Oligomerization and higher-order assembly contribute to sub-cellular localization of a bacterial scaffold

Abstract

In Caulobacter crescentus, the PopZ polar scaffold protein supports asymmetric cell division by recruiting distinct sets of binding partners to opposite cell poles. To understand how polar organizing centres are established by PopZ, we investigated a set of mutated PopZ proteins for defects in sub-cellular localization and recruitment activity. We identified a domain within the C-terminal 76 amino acids that is necessary and sufficient for accumulation as a single subcellular focus, a domain within the N-terminal 23 amino acids that is necessary for bipolar targeting, and a linker domain between these localization determinants that tolerates large variation. Mutations that inhibited dynamic PopZ localization inhibited the recruitment of other factors to cell poles. Mutations in the C-terminal domain also blocked discrete steps in the assembly of higher-order structures. Biophysical analysis of purified wild type and assembly defective mutant proteins indicates that PopZ self-associates into an elongated trimer, which readily forms a dimer of trimers through lateral contact. The final six amino acids of PopZ are necessary for connecting the hexamers into filaments, and these structures are important for sub-cellular localization. Thus, PopZ undergoes multiple orders of self-assembly, and the formation of an interconnected superstructure is a key feature of polar organization in Caulobacter.

Figure 1

Subcellular localization of PopZ as a function of the cell cycle and PopZ sequence. A. A schematic of the Caulobacter cell cycle. Circles and theta structures within the cell indicate the replicating chromosome. Following the initiation of DNA replication, a new focus of PopZ assembles at the pole opposite the stalk, where it is required for tethering the newly duplicated ParB/parS centromere. B. PopZ primary sequence, with shaded regions indicating the degree of conservation in Alphaproteobacteria. Black bars under the sequence represent predicted α-helices. The R1, R2, and R3 regions of the PopZ protein are indicated above the sequence as red, green, and blue bars, respectively.

Figure 2

The C-terminal region of PopZ is sufficient for sub-cellular localization and oligomer formation. A. Schematic of individual PopZ regions. B. Images of strains expressing full length mCherry-PopZ (GB1078), mCherry-PopZ R1 (GB1079), mCherry-PopZ R2 (GB1080), and mCherry-PopZ R3 (GB750) in a ΔpopZ background. mCherry fluorescence (red) overlays the phase contrast image (grayscale). Bar = 1 µm. C. Electrophoretic migration of individual PopZ regions. Whole cell lysates of strains expressing no PopZ (GB255), mCherry-PopZ (GB1078), mCherry-PopZ R1(GB1079), mCherry-PopZ R2 (GB1080), and mCherry-PopZ R3 (GB750) were resolved on native and denaturing gels, then probed with anti-mCherry sera by immunoblotting. Cross-reactive bands are indicated by an asterisk. D. Electrophoretic migration of co-expressed PopZ variants. A PopZ-FLAG fusion was expressed by itself (GB135) and in parallel with either mCherry-PopZ R1 (GB757) or mCherry-PopZ R3 (GB758). Whole cell lysates were resolved on native and denaturing gels, then probed with anti-FLAG sera by immunoblotting.

Figure 3

The C-terminal region of PopZ is necessary for sub-cellular localization and oligomer formation. A. Schematic of PopZ truncations. B. Images of strains expressing full length mVenus-PopZ (AP323) (WT), mVenus-PopZΔ_134–177 (AP303), mVenus-PopZΔ_160–177 (AP322), and mVenus-PopZΔ_172–177 (AP305) in a ΔpopZ background. Venus fluorescence (green) overlays the phase contrast image (grayscale). C. Percent of polar localized PopZ signal in B. D. Average cell length of strains expressing WT PopZ (GB699), no PopZ (GB255), PopZΔ_134–177 (GB885), PopZΔ_160–177 (GB886), and PopZΔ_172–177 (GB888) in a ΔpopZ background. E. Localization of SpmX-mCherry in PopZ variant backgrounds. Strains in B are modified to express SpmX-mCherry from the chromosomal spmX promoter. Images from AP253, AP236, AP280, AP300, and AP282 are presented. SpmX-mCherry fluorescence (red) overlays the phase contrast image (grayscale). F. Percent of polar localized SpmX-mCherry signal in PopZ variant strains, shown in E. G. Electrophoretic migration of PopZ truncations. Whole cell lysates of strains in panel D were resolved on native and denaturing gels, then probed with anti-PopZ sera by immunoblotting. In images, Bar = 1µm. In graphs, error bars represent SEM from 2 separate experiments of 30–60 cells each.

Figure 4

The middle region of PopZ (R2) is a required spacer between N and C termini. A. Schematic of internal PopZ deletions. B. Images of strains expressing mVenus-PopZ (AP323) (WT), mVenus-PopZΔ_1–80 (AP321), mVenus-PopZΔ_24–102 (AP324), mVenus-PopZΔ_24–81 (AP327), mVenus-PopZΔ_81–102 (AP320), and mVenus-PopZΔ_48–102 (AP301) in a ΔpopZ background. Venus fluorescence (green) overlays the phase contrast image (grayscale). C. Images of strains expressing scrambled R2 regions in a ΔpopZ spmX-mCherry background. mVenus-PopZΔ_{48–102+24–47scr} (AP343) has the linker sequence EEDPPPADAAAPPAPEAVPPEPPE and mVenus-PopZΔ_{24–81+82–102scr} (AP344) has the linker sequence PDDEPEFETPPPERAYAPSVA. Venus fluorescence (green) overlays the phase contrast image (grayscale). D. Percent of polar localized PopZ signal in B and C. E. Average cell length of strains expressing WT PopZ (GB699), no PopZ (GB255), PopZΔ_1–80 (GB1115), PopZΔ_24–102 (GB1117), PopZΔ_24–81 (GB1116), PopZΔ_81–102 (GB1119), and PopZΔ_48–102 (GB1118) in a ΔpopZ background. F. Percent of polar localized SpmX-mCherry signal in PopZ variant strains. Strains in B are modified to express SpmX-mCherry from the chromosomal spmX promoter. Data from AP253, AP236, AP299, AP254, AP257, AP298, AP342, AP343, and AP344 are presented. Corresponding images are in Supplementary Figure S5. G. Electrophoretic migration of PopZ variants. Whole cell lysates of strains in panel E were resolved on native and denaturing gels, then probed with anti-PopZ sera by immunoblotting. In images, Bar = 1µm. In graphs, error bars represent SEM from 2 separate experiments of 30–60 cells each.

