The response regulator SprE (RssB) is required for maintaining poly(A) polymerase I-degradosome association during stationary phase

Abstract

Poly(A) polymerase I (PAP I) is the enzyme responsible for the addition of poly(A) tails onto RNA molecules in Escherichia coli. Polyadenylation is believed to facilitate the destruction of such RNAs by the mRNA degradosome. Recently, it was discovered that the stationary-phase regulatory protein SprE (RssB) has a second function in the control of polyadenylation that is distinct from its known function in the regulated proteolysis of RpoS. In the work presented herein, we used a targeted proteomic approach to further investigate SprE's involvement in the polyadenylation pathway. Specifically, we used cryogenic cell lysis, immunopurifications on magnetic beads, and mass spectrometry to identify interacting partners of PAP I-green fluorescent protein. We provide the first in vivo evidence that PAP I interacts with the mRNA degradosome during both exponential and stationary phases and find that the degradosome can contain up to 10 different proteins under certain conditions. Moreover, we demonstrate that the majority of these PAP I interactions are formed via protein-protein interactions and that SprE plays an important role in the maintenance of the PAP I-degradosome association during stationary phase.

FIG. 1.

Strategy to isolate PAP I-GFP complexes. An E. coli strain that expressed a GFP-tagged PAP I was constructed as described in Materials and Methods. PAP I-GFP was visualized by fluorescence microscopy to determine intracellular localization. Cells were frozen and subjected to cryogenic cell lysis, which allowed for efficient lysis of >95% of cells, as determined by light microscopy. Proteins were isolated on magnetic beads coated with high-purity anti-GFP antibodies, resolved by SDS-PAGE, and identified by MS and MS/MS analyses using a MALDI LTQ Orbitrap mass spectrometer.

FIG. 2.

PAP I-GFP interactions in the presence and absence of SprE. Representative immunopurifications of VC239 (MC4100/ppcnB-GFP), or VC244 (ΔsprE/ppcnB-GFP), where cells were grown to either exponential or stationary phase, are shown. Interacting partners that were not detected in control immunopurifications (see Fig. S1 and Table S2 in the supplemental material) and were reproducibly isolated in triplicate experiments are labeled. Prominent bands marked with asterisks represent nonspecific associations as determined from control experiments; these proteins are listed in Table S2 in the supplemental material. The left inset illustrates examples of MS (top left) and MS/MS (bottom left) analyses for the identification of PAP I from wild-type cells. Selected singly charged PAP I peptides derived from trypsin digestion are indicated (T⁺_n, where n is the peptide number).

FIG. 3.

Relative quantification of PAP I's interacting partners using guanidination. (A) Workflow of the guanidination procedure, described in detail in Materials and Methods. After in-gel digestion with trypsin, the guanidination reaction was carried out using differentially labeled N-O-methylisourea. The wild-type peptides were labeled with ¹⁴N, which shifts the mass of lysine-containing peptides by +42 Th, and the ΔsprE peptides were labeled with ¹⁵N, for a shift of +44 Th. After the samples were mixed, relative quantification was performed by comparing the intensities of the light and heavy monoisotopic peaks. A parallel experiment was performed using reverse labeling. IP, immunopurification. (B) Sample spectra of guanidinated PAP I and HrpA at the MS and MS/MS level. On the MS panels, representative arginine-containing peptides are labeled. The T*⁺ designation represents the tryptic fragments containing modified lysine residues. The enlarged regions show the peptide of interest, with the +2-Th shift between heavy and light peaks. The insets in the MS/MS spectra illustrate the +2-Th shift between y ions. K* refers to homoarginine. (C) The relative quantification of PAP I interacting partners in the ΔsprE background. Since the +2-Th-shifted peak represents a mixture of the monoisotopic ΔsprE peptides and the third isotope of the wild-type peaks, all intensities of the heavy peptides were adjusted using the formula (ΔsprE_obs − ISO_exp) + [PAP I^WT_obs − (PAP I^ΔSprE_obs − ISO_exp)], where ΔsprE_obs is the intensity of any peptide from the ΔsprE samples (obs, observed) and ISO_exp is the predicted isotopic peak intensity (exp, expected). All intensities were adjusted to that of PAP I to correct for differences in starting material (see Fig. S3 in the supplemental material). (D) Results of the reverse labeling experiment, where wild-type peptides were derivatized with [¹⁵N]O-methylisourea and ΔsprE peptides with [¹⁴N]O-methylisourea. The isotopic correction described in Fig. S3 in the supplemental material was therefore not necessary. The intensities of the peaks from the wild-type peptides were set at 100%, and the intensities of the ΔsprE peptides are reported relative to that. All intensities were adjusted to that of PAP I to correct for differences in starting material. Error bars show standard deviations. WT, wild type.

FIG. 4.

Results of PAP I-GFP immunopurification (IP) from ΔrpoS cells. A control PAP I-GFP immunopurification was performed from VC252 (ΔrpoS/ppcnB-GFP) during stationary phase. Interacting partners are labeled, and a complete list of proteins is given in Table S7 in the supplemental material.

FIG. 5.

Summary of PAP I-GFP interacting partners. A summary of the results of the four immunopurifications illustrated in Fig. 2 represented as a Venn diagram. During stationary phase, in the absence of SprE, the only interacting partners of PAP I-GFP isolated were RNase E and Hfq. During exponential phase, the rest of the degradosome components were isolated, with the exception of SrmB. In a wild-type background, SrmB could be isolated in exponential phase, along with the other degradosome components. HrpA and RNase R were additionally isolated during stationary phase in the wild-type case.