Novel Filaments 5 nm in Diameter Constitute the Cytosolic Ring of the Plastid Division Apparatus

Abstract

The plastid division apparatus (called the plastid-dividing ring) has been detected in several plant and algal species at the constricted region of plastids by transmission electron microscopy. The apparatus is composed of two or three rings: an outer ring in the cytosol, an inner ring in the stroma, and a middle ring in the intermembrane space. The components of these rings are not clear. FtsZ, which forms the bacterial cytokinetic ring, has been proposed as a component of both the inner and outer rings. Here, we present the ultrastructure of the outer ring at high resolution. To visualize the outer ring by negative staining, we isolated dividing chloroplasts from a synchronized culture of a red alga, Cyanidioschyzon merolae , and lysed them with nonionic detergent Nonidet P-40. Nonidet P-40 extracted primarily stroma, thylakoids, and the inner and middle rings, leaving the envelope and outer ring largely intact. Negative staining revealed that the outer ring consists of a bundle of 5-nm filaments in which globular proteins are spaced 4.8 nm apart. Immunoblotting using an FtsZ-specific antibody failed to show immunoreactivity in the fraction containing the filament. Moreover, the filament structure and properties are unlike those of known cytoskeletal filaments. The bundle of filaments forms a very rigid structure and does not disassemble in 2 M urea. We also identified a dividing phase-specific 56-kD protein of chloroplasts as a candidate component of the ring. Our results suggest that the main architecture of the outer ring did not descend from cyanobacteria during the course of endosymbiosis but was added by the host cell early in plant evolution. INTRODUCTIONEukaryotic cells contain organelles that are duplicated and inherited by daughter cells during cell division. Among these organelles, mitochondria and plastids are unique in that they contain DNA-protein complexes (nucleoids) and machinery sufficient for protein synthesis. It is generally believed that mitochondria and plastids arose from prokaryotic endosymbionts during eukaryotic evolution. The endosymbiotic theory states that the progenitor of mitochondria was an ␣ -proteobacterium and that the plastid ancestor was a cyanobacterium (Gray, 1992). Mitochondria and plastids multiply by the division of preexisting organelles, as do bacteria (Leech et al., 1981;Kuroiwa, 1982Kuroiwa, , 1991Possingham and Lawrence, 1983; Boffey and Lloyd, 1988). Of the characterized mitochondrial and plastid genomes, however, none has a complete set of genes sufficient for self-replication. Mitochondria that cannot synthesize proteins as a result of large genome deletions (petite mutant) (Attardi and Schatz, 1988) and plastids that lack ribosomes (Hashimoto and Possingham, 1989) still can proliferate. Therefore, it is believed that products from the host (nuclear) genomes perform mitochondrial and plastid division. Nuclear regulation of organelle division remains poorly understood, but in plastid division both the plastid-dividing ring (PD ring) (summarized in Kuroiwa et ...

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

“…First, they indicate that FtsZ2 is not a component of the outer PD ring. This is consistent with a recent report by Miyagishima et al (2001) indicating that FtsZ could not be detected in outer PD ring extracts from red algae. Based on these and other data, these investigators hypothesized that the outer PD ring is not derived from the endosymbiotic progenitor of chloroplasts, as are the FtsZ rings, but rather from the host cell.…”

“…However, inconsistent with the hypothesized localization of FtsZ2 in the outer PD ring, subsequent submissions of other plant FtsZ2 sequences revealed that the extreme amino termini of most FtsZ2 family members, including AtFtsZ2-2, were longer than that predicted for AtFtsZ2-1 (Osteryoung and McAndrew, 2001), suggesting that the AtFtsZ2-1 ORF used in our previous analysis (Osteryoung et al, 1998) might be incomplete, lacking the transit peptide. In addition, two recent studies demonstrated that nuclearencoded FtsZ2-like proteins from the moss Physcomitrella patens were localized in the chloroplast when expressed as green fluorescent protein fusion proteins in vivo (Kiessling et al, 2000) and that FtsZ could not be identified immunologically in outer PD ring preparations from red algae (Miyagishima et al, 2001). Furthermore, genes encoding both FtsZ1 and FtsZ2 proteins have only been identified in higher plant genomes; thus, the relationship of the findings in moss and algae to the organization of the plastid division apparatus in higher plants remains unclear.…”

ARC5, a cytosolic dynamin-like protein from plants, is part of the chloroplast division machinery

“…Interestingly, the topology of the ARC5-containing ring is similar to that of the outer PD ring (15,50). Like ARC5, which lacks homologues in cyanobacteria and was probably derived from the eukaryotic host cell, the outer PD ring has been proposed to be of eukaryotic origin (19).…”

FtsZ and the division of prokaryotic cells and organelles

“…The protein machine of chloroplasts, unlike that of bacteria, can be observed in electron micrographs of thin sections. One possible reason for its visibility is that this machine consists of at least three layers: an innermost ring comprising FtsZ, followed by the plastid dividing ring that is visible by negative staining but is of unknown composition 102 , and an outermost ring on the outside of the organelle that contains a dynamin-like protein (DRP) 103,104 . Dynamins are eukaryotic GTPases that assemble on membranes and mediate membrane fission 97 ; dynamin also has a role in cytokinesis of animal and plant cells 105,106 .…”