Colocalization of Plastid Division Proteins in the Chloroplast Stromal Compartment Establishes a New Functional Relationship between FtsZ1 and FtsZ2 in Higher Plants

Abstract

Chloroplast division is driven by a macromolecular complex containing components that are positioned on the cytosolic surface of the outer envelope, the stromal surface of the inner envelope, and in the intermembrane space. The only constituents of the division apparatus identified thus far are the tubulin-like proteins FtsZ1 and FtsZ2, which colocalize to rings at the plastid division site. However, the precise positioning of these rings relative to the envelope membranes and to each other has not been previously defined. Using newly isolated cDNAs with open reading frames longer than those reported previously, we demonstrate here that both FtsZ2 proteins in Arabidopsis, like FtsZ1 proteins, contain cleavable transit peptides that target them across the outer envelope membrane. To determine their topological arrangement, protease protection experiments designed to distinguish between stromal and intermembrane space localization were performed on both in vitro imported and endogenous forms of FtsZ1 and FtsZ2. Both proteins were shown to reside in the stromal compartment of the chloroplast, indicating that the FtsZ1-and FtsZ2-containing rings have similar topologies and may physically interact. Consistent with this hypothesis, double immunofluorescence labeling of various plastid division mutants revealed precise colocalization of FtsZ1 and FtsZ2, even when their levels and assembly patterns were perturbed. Overexpression of FtsZ2 in transgenic Arabidopsis inhibited plastid division in a dose-dependent manner, suggesting that the stoichiometry between FtsZ1 and FtsZ2 is an important aspect of their function. These studies raise new questions concerning the functional and evolutionary significance of two distinct but colocalized forms of FtsZ in plants and establish a revised framework within which to understand the molecular architecture of the plastid division apparatus in higher plants.Evolved from prokaryotic endosymbionts (Martin and Herrmann, 1998;Gray, 1999;McFadden, 1999), plastids divide by binary fission, whereby a constriction forms at the division plane, progressively tightening to separate the new organelles. Although plastids differ structurally from their cyanobacterial ancestors (Douglas, 1998), the division processes in both share mechanistic similarities. In bacteria, the tubulin-like GTPase FtsZ assembles into a dynamic circular structure termed the Z-ring at the cell center, forming a cytoskeletal framework to which all other cell division proteins are recruited (for review, see Rothfield et al., 1999) and probably providing the force that powers the contractile machinery (Lu et al., 2000;Erickson, 2001). It is now established that homologs of FtsZ also are essential for chloroplast division in land plants (Osteryoung and Vierling, 1995;Osteryoung et al., 1998; Strepp et al., 1998). In vascular plants, plastid division entails the participation of two distinct, highly conserved nuclear-encoded FtsZ protein families, FtsZ1 and FtsZ2 (Osteryoung et al., 1998;Osteryoung and McAndrew, 2001). Immu...

An Arabidopsis Homolog of the Bacterial Cell Division Inhibitor SulA Is Involved in Plastid Division[W]

“…Three independent T2 lines were analyzed for both AtFtsZ1-1 and AtFtsZ2-1. We observed more or less severe inhibition of plastid division in the transgenic plants, consistent with previous results (Osteryoung et al, 1998;McAndrews et al, 2001). Figure 6 shows control cells (mesophyll, Figure 6A; bundle sheath, Figure 6B) and cells displaying little or no plastid division defects ( Figures 6C, 6G, and 6H) (;55% of the observed plants, Table 2), intermediate phenotypes (15% of the plants), or very severe phenotypes ( Figures 6D, 6E, 6F, 6I, and 6J) (22% of the plants).…”

“…It has previously been reported that overexpression of either AtFtsZ1-1 or AtFtsZ2-1 results in a dominant negative effect causing severe plastid division defects McAndrews et al, 2001). In these experiments, plastid division was more or less severely affected in the different lines.…”

“…Various studies have demonstrated that the stoichiometry of components of the plastid division machinery influences its functionality. Indeed overexpression of the plastid division protein AtFtsZ1-1, AtFtsZ2-1, or ARC6 inhibits plastid division dramatically McAndrews et al, 2001; Plants were considered to be WTL if no division defects could be observed in a whole cotyledon; variability of phenotypes amongst the plants that showed plastid division defects was not taken into account. , 2003).…”

“…In these experiments, plastid division was more or less severely affected in the different lines. Indeed, ;20% of the plants were similar to the wild type, whereas ;40% could contain as few as a single greatly enlarged plastid in mesophyll cells and ;40% had an intermediate phenotype (Osteryoung et al, 1998;McAndrews et al, 2001). These authors have shown that the severity of plastid division defects correlates with the protein abundance.…”

“…FtsZ is a tubulin-related protein (Erickson, 1997) that polymerizes at midcell, forming a ring that shrinks until division is completed (Rothfield and Justice, 1997). Involvement of FtsZ homologs in plastid division has been clearly established in Arabidopsis (AtFtsZ1-1 and AtFtsZ2-1) and the moss Physcomitrella patens (Osteryoung et al, 1998;Strepp et al, 1998;McAndrews et al, 2001).…”