Analysis of single-molecule images for the subcellular localization and dynamics of proteins.

(A) Single-molecule image analysis. Spots were detected in each frame (highlighted with yellow circles), and tracks were created across frames (different colors were chosen for different tracks). (B) Cell detection. Cell outlines were determined from bright-field images. Only non-dividing cells were analyzed (indicated by white outlines). (C) Normalized position of spots of RNE along the short (x) and the long (y) axes of an example cell. Red spots are inside the cell endcaps, and cyan spots are in the cylindrical region of the cell. (D) xNorm histogram of RNE and LacY. Only spots in the cylindrical region of cells (like cyan spots in C) were included, totaling n = 143,000 spots. The standard error of the mean (SEM) calculated from bootstrapping is displayed as a shaded area but is smaller than the line width (see Fig. S1A for details). (E) The membrane binding percentage (MB%) of RNE, LacY, and LacZ. Error bars are from the 95% confidence interval. (F) Histogram of absolute xNorm and model fitting of RNE, LacY, and LacZ to determine MB%. Orange highlights indicate the range of xNorm expected based on the standard deviations in the parameter values estimated by MCMC. The white scale bars in panels A-B are 1 µm. See Table S6 for data statistics.

Mutations in the MTS affecting the localization of RNE.

(A) Linear representation of the RNE monomer. The NTD and the CTD are defined as regions flanking the MTS. Numbers indicate the amino acid residues. (B) xNorm histograms of cytoplasmic RNE mutants. (C) Helical wheel diagram of the MTS region of RNE (residue 568-582). (D) xNorm histogram of RNE MTS point mutants. (E) MB% of RNE MTS point mutants. Error bars indicate the 95% confidence interval. In panels B and D, the SEM from bootstrapping is shown but is smaller than the line width. See Table S6 for data statistics.

RNE diffusion, influenced by membrane binding and interactions with mRNAs.

(A) MSD versus time delay (τ) for RNE. Ensemble-averaged time-averaged (EATA) MSD was calculated by averaging the time-averaged MSD of individual tracks. (B) Mean diffusion coefficients of various RNE mutants, lacking the MTS and/or the CTD. (C-E) Change in the mean diffusion coefficient of RNE (C), LacY (D), and ribosome L1 protein (E) when cellular RNAs were depleted by rifampicin treatment. Error bars in panels B-E represent the SEM. See Table S6 for data statistics.

Localization and diffusion of membrane-binding motifs.

(A) Cartoon schematic of the membrane-binding motifs used in this study (not to scale). The orange circles indicate mEos3.2 used for imaging. (B) xNorm histograms of membrane-binding motifs. The SEM from bootstrapping is displayed but smaller than the line width. Data are from at least 107,000 spots. (C) Mean diffusion coefficients of membrane-binding motifs. Error bars are the SEM from at least 3,000 tracks. (D) Estimated mass of membrane-binding motifs based on the amino acid sequence including linkers and mEos3.2. (E) Diffusion coefficients of the membrane-binding motifs obtained from all-atom MD simulation. (F) Representative simulation snapshots of the membrane-binding motifs embedded in the E. coli membrane. Proteins are displayed in purple, and lipid tails are shown in cyan. Nitrogen and phosphorus atoms of the lipid head groups are represented in the van der Waals form in blue and grey, respectively. See Table S6 for data statistics.

Localization and diffusion of chimeric RNE with or without the CTD.

(A-B) Cartoon schematic of RNE chimeric variants with the CTD (A) and without the CTD (B). They are not to scale. (C-D) xNorm histograms of chimeric RNE localization compared with that of LacY. The SEM from bootstrapping is displayed but smaller than the line width. (E-F) MB% of chimeric RNE mutants without the CTD (E) or with the CTD (F) with various membrane-binding motifs. Error bars are from a 95% confidence interval. (G-H) Mean diffusion coefficients of chimeric RNE without the CTD (G) or with the CTD (H). Error bars are the SEM. Each data set contains at least 70,000 tracks for diffusion or 72,000 spots for xNorm. See Table S6 for data statistics.

lacZ mRNA degradation rates in RNE mutant strains.

(A-B) lacZ mRNA levels in WT RNE (A, strain SK595) and in RNE-LacY2-CTD (B, strain SK505) when lacZ transcription was induced with 0.2 mM IPTG at t = 0 s and re-repressed with 500 mM glucose at t = 75 s. Blue and yellow regions indicate the time windows used to measure kd1 and kd2, respectively, by exponential fitting of 5’ lacZ mRNA (Z5) in individual replicates. (C-F) Co-transcriptional and post-transcriptional lacZ mRNA degradation rates, kd1 (C, E) and kd2 (D, F), respectively, in various RNE mutants containing different membrane-binding motifs, either with the CTD (solid bars, C-D) or ΔCTD (light bars, E-F). The dotted lines indicate the kd1 and kd2 values of cytoplasmic RNE ΔMTS (strain SK339 in C-D)15 or RNE ΔMTS ΔCTD (strain SK370, in E-F). In all panels, error bars represent the standard deviations from 2-3 biological replicates. Two-sample t-tests were performed relative to the MTS case in each graph (See Table S7 for the p values).