Figure 5

The N-terminal R1 region of PopZ is necessary for bi-polar distribution. A. Schematic of N-terminal PopZ amino acid substitutions. B. Images of strains expressing mVenus-PopZ (AP323) (WT), mVenus-PopZ E12A (AP316), mVenus-PopZ I13A (AP313), mVenus-PopZ I17A (AP314), and mVenus-PopZ R19A (AP315) in a ΔpopZ background. Venus fluorescence (green) overlays the phase contrast image (grayscale). C. Percent of polar localized PopZ signal in B.D. Average cell length in strains expressing WT PopZ (GB699), no PopZ (GB255), PopZ E12A (GB899), PopZ I13A (GB897), PopZ I17A (GB898), and PopZ R19A (GB900). E. Percent of polar localized SpmX-mCherry signal in PopZ variant strains. Strains in B are modified to express SpmX-mCherry from the chromosomal spmX promoter. Data from AP253, AP236, AP292, AP293, AP290, and AP291 are presented. Corresponding images are in Supplementary Figure S5.F. Percent of cells exhibiting bi-polar localization of Venus-PopZ, in strains shown in panel B. G. Electrophoretic migration of PopZ N-terminal mutant proteins. Whole cell lysates of strains in panel D were resolved on native and denaturing gels, then probed with anti-PopZ sera by immunoblotting. In images, Bar = 1µm. In graphs, error bars represent SEM from 2 separate experiments of 30–60 cells each.

Figure 6

Amino acid substitutions in the C-terminal R3 region of PopZ affect both localization and oligomer formation. A. Schematic of C-terminal PopZ amino acid substitutions. B. Images of strains expressing mVenus-PopZ (AP323) (WT), mVenus-PopZ P146A (AP306), mVenus-PopZ D153A (AP308), mVenus-PopZ L156A (AP309), mVenus-PopZ V160A (AP310), mVenus-PopZ V164A (AP311), and mVenus-PopZ E167A (AP312) in a ΔpopZ background. C. Percent of polar localized PopZ signal in B. D. Average cell length of strains expressing WT PopZ (GB699), no PopZ (GB255), PopZ P146A (GB890), PopZ D153A (GB892), PopZ L156A (GB893), PopZ V160A (GB894), PopZ V164A (GB895), and PopZ E167A (GB896) in a ΔpopZ background. E. Percent of polar localized SpmX-mCherry signal in PopZ variant strains. Strains in B are modified to express SpmX-mCherry from the chromosomal spmX promoter. Data from AP253, AP236, AP283, AP285, AP286, AP287, AP288, and AP289 are presented. Corresponding images are in Supplementary Figure S5. F. Electrophoretic migration of PopZ N-terminal mutant proteins. Whole cell lysates of strains in panel D were resolved on native and denaturing gels, then probed with anti-PopZ sera by immunoblotting. In images, Bar = 1µm. In graphs, error bars represent SEM from 2 separate experiments of 30–60 cells each.

Figure 7

Biophysical analysis of PopZ complexes. A. Recombinant wildtype 6His-PopZ protein and the indicated 6-His tagged mutant forms were purified from E. coli, resolved by native gel electrophoresis, and subsequently stained with Coomassie blue. B. SEC-LS analysis. The mobility of 6His-tagged proteins (X-axis) was determined by measuring protein concentration in fractions eluting from the column (narrow lines). The peak concentration is indicated by a triangle below the X-axis. Fractions in the vicinity of the peak were analyzed by MALLS to determine the molecular mass of the particles (Y-axis, thick lines). Three separate column runs were performed for each 6His-PopZ variant, with increasing amounts of protein loaded. Darker shading represents data from runs with more protein. In a separate run, apoferritin was used as a standard. C. TEM microscopy on purified 6His tagged proteins, prepared at a concentration of 0.11 mg/ml. Scale bar 50nm. D. Co-assembly of purified proteins. 6-His tagged proteins were denatured in 5M urea and then refolded by dialysis before running on a native gel and staining with Coomassie blue. Proteins were mixed at the following ratios: 2:1 WT:P146A (lane 5), 2:1 WT:Δ172–177 (lane 6), 2:1 Δ172–177:P164A (lane 7).

Fig. 8

Functionally distinct regions of PopZ are responsible for control of cell division and the assembly of PopZ superstructures. A. Schematic diagram of the PopZ protein sequence. Distinct functional regions, based on areas of conserved sequence, predicted positions of alpha helices, and our functional data, are color coded. B. A model showing the relationship between PopZ assembly and sub-cellular localization. The first step is the self-association of monomers into rod-shaped trimers, which subsequently dimerize through lateral contact to form hexamers. End-to-end contacts between hexamers produce filaments, and in vivo, these filaments accumulate at cell poles. Each step in this process can be blocked by a mutation in PopZ, as indicated. Each of the forms is metastable and its frequency is influenced by protein concentration and conditions in the buffer or cell extract.