A novel amphiphilic motif at the C-terminus of FtsZ1 facilitates chloroplast division

Abstract

In bacteria and chloroplasts, the GTPase filamentous temperature-sensitive Z (FtsZ) is essential for division and polymerizes to form rings that mark the division site. Plants contain two FtsZ subfamilies (FtsZ1 and FtsZ2) with different assembly dynamics. FtsZ1 lacks the C-terminal domain of a typical FtsZ protein. Here, we show that the conserved short motif FtsZ1Carboxyl-terminus (Z1C) (consisting of the amino acids RRLFF) with weak membrane-binding activity is present at the C-terminus of FtsZ1 in angiosperms. For a polymer-forming protein such as FtsZ, this activity is strong enough for membrane tethering. Arabidopsis thaliana plants with mutated Z1C motifs contained heterogeneously sized chloroplasts and parallel FtsZ rings or long FtsZ filaments, suggesting that the Z1C motif plays an important role in regulating FtsZ ring dynamics. Our findings uncover a type of amphiphilic beta-strand motif with weak membrane-binding activity and point to the importance of this motif for the dynamic regulation of protein complex formation.

Figure 1

FtsZ1 in angiosperms contains a conserved motif at its C-terminus. A, Sequence alignment of the C-terminal regions of FtsZ1 in five angiosperms. These C-terminal regions contain a conserved motif. B, Diagram of the evolution of the C-terminal motif in FtsZ1. FtsZ1 and FtsZ2 evolved from bacterial FtsZ and started to differentiate in green algae. As plants evolved, a conserved region appeared at the C-terminus of FtsZ1 in angiosperms (highlighted in red). The pink, violet, and orange lines indicate bacterial FtsZ, plant FtsZ2, and plant FtsZ1, respectively. The black triangle indicates the increasing conservation of the C-terminus of FtsZ1.

Figure 2

The C-terminus of FtsZ1 is important for its function. A, Chloroplasts in mesophyll cells from the wild-type (WT), cpd201 mutant, and a complemented line (COM). cpd201 is an Arabidopsis mutant of FtsZ1 that encodes a protein that lacks the last 18 amino acids. Bar = 20 µm. The bar applies to all three images. B, Diagram of the structure of Arabidopsis FtsZ1-1 and this gene in the cpd201 mutant. FtsZ1-1 contains six exons and five introns, which are represented by filled boxes and lines, respectively. The mutation site of cpd201 is indicated by an arrow. The black and red asterisks represent the stop codon in the wild type and cpd201 mutant, respectively. C, Immunoblot analysis of FtsZ1 probed with anti-FtsZ1 antibodies reveals a truncated form of FtsZ1 in cpd201. Coomassie brilliant blue (CBB) staining was used as a loading control. D, Relationship between chloroplast number and cell area in the WT, cpd201, and complemented line (n > 30 cells per sample) shown in A. The best-fit lines had slopes of 0.0178 (R² = 0.7158), 0.0092 (R²  = 0.2842), and 0.0172 (R² = 0.8068) for the WT, cpd201, and complemented line, respectively. Three biological replicates were performed, with similar results.

Figure 3

The last five amino acids of the C-terminus of FtsZ1 are important for its function. A, Chloroplasts in mesophyll cells from the wild type, ftsz1, and transgenic plant P_FtsZ1:FtsZ1^5A in the ftsz1 background. Bar = 10 µm. All the images have the same magnification. B, Immunoblot analysis of FtsZ1 accumulation in the plants shown in (A) probed with anti-FtsZ1 antibodies. CBB staining was used as a loading control. C, Relationship between chloroplast number and cell area in wild type, ftsz1, and two transgenic lines (n > 30 cells per sample). The best-fit lines had slopes of 0.0155 (R² = 0.7258), 0.0024 (R² =  0.0521), 0.0043 (R² = 0.1159), and 0.0077 (R² = 0.1636) for the WT, ftsz1, and two transgenic plants, respectively. Three biological replicates were performed, with similar results.

Figure 4

The C-terminal region of FtsZ1 is important for the proper formation of FtsZ rings. Immunofluorescence staining of FtsZ in wild type, cpd201, and ftsz1 plants. Antibodies against full-length FtsZ2-1 and a conventional fluorescence microscope were used. The background green fluorescence is from chlorophyll. All the images have the same magnification. Bar = 10 µm. The bar refers to all images shown here.

