A ribosome–nascent chain sensor of membrane protein biogenesis in Bacillus subtilis

Abstract

Proteins in the YidC/Oxa1/Alb3 family have essential functions in membrane protein insertion and folding. Bacillus subtilis encodes two YidC homologs, one that is constitutively expressed (spoIIIJ/yidC1) and a second (yqjG/yidC2) that is induced in spoIIIJ mutants. Regulated induction of yidC2 allows B. subtilis to maintain capacity of the membrane protein insertion pathway. We here show that a gene located upstream of yidC2 (mifM/yqzJ) serves as a sensor of SpoIIIJ activity that regulates yidC2 translation. Decreased SpoIIIJ levels or deletion of the MifM transmembrane domain arrests mifM translation and unfolds an mRNA hairpin that otherwise blocks initiation of yidC2 translation. This regulated translational arrest and yidC2 induction require a specific interaction between the MifM C-terminus and the ribosomal polypeptide exit tunnel. MifM therefore acts as a ribosome-nascent chain complex rather than as a fully synthesized protein. B. subtilis MifM and the previously described secretion monitor SecM in Escherichia coli thereby provide examples of the parallel evolution of two regulatory nascent chains that monitor different protein export pathways by a shared molecular mechanism.

Recruitment of a species-specific translational arrest module to monitor different cellular processes

“…The results presented above show that in vitro translation of MifM is arrested at a point very close to the C terminus as previously shown to be the case in vivo (5). To substantiate that the arrest we observed in vitro was physiologically relevant, we first examined an arrest-compromised mutant of MifM with Ile70Ala alteration (5).…”

“…The species specificity we observed substantiates the divergent evolution of the ribosome-stalling sequences (2,5,17), which are likely to have been "tailor-made" to fit the native ribosome of the organism. Nevertheless, we were able to engineer the MifM polypeptide, which originally monitors protein insertion into the membrane, to monitor protein secretion by replacing its membrane-anchoring sequence by an export signal sequence.…”

“…These studies revealed that the ribosome cannot necessarily complete translation of messages for any given amino acid sequences without any delay. Remarkably, programmed elongation arrest can serve as a novel mechanism of cellular regulation, for instance to monitor protein localization, metabolite concentration, and reception of an antibiotic (1)(2)(3)(4)(5)(6). Although regulatory nascent peptides having divergent stall-inducing amino acid sequences have been identified from different living organisms, their species-specificity and exchangeability among different systems have not been explored.…”

“…Therefore, the elongation arrest, which is transient in normal cells but prolonged in secretion-compromised cells, assures the basal level expression of secA as well as its up-regulation in response to a secretion defect (13). MifM monitors YidC-dependent membrane biogenesis pathway in B. subtilis, which has two YidC homologs, the primary SpoIIIJ (YidC1) and the secondary YidC2 (5,14). It is encoded by the open reading frame upstream of yidC2 and contains a hydrophobic sequence at the N-terminal region, which is guided by SpoIIIJ for insertion into the membrane and/or subsequent folding (15).…”

“…The stalled ribosome then uncovers the SD sequence of yidC2. Because the SpoIIIJ-mediated insertion of MifM leads to the release of elongation arrest, yidC2 is induced when the SpoIIIJ function is impaired (5). SecM and MifM share no appreciable sequence similarity and monitor different protein localization pathways in different organisms, yet their modes of actions are strikingly similar.…”