alpha-Amylases