The Arabidopsis thaliana chloroplast division protein FtsZ1 counterbalances FtsZ2 filament stability in vitro.

Bacterial cell and chloroplast division are driven by a contractile "Z ring" composed of the tubulin-like cytoskeletal GTPase FtsZ. Unlike bacterial Z rings, which consist of a single FtsZ, the chloroplast Z ring in plants is composed of two FtsZ proteins, FtsZ1 and FtsZ2. Both are required for chloroplast division in vivo, but their biochemical relationship is poorly understood. We used GTPase assays, light scattering, transmission electron microscopy, and sedimentation assays to investigate the assembly behavior of purified Arabidopsis thaliana (At) FtsZ1 and AtFtsZ2 both individually and together. Both proteins exhibited GTPase activity. AtFtsZ2 assembled relatively quickly, forming protofilament bundles that were exceptionally stable, as indicated by their sustained assembly and slow disassembly. AtFtsZ1 did not form detectable protofilaments on its own. When mixed with AtFtsZ2, AtFtsZ1 reduced the extent and rate of AtFtsZ2 assembly, consistent with its previously demonstrated ability to promote protofilament subunit turnover in living cells. Mixing the two FtsZ proteins did not increase the overall GTPase activity, indicating that the effect of AtFtsZ1 on AtFtsZ2 assembly was not due to a stimulation of GTPase activity. However, the GTPase activity of AtFtsZ1 was required to reduce AtFtsZ2 assembly. Truncated forms of AtFtsZ1 and AtFtsZ2 consisting of only their conserved core regions largely recapitulated the behaviors of the full-length proteins. Our in vitro findings provide evidence that FtsZ1 counterbalances the stability of FtsZ2 filaments in the regulation of chloroplast Z-ring dynamics and suggest that restraining FtsZ2 self-assembly is a critical function of FtsZ1 in chloroplasts.

Author Correction: Chloroplast FtsZ assembles into a contractible ring via tubulin-like heteropolymerization.

In the version of this Article originally published, the authors incorrectly referred to the fluorescent protein Venus being used in their study; the actual one used was enhanced yellow fluorescence protein (eYFP).

The chloroplast division protein ARC6 acts to inhibit disassembly of GDP-bound FtsZ2.

Chloroplasts host photosynthesis and fulfill other metabolic functions that are essential to plant life. They have to divide by binary fission to maintain their numbers throughout cycles of cell division. Chloroplast division is achieved by a complex ring-shaped division machinery located on both the inner (stromal) and the outer (cytosolic) side of the chloroplast envelope. The inner division ring (termed the Z ring) is formed by the assembly of tubulin-like FtsZ1 and FtsZ2 proteins. ARC6 is a key chloroplast division protein that interacts with the Z ring. ARC6 spans the inner envelope membrane, is known to stabilize or maintain the Z ring, and anchors the Z ring to the inner membrane through interaction with FtsZ2. The underlying mechanism of Z ring stabilization is not well-understood. Here, biochemical and structural characterization of ARC6 was conducted using light scattering, sedimentation, and light and transmission EM. The recombinant protein was purified as a dimer. The results indicated that a truncated form of ARC6 (tARC6), representing the stromal portion of ARC6, affects FtsZ2 assembly without forming higher-order structures and exerts its effect via FtsZ2 dynamics. tARC6 prevented GDP-induced FtsZ2 disassembly and caused a significant net increase in FtsZ2 assembly when GDP was present. Single particle analysis and 3D reconstruction were performed to elucidate the structural basis of ARC6 activity. Together, the data reveal that a dimeric form of tARC6 binds to FtsZ2 filaments and does not increase FtsZ polymerization rates but rather inhibits GDP-associated FtsZ2 disassembly.

Conserved Dynamics of Chloroplast Cytoskeletal FtsZ Proteins Across Photosynthetic Lineages.

