Peptide Signals Encode Protein Localization

Strains and plasmids.The C. crescentus strain used in this work was CB15N (14). A plasmid library was constructed by amplifying the egfp gene (Clontech) by PCR using the primers GGAATTCCATATG(N)₆₀GTGAGCAAGGGCGAGGAGCTGTTC and CTAGTTCTAGATTACTTGTACAGC and cloning the product into pJS14 (33) under control of a xylose-inducible promoter (24) to generate N₆₀ -egfp. Escherichia coli cells were transformed with the plasmid library and grown as described previously (9), resulting in ∼2 × 10⁵ colonies. E. coli colonies were pooled, and plasmid DNA was prepared and mobilized into C. crescentus by electroporation. To generate LP2-mCherry, the mCherry coding sequence was cloned into pJS14, and a DNA fragment containing the xylose-inducible promoter and the LP2 sequence was amplified by PCR and cloned into a BamHI site at the 5′ end of mCherry. Due to the BamHI site, the LP2-mCherry protein contains a glycine-serine linker between LP2 and mCherry.

Screen for localization signals.Cells from the C. crescentus library were grown to an optical density at 660 nm of 0.30 to 0.40 in PYE medium (11) with 0.3% xylose to induce expression of N₆₀ -egfp and visualized using epifluorescence microscopy. Localization patterns were confirmed by growing independent cultures in PYE with xylose, or in M2X (24), and examining the cells with epifluorescence microscopy. In all cases, the localization patterns were unchanged in different media. Plasmid DNA was prepared from cells containing localized fluorescence signal and sequenced to identify the N-terminal peptide fused to green fluorescent protein (GFP). The effect of A22 was assayed by adding the inhibitor to a final concentration of 10 μg/ml for 1 hour prior to observation.

Microscopy.Cells were immobilized on 1% agar and imaged using an Eclipse E600 microscope (Nikon) with a 100× Plan Fluor oil numerical aperture 1.30 objective in conjunction with a CoolSNAP fx charge-coupled device camera (Photometrics) controlled by Image-Pro Discovery software (Media Cybernetics). Images were deconvolved using the 2D Blind Deconvolution algorithm in AutoQuant software (Media Cybernetics).

For optical sectioning, an IX70 laser scanning confocal microscope with a 100× UPlanFL oil numerical aperture 1.3 objective and Fluoview software (Olympus) was used to obtain a Z-series of confocal images at increments of 0.15 μM. Each series of optical sections was deconvolved through 30 iterations using AutoQuant software.

Identification of peptide localization signals.To screen for peptides that contain a localization signal, a library of genes containing 20 redundant codons fused to the 5′ end of a gene encoding GFP was expressed in C. crescentus, and the cells were screened for localized GFP fluorescence. Cells producing GFP without an N-terminal peptide extension had fluorescence distributed evenly throughout the cell (Fig. 1A), as did the vast majority of clones from the library, demonstrating that GFP has no inherent localization signals. Five of the ∼3,000 clones had specific patterns of fluorescence within the cells, and three of these were investigated in more detail.

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FIG. 1.

Peptides can specify discrete locations within the bacterial cell. C. crescentus cells producing GFP (A) or GFP with a 20-amino-acid peptide at the N terminus (B to D) were imaged by epifluorescence (left panels) or differential interference contrast (right panels) microscopy. The position of the focus of fluorescence in cells producing LP3-GFP, shown by schematic diagram (D, bottom left), was measured as a fraction of the distance from the stalked pole (fractional distance = 0) to the flagellar pole (fractional distance = 1.0), and a histogram is shown (D, bottom right).

Epifluorescence images of cells producing LP1-GFP showed a pattern of three to four bands extending across the short axis of the cell (Fig. 1B). Similar patterns have been observed in fluorescence images by proteins localized in a three-dimensional helical structure (1, 8, 16, 31, 32, 34). To determine if LP1-GFP was localized in a three-dimensional structure, optical sections were obtained by taking confocal images at 0.15-μm increments along the z axis (Fig. 2). These optical sections revealed two helices of fluorescence spanning the length of the cell.

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FIG. 2.

LP1-GFP localizes to two helices in C. crescentus. Confocal microscopy was used to acquire a Z-series of fluorescence images of C. crescentus producing LP1-GFP. Schematic diagrams indicate the relative position of the optical section. A differential interference contrast image (top) shows the cell boundaries.

