Polyadenylation is known to enhance RNA degradation, and this has led to the prediction that PAP I and Hfq may be associated with the RNA degradosome. We have confirmed and extended this prediction. We have shown that PAP I and Hfq are associated with the RNA degradosome in vivo
in both exponential and stationary phase (Fig. ). Our results further show that, besides PAP I and Hfq (35
), the degradosome contains not only the four core components RNase E, PNPase, RhlB, and enolase but also three additional proteins, the RNA helicases SrmB (23
), HrpA (24
), and RNase R (1
). Indeed, under certain conditions, such as cold shock, the RNA degradosome may contain as many as 10 different proteins. While previous reports have provided insights into various components and features of the degradosome (9
), our results provide, to our knowledge, the most comprehensive characterization of the in vivo
degradosome to date.
FIG. 5. Summary of PAP I-GFP interacting partners. A summary of the results of the four immunopurifications illustrated in Fig. represented as a Venn diagram. During stationary phase, in the absence of SprE, the only interacting partners of PAP (more ...)
Strikingly, we have discovered that the adaptor protein SprE is required to maintain the association of PAP I and Hfq with the degradosome during stationary but not exponential growth phase (Fig. ). Previously, we had shown that SprE was required for the change in the intracellular localization of PAP I that occurs during stationary phase (7
). We suspect that these two observations are functionally related and likely reflect an important role for SprE in stationary-phase physiology through its effects on polyadenylation and mRNA stability.
In stationary phase, in the absence of SprE, PAP I remains membrane associated (7
) and, as we have shown here, in a tight complex with Hfq, but the association of PAP I and Hfq with the degradosome is lost. Although it is clear that the core degradosome members are not degraded, we do not know if they remain associated with each other in a ΔsprE
background, nor do we know their cellular localization during stationary phase. Performing immunopurifications and localization studies with a GFP-tagged variant of another complex member would address these issues. In addition, we do not yet understand the phenotypic consequences of these changes in degradosome membership. Microarray analysis might reveal SprE-dependent changes in the stability of important mRNAs during stationary phase.
We showed that the PAP I-degradosome association is independent of the alternate sigma factor RpoS and, therefore, is distinct from SprE's known function in the regulated proteolysis of RpoS. So, how does SprE regulate the PAP I-degradosome association in stationary phase? The simplest explanation is that SprE is the glue that holds the complex together under these conditions. If this were true, then we should have detected SprE in the complex during stationary phase. While the evidence supporting its presence in this complex is weak (only one SprE peptide confirmed by MS/MS analyses), we think this is real because it was reproducibly detected (in all six experiments) only during stationary phase. The fact that the PAP I-degradosome association was still maintained in exponential phase in the absence of SprE indicates that SprE is not the main scaffold for this interaction. Thus, we think that SprE may interact with the degradosome, but probably it is not the glue holding it together. It seems more likely that its interaction is weak or transient.
It is important to note that the PAP I isolations were performed under particularly stringent lysis buffer conditions (i.e., incorporating high concentrations of both Triton X-100 and deoxycholate detergents and salt). Our previous experience has indicated that the lysis buffer conditions are critical for obtaining a balance between the efficient isolation of a tagged protein and the maintenance of interactions (18
). During our optimizations of the PAP I isolation, we noticed that the efficient purification of PAP I required stringent conditions, likely due to the necessity to penetrate the E. coli
cell wall and obtain access to the cytoplasmic PAP I-GFP. As a comparison, this lysis condition was more stringent than those we utilized for isolating members of the nuclear pore complex in yeast (18
), postsynaptic densities from mice cerebella (45
), or cytomegalovirus virion assemblies in human cells (36
), all known to present special challenges. We have previously utilized these harsh lysis conditions only when accessing virally induced vesicles in mammalian cells (17
). While these stringent conditions demonstrate that the observed interactions between PAP I and the degradosome members are strong, it is likely that they have also led to the disruption of weak-affinity or transient interactions. Other approaches incorporating reversible cross-linking and reciprocal isolations could be implemented to stabilize such interactions, potentially revealing the presence of SprE.
In summary, our targeted proteomic approach incorporating a GFP-tagged PAP I, cryogenic cell lysis, affinity purification, and mass spectrometry has allowed us to capture a more complete picture of the RNA degradation machinery. Our results provide the first evidence for the in vivo association of PAP I and Hfq with the degradosome and uncover a role for SprE in maintaining this important complex during stationary phase.