The cytoskeletal Filamenting temperature-sensitive Z (FtsZ) ring is critical for cell division in bacteria and chloroplast division in photosynthetic eukaryotes. While bacterial FtsZ rings are composed of a single FtsZ, except in the basal glaucophytes, chloroplast division involves two heteropolymer-forming FtsZ isoforms: FtsZ1 and FtsZ2 in the green lineage and FtsZA and FtsZB in red algae. FtsZ1 and FtsZB probably arose by duplication of the more ancestral FtsZ2 and FtsZA, respectively. We expressed fluorescent fusions of FtsZ from diverse photosynthetic organisms in a heterologous system to compare their intrinsic assembly and dynamic properties. FtsZ2 and FtsZA filaments were morphologically distinct from FtsZ1 and FtsZB filaments. When coexpressed, FtsZ pairs from plants and algae colocalized, consistent with heteropolymerization. Fluorescence recovery after photobleaching experiments demonstrated that subunit exchange was greater from FtsZ1 and FtsZB filaments than from FtsZ2 and FtsZA filaments and that FtsZ1 and FtsZB increased turnover of FtsZ2 and FtsZA, respectively, from heteropolymers. GTPase activity was essential only for turnover of FtsZ2 and FtsZA filaments. Cyanobacterial and glaucophyte FtsZ properties mostly resembled those of FtsZ2 and FtsZA, though the glaucophyte protein exhibited some hybrid features. Our results demonstrate that the more ancestral FtsZ2 and FtsZA have retained functional attributes of their common FtsZ ancestor, while eukaryotic-specific FtsZ1 and FtsZB acquired new but similar dynamic properties, possibly through convergent evolution. Our findings suggest that the evolution of a second FtsZ that could copolymerize with the more ancestral form to enhance FtsZ-ring dynamics may have been essential for plastid evolution in the green and red photosynthetic lineages.

Correction: Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga Nannochloropsis oceanica CCMP1779.

[This corrects the article DOI: 10.1371/journal.pgen.1003064.].

Chloroplast FtsZ assembles into a contractible ring via tubulin-like heteropolymerization.

Chloroplast division is driven by a ring containing FtsZ1 and FtsZ2 proteins, which originated from bacterial FtsZ, a tubulin-like protein; however, mechanistic details of the chloroplast FtsZ ring remain unclear. Here, we report that FtsZ1 and FtsZ2 can heteropolymerize into a contractible ring ex vivo. Fluorescently labelled FtsZ1 and/or FtsZ2 formed single rings in cells of the yeast Pichia pastoris. Photobleaching experiments indicated that co-assembly of FtsZ1 and FtsZ2 imparts polarity to polymerization. Assembly of FtsZ chimaeras revealed that the protofilaments assemble via heteropolymerization of FtsZ2 and FtsZ1. Contraction of the ring was accompanied by an increase in the filament turnover rate. Our findings suggest that the evolutionary duplication of FtsZ in plants may have increased the mobility and kinetics of FtsZ ring dynamics in chloroplast division. Thus, the gene duplication and heteropolymerization of chloroplast FtsZs may represent convergent evolution with eukaryotic tubulin.

Functional Analysis of the Chloroplast Division Complex Using Schizosaccharomyces pombe as a Heterologous Expression System.

Chloroplast division is driven by a macromolecular complex that assembles at the midplastid. The FtsZ ring (Z ring) is the central structure in this complex, and is composed of the functionally distinct cytoskeletal proteins FtsZ1 and FtsZ2. Recent studies in the heterologous Schizosaccharomyces pombe system showed that Arabidopsis FtsZ1 and FtsZ2 filaments have distinct assembly and turnover characteristics. To further analyze these FtsZs, we employed this system to compare the assembly and dynamic properties of FtsZ1 and FtsZ2 lacking their N- and/or C-termini with those of their full-length counterparts. Our data provide evidence that the N-terminus of FtsZ2 is critical for its structural dominance over FtsZ1, and that the N- and C-termini promote polymer bundling and turnover of both FtsZs and contribute to their distinct behaviors. We also assessed how ARC6 affects FtsZ2 filament dynamics, and found that it interacts with and stabilizes FtsZ2 filaments in S. pombe independent of its presumed Z-ring tethering function in planta. Finally, we generated FtsZ1-FtsZ2 coexpression constructs to facilitate reconstitution of more complex interaction networks. Our experiments yield new insight into factors influencing FtsZ behavior and highlight the utility of S. pombe for analyzing chloroplast FtsZs and their assembly regulators.

Roles of Arabidopsis PARC6 in Coordination of the Chloroplast Division Complex and Negative Regulation of FtsZ Assembly.