The epifluorescence images of cells producing LP2-GFP showed an array of spots (Fig. 1C), a pattern which is also consistent with a three-dimensional structure (1, 8, 16, 31, 32, 34). Optical sections of these cells revealed a helix of GFP fluorescence along the long axis of the cell (Fig. 3). The pattern produced by LP2-GFP is clearly distinct from the pattern produced by LP1-GFP, demonstrating that 20-amino-acid peptides target GFP to at least two different helical structures in the cell.

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FIG. 3.

LP2-GFP localizes to a helix in C. crescentus. Confocal microscopy was used to acquire a Z-series of fluorescence images of C. crescentus producing LP2-GFP. Schematic diagrams indicate the relative position of the optical section. A differential interference contrast image (top) shows the cell boundaries.

A third fusion protein, LP3-GFP, produced a single focus of fluorescence signal near the middle of each cell (Fig. 1D). To more precisely determine the position of the LP3-GFP focus, the distance from the stalked pole was measured in 54 cells. In 87% of the cells the focus was one-fourth to one-half of the distance from the stalked pole to the flagellar pole (Fig. 1D). Similar patterns have been observed for the chromosomal replication machinery and proteins required for cytokinesis and chromosome segregation (27, 32), although it is not clear that LP3-GFP colocalizes with any of these structures.

Helices do not require MreB filaments.Helical patterns have been observed for the actin-like protein MreB in several species of bacteria, including C. crescentus (16, 32). When LP1-GFP or LP2-GFP was produced in cells that contained MreB fused to the red fluorescent protein mCherry, the GFP and mCherry fluorescence signals were not colocalized (data not shown), indicating that the LP1 and LP2 peptides were not directing GFP to the MreB helix. The MreB filaments can be disrupted in vivo by the addition of the small molecule A22 (17). When A22 was added to cells producing LP1-GFP or LP2-GFP, the GFP helices remained intact for more than 1 hour (Fig. 4). Under the same conditions, the mCherry-MreB signal was completely dispersed. These results confirm that localization mediated by LP1 and LP2 does not require MreB filaments. Electron tomography studies of C. crescentus showed several MreB-independent filaments, although the composition of these filaments has not been determined (7). LP1 and LP2 may be targeting GFP to one of these filaments or to a previously unobserved structure.

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FIG. 4.

LP peptide-directed protein localization is not affected by the MreB inhibitor A22. C. crescentus cells producing mCherry-MreB (A) or GFP with a localizing peptide at the N terminus (B and C) were imaged by epifluorescence (left panels) and differential interference contrast (right panels) microscopy before (−A22) and after (+A22) treatment with A22 for 1 h.

Localization is independent of GFP.It is likely that GFP does not contribute to the localization signal in the LP fusion proteins, because GFP with no N-terminal peptide has no localization and the vast majority of 20-mer peptides do not induce localization, whereas the three LP peptides each produce a different pattern. To determine if other proteins can also be localized by LP signals, LP2 was fused to mCherry, which has little sequence similarity to GFP (29). Localization of LP2-mCherry was indistinguishable from that of LP2-GFP (Fig. 5), demonstrating that the information contained in the LP2 sequence is sufficient for localization of two distinct proteins.

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FIG. 5.

LP2 localizes mCherry to the same pattern as GFP. LP2-GFP and LP2-mCherry were imaged by epifluorescence (left panels) and differential interference contrast (right panels) microscopy. The localization patterns are indistinguishable, indicating that LP2 is sufficient to localize two different proteins to the same pattern in C. cresentus.

Protein localization produced by locons.The precise localization patterns produced by the LP peptides demonstrate that protein localization can be encoded in a short amino acid sequence. We suggest naming these signals “locons,” by analogy with the “degron” degradation signals (37). Although the possibility that the LP1 and LP2 peptides polymerize into filaments has not been excluded, the small size of these peptides limits the surface area available for protein-protein interactions and makes polymerization improbable. The sequences of the LP peptides suggest that they do not interact with known protein secretion systems. The signal sequence prediction programs PSORT (15), SignalP (3), and TatP (4) all predict that each of the LP-GFP fusion proteins is cytoplasmic. It is more likely that the localizing peptides bind to unknown structures in the cell or interact with an uncharacterized protein sorting system. No C. crescentus protein contains an exact match to one of the complete LP peptides, but many proteins have seven to nine residues in common with one of the peptides, raising the possibility that localization signals for endogenous proteins are contained in a shorter peptide sequence or are partially redundant. The discovery and further characterization of the LP localization signals will permit the placement of biophysical probes and other molecules at particular sites within the cell and allow protein localization to be predicted from bioinformatic data.