Critical Regions of secM That Control Its Translation and Secretion and Promote Secretion-Specific secA Regulation

Abstract

SecA is an essential ATP-driven motor protein that binds to presecretory or membrane proteins and the translocon and promotes the translocation or membrane integration of these proteins. secA is subject to a protein secretion-specific form of regulation, whereby its translation is elevated during secretion-limiting conditions. A novel mechanism that promotes this regulation involves translational pausing within the gene upstream of secA, secM. The secM translational pause prevents formation of an RNA helix that normally blocks secA translational initiation. The duration of this pause is controlled by the rate of secretion of nascent SecM, which in turn depends on its signal peptide and a functional translocon. We characterized the atypical secM signal peptide and found that mutations within the amino-terminal region specifically affect the secM translational pause and secA regulation, while mutations in the hydrophobic core region affect SecM secretion as well as translational pausing and secA regulation. In addition, mutational analysis of the 3 end of secM allowed us to identify a conserved region that is required to promote the translational pause that appears to be operative at the peptide level. Together, our results provide direct support for the secM translational pause model of secA regulation, and they pinpoint key sequences within secM that promote this important regulatory system.In bacteria nascent or fully synthesized presecretory or membrane proteins are selectively targeted to the translocon by interactions with SecB and SecA or the signal recognition particle and its receptor (3,24,40,47,50). These pathways converge at the translocon, which consists of the integral membrane proteins SecYEG and SecDFyajC and the peripheral membrane protein SecA ATPase. SecYE forms the preprotein channel and SecA receptor (10,20,27,30), while SecG and SecDFyajC greatly enhance the rate of protein translocation by regulating SecA membrane cycling (11,28,34). SecA is central to protein translocation since it binds to the signal peptides or transmembrane segments of presecretory and membrane proteins, interacts with the SecB chaperone to promote release of the bound preprotein, and acts as a motor protein to drive protein translocation at the translocon (for a review, see reference 26). Considerable evidence suggests that SecA undergoes ATP-driven cycles of insertion and retraction at SecYE, thereby promoting the stepwise translocation of proteins across the plasma membrane (12, 13, 52).The selectivity of the translocon for its protein cargo is remarkable, since erroneous translocation of cytoplasmic proteins is essentially undetectable. Current evidence suggests that the translocon possesses a proofreading activity that is responsible for aborting the translocation of preproteins that lack a functional signal peptide (for a review, see reference 7). prl alleles of secA, secY, secE, and secG have been isolated that allow translocation of preproteins with a defective signal peptide (4,15,17,23,48). A recent study sugge...

Translation arrest of SecM is essential for the basal and regulated expression of SecA

“…This feature of mRNA is well corroborated by the properties of SecM, which monitors its own secretion status to affect effectiveness of the elongation arrest at the C-terminal region. This event will generate the stalled ribosome, which disrupts the mRNA secondary structure and thereby facilitates entry of new ribosomes for secA translation (9,11,12,15). Although the ribosome stall might also activate a recently described ribonucleolytic quality control mechanism (33)(34)(35), its significance in the regulation is unknown.…”

“…In normal cells, in which the N-terminal part of the nascent SecM is pulled by the Sec translocase, the elongation arrest is soon released, whereas the arrest is strikingly prolonged under conditions in which the translocation machinery or the SecM signal sequence is functionally defective (11,14,15). Although it is believed that the stalled ribosome will disrupt the secondary structure of the secM-secA mRNA to allow the easier entry of new ribosomes that translate secA, the importance of SecM elongation arrest in basal and regulated secA expression has not been established.…”

SsrA Tagging of Escherichia coli SecM at Its Translation Arrest Sequence

“…Immunoprecipitates were subjected to SDS-PAGE, and labeled proteins were visualized on storage phosphor screens. the periplasm (7,39). Since SsrA tagging of proteins can be induced by ribosome stalling, we hypothesized that elongationarrested SecM protein is subject to SsrA tagging.…”

Translocon “Pulling” of Nascent SecM Controls the Duration of Its Translational Pause and Secretion-Responsive secA Regulation

“…However, since the translational pause site is near the end of secM and the inserted sequence would disrupt the repressor helix on secM-secA mRNA (since the 5Ј end of the repressor helix overlaps the 3Ј end of the pause site) (12,21), this option was not pursued further.…”

“…Additional studies have defined the secM translational pause site and have implicated this sequence and portions of the peptide exit channel of the ribosome in promoting the observed translational pause (12,21). By contrast, the mechanism regulating the release of the secM translational pause remains less defined.…”