The plastid division proteins, FtsZ1 and FtsZ2, differ in their biochemical properties and sub-plastidial localization

Abstract

Plastid division in higher plants is morphologically similar to bacterial cell division, with a process termed binary fission involving constriction of the envelope membranes. FtsZ proteins involved in bacterial division are also present in higher plants, in which the ftsZ genes belong to two distinct families: ftsZ1 and ftsZ2. However, the roles of the corresponding proteins FtsZ1 and FtsZ2 in plastid division have not been determined. Here we show that the expression of plant FtsZ1 and FtsZ2 in bacteria has different effects on cell division, and that distinct protein domains are involved in the process. We have studied the assembly of purified FtsZ1 and FtsZ2 using a chemical cross-linking approach followed by PAGE and electron microscopy analyses of the resulting polymers. This has revealed that FtsZ1 is capable of forming long rod-shaped polymers and rings similar to the bacterial FtsZ structures, whereas FtsZ2 does not form any organized polymer. Moreover, using purified sub-plastidial fractions, we show that both proteins are present in the stroma, and that a subset of FtsZ2 is tightly bound to the purified envelope membranes. These results indicate that FtsZ2 has a localization pattern distinct from that of FtsZ1, which can be related to distinct properties of the proteins. From the results presented here, we propose a model for the sequential topological localization and functions of green plant FtsZ1 and FtsZ2 in chloroplast division.

Figure 1. Effects of His₆-tagged FtsZ1, FtsZ2 and C-terminally truncated FtsZ2 (FtsZ2D) on E. coli M15 cell growth and division

(A) Cells were grown to early exponential phase before the addition, or not, of IPTG (1 mM final), and overexpression of the proteins was determined using an anti-histidine antibody after 3 h of growth in the absence (−) or presence (+) of IPTG. (B) Aliquots were removed for absorbance reading at 600 nm. Cells were photographed after 3 h of growth in the absence (C) or presence of 1 mM IPTG inducing the expression of FtsZ1 (D), FtsZ2 (E) and truncated FtsZ2 (F).

Figure 2. Cross-linking of FtsZ1 and FtsZ2 polymerization products

(A) Reaction mixtures (60 μl) containing FtsZ1 or FtsZ2 were incubated in polymerization buffer for 20 min without treatment (lane 2) or after chemical cross-linking (lane 3) with the addition of glutaraldehyde. FtsZ protein (1 μg; lane 1) together with samples of the polymerization reaction (5 μl) were resolved on an SDS/8%-glutaraldehyde gel and subsequently blotted on to Immobilon-P membranes and probed with the indicated antibodies. (B) Kinetics of FtsZ1 and FtsZ2 polymerization. Polymerization reactions were performed with the addition of glutaraldehyde. Aliquots of the reaction mixture were withdrawn at different times (min; shown above each lane) and polymer formation was monitored as described above with anti-FtsZ1 or anti-FtsZ2 antibody. MM, molecular mass.

Figure 3. TEM observation of FtsZ1 and FtsZ2 assembly

Typical TEM images are shown of the products of plant FtsZ1 (A–D) and FtsZ2 (E) polymerization reactions analysed in Figure 2. Structures were visualized after negative staining. The scale bars correspond to 50 nm in (A), (B) and (E), and to 20 nm in (C) and (D).

Figure 4. FtsZ2 promotes the assembly of FtsZ1 in the absence of GTP

Reaction mixtures containing FtsZ1, FtsZ2 or FtsZ1 plus FtsZ2 were incubated in polymerization buffer in the presence (+) or absence (−) of GTP for 30 min. Products were analysed as described in the legend to Figure 2 with anti-FtsZ1 or anti-FtsZ2 antibodies.

Figure 5. Chloroplast sublocalization of plant FtsZ1 and FtsZ2 proteins

(A) Western blot analysis of protein fractions (30 μg of each) from chloroplasts (lane 1), thylakoids (lane 2), stroma (lane 3), IEM (lane 4) and OEM (lane 5) with antibodies against plant FtsZ1 and FtsZ2 proteins, IE37, OE24 and KARI (a stromal protein). (B) Purified plastid envelopes (30 μg of protein) were incubated for 30 min in the indicated media and the proteins were separated into insoluble (P) and soluble (S) fractions. These fractions, together with purified envelope proteins (Ev), were fractionated by SDS/PAGE, blotted and reacted with the anti-FtsZ2 antibody. (C) Purified envelopes from control chloroplasts (lane 1) and from chloroplasts treated with 100 μg·ml⁻¹ (lane 2) or 600 μg·ml⁻¹ (lane 3) thermolysin were probed with antibodies against FtsZ2, IE37, OE24 and KARI.

Figure 6. Sub-plastidial localization of FtsZ2 using immunogold techniques

Intact spinach chloroplasts were immobilized, fixed and observed under electron microscopy (A) or incubated successively with anti-FtsZ2 antibody and goat anti-(rabbit IgG) conjugated to colloidal gold. Panels (B) and (C) show FtsZ2 labelling within the chloroplast and in contact with the chloroplast envelope respectively. Higher-magnification images of the gold clusters indicated with the arrows are shown in the bottom left corners in (B) and (C). The IEM and OEM are indicated by arrowheads in (C). The scale bars correspond to 1 μm in (A) and 50 nm in (B) and (C).

Figure 7. Proposed sequence of events during chloroplast division

Stage I: localization of FtsZ2 at a contact site between the OEM and IEM. FtsZ2, in association or not with other proteins, forms a protein bridge spanning the two membranes and connecting the outside surface of the chloroplast to the stroma (black rectangles). Stage II: interaction of FtsZ1 (grey circles) with the membrane-bound FtsZ2 protein complex. Stage III: formation of the FtsZ1 ring. Stage IV: FtsZ2 (black circles) interacts with the FtsZ1 ring, allowing the recruitment of new proteins and the formation of the inner PD-ring. Concomitantly, or soon afterwards, the outer PD-ring and the dynamin ring are formed on the cytoplasmic side of the chloroplast. IMS, inner membrane space.