Figure 5

The C-terminal region of FtsZ1 has membrane-binding activity in chloroplasts. A, Immunoblot assay. One or two FtsZ1 C-terminal regions (35 amino acids) were fused with chloroplast-targeted GFP. These constructs were transiently expressed in N. benthamiana leaves. Proteins of isolated chloroplasts were separated into soluble and pellet fractions. The membrane protein YFP-PDV2 and the soluble protein Rubisco were used as controls. Both GFP and YFP were detected with YFP antibodies, and Rubisco was detected by CBB staining. B, Liposome co-sedimentation assay of GFP, GFP-Z1C35, and GFP-2Z1C35. The proteins were incubated with (+) liposomes (SUVs) or without (–) liposomes. Supernatant (S) and pellet (P) were separated by centrifugation and resolved by SDS–PAGE. Anti-His antibodies were used for the immunoblot assay. Purified GFP-2Z1C35 protein was partially degraded. C, A diagram summarizing the results is shown in (A) and (B). GFP fused with one or two Z1C35 fragments are able to bind to the membrane.

Figure 6

The C-terminal region of FtsZ1 has membrane-binding activity in E. coli. The effects of the C-terminal region of FtsZ1 and various mutants on the membrane binding of GFP-FtsZ2-1 in E. coli. In this assay, fusion proteins that polymerize and bind to the membrane form helices in the E. coli cells. The insets in the upper-right corners of the images show three times magnified views of the regions highlighted by white boxes. BF, bright field; Bar = 5 µm. A, The last five amino acids of FtsZ1 are essential for its membrane binding. FtsZ2-1-FZ1 C35, the last 35 amino acids of FtsZ1 fused to the C-terminus of FtsZ2-1; C35^5A, the last five amino acids of FtsZ1 were substituted with five alanines; MTS(EcMinD), membrane-tethering sequence of MinD in E. coli. B, A detailed analysis of the C-terminus of FtsZ1 with various mutations. FZ1 C10, the last 10 amino acids of FtsZ1. A diagram of the mutations in various mutants, such as substitutions and insertions, is shown below the images. Blue font indicates the mutations. C, Co-expression of GFP-FtsZ2-1 and FtsZ1 or FtsZ1^5A in E. coli. In FtsZ1^5A, the last five amino acids of FtsZ1 were changed to five alanines.

Figure 7

FtsZ1 brings FtsZ2 filaments to the chloroplast envelope. FtsZ proteins with YFP or GFP tags were transiently expressed in N. benthamiana leaves. Proteins expressed are indicated on the left. GFP-FtsZ1 has the chloroplast transit peptide of FtsZ1 at the N-terminus. To get a clear view, protoplasts were isolated for observation. Some confocal Z-stack slices of the copolymers are shown in Supplemental Figures S8 and S9. Bar = 10 µm.

Figure 8

Secondary structure analysis of the C-terminal end of FtsZ1. A, CD spectrum of peptide analogs of the extreme C-terminal 19 amino acids of FtsZ1 (C19) and the two insertion mutants C19^+RL and C19^+AA as shown in Figure  6B, with or without SUVs. Peptide sequences are given above the spectra. MRE is the mean residue ellipticity in units of degrees cm2 dmol-1. B, Determination of the secondary structure constitutions in peptides analyzed in (A) using the protein secondary structure analysis tool of J-1500. C, Helical wheel prediction (https://heliquest.ipmc.cnrs.fr/) of C10, C10^+RL, and C10^+AA peptides of FtsZ1 as shown in Figure  6B. Only 10 or 12 amino acids were used in the analysis.

Figure 9

Working model of the structure and activity of the C-terminus of FtsZ1. The Z1C motif helps the FtsZ2/FtsZ1 copolymer bind to the chloroplast inner envelope. Upper half of the diagram: The hydrophobic amino acids “LFF” at the C-terminus bind to the inside of the membrane, while the basic amino acids “RR” bind to the hydrophilic surface of the membrane. Lower half of the diagram: the Z1C motif helps FtsZ filaments bind to the inner envelope membrane of the chloroplast. When only one FtsZ1 protein is present, its binding ability is not very strong. When multiple FtsZ1 proteins are present in FtsZ1 and FtsZ2 copolymers, the Z1C motif helps FtsZ filaments bind to the membrane. When the C-terminus of FtsZ1 is removed (FtsZ1△C) or the last five amino acids at its C-terminus mutated (FtsZ1^5A), FtsZ1 loses its ability to bind to the membrane. OEM, outer envelope membrane; IMS, inter-membrane space; IEM, inner envelope membrane.