Chloroplast division is driven by the simultaneous constriction of the inner FtsZ ring (Z ring) and the outer DRP5B ring. The assembly and constriction of these rings in Arabidopsis (Arabidopsis thaliana) are coordinated partly through the inner envelope membrane protein ACCUMULATION AND REPLICATION OF CHLOROPLASTS6 (ARC6). Previously, we showed that PARC6 (PARALOG OF ARC6), also in the inner envelope membrane, negatively regulates FtsZ assembly and acts downstream of ARC6 to position the outer envelope membrane protein PLASTID DIVISION1 (PDV1), which functions together with its paralog PDV2 to recruit DYNAMIN-RELATED PROTEIN 5B (DRP5B) from a cytosolic pool to the outer envelope membrane. However, whether PARC6, like ARC6, also functions in coordination of the chloroplast division contractile complexes was unknown. Here, we report a detailed topological analysis of Arabidopsis PARC6, which shows that PARC6 has a single transmembrane domain and a topology resembling that of ARC6. The newly identified stromal region of PARC6 interacts not only with ARC3, a direct inhibitor of Z-ring assembly, but also with the Z-ring protein FtsZ2. Overexpression of PARC6 inhibits FtsZ assembly in Arabidopsis but not in a heterologous yeast system (Schizosaccharomyces pombe), suggesting that the negative regulation of FtsZ assembly by PARC6 is a consequence of its interaction with ARC3. A conserved carboxyl-terminal peptide in FtsZ2 mediates FtsZ2 interaction with both PARC6 and ARC6. Consistent with its role in the positioning of PDV1, the intermembrane space regions of PARC6 and PDV1 interact. These findings provide new insights into the functions of PARC6 and suggest that PARC6 coordinates the inner Z ring and outer DRP5B ring through interaction with FtsZ2 and PDV1 during chloroplast division.

FtsZ in chloroplast division: structure, function and evolution.

FtsZ is a key cytoskeletal component of the chloroplast division machinery that arose from the related cell division FtsZ in the cyanobacterial ancestor of chloroplasts. FtsZ is widely conserved in photosynthetic eukaryotes, where it forms a ring inside the organelle at the chloroplast division site. A distinctive feature of chloroplast division systems is the evolution of two phylogenetically and structurally distinct FtsZ families by independent gene duplications in different photosynthetic lineages. While many functional aspects of these proteins remain unknown, recent studies on the biochemical and dynamic properties of FtsZs from land plants, in combination with ongoing research on bacterial FtsZs, have begun to suggest mechanisms by which two functionally distinct FtsZ proteins may cooperate to drive chloroplast division.

Chloroplast division protein ARC3 regulates chloroplast FtsZ-ring assembly and positioning in arabidopsis through interaction with FtsZ2.

Chloroplast division is initiated by assembly of a mid-chloroplast FtsZ (Z) ring comprising two cytoskeletal proteins, FtsZ1 and FtsZ2. The division-site regulators ACCUMULATION AND REPLICATION OF CHLOROPLASTS3 (ARC3), MinD1, and MinE1 restrict division to the mid-plastid, but their roles are poorly understood. Using genetic analyses in Arabidopsis thaliana, we show that ARC3 mediates division-site placement by inhibiting Z-ring assembly, and MinD1 and MinE1 function through ARC3. ftsZ1 null mutants exhibited some mid-plastid FtsZ2 rings and constrictions, whereas neither constrictions nor FtsZ1 rings were observed in mutants lacking FtsZ2, suggesting FtsZ2 is the primary determinant of Z-ring assembly in vivo. arc3 ftsZ1 double mutants exhibited multiple parallel but no mid-plastid FtsZ2 rings, resembling the Z-ring phenotype in arc3 single mutants and showing that ARC3 affects positioning of FtsZ2 rings as well as Z rings. ARC3 overexpression in the wild type and ftsZ1 inhibited Z-ring and FtsZ2-ring assembly, respectively. Consistent with its effects in vivo, ARC3 interacted with FtsZ2 in two-hybrid assays and inhibited FtsZ2 assembly in a heterologous system. Our studies are consistent with a model wherein ARC3 directly inhibits Z-ring assembly in vivo primarily through interaction with FtsZ2 in heteropolymers and suggest that ARC3 activity is spatially regulated by MinD1 and MinE1 to permit Z-ring assembly at the mid-plastid.

Genome, functional gene annotation, and nuclear transformation of the heterokont oleaginous alga Nannochloropsis oceanica CCMP1779.

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogen-depleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica-specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus.

Distinct functions of chloroplast FtsZ1 and FtsZ2 in Z-ring structure and remodeling.

FtsZ, a cytoskeletal GTPase, forms a contractile ring for cell division in bacteria and chloroplast division in plants. Whereas bacterial Z rings are composed of a single FtsZ, those in chloroplasts contain two distinct FtsZ proteins, FtsZ1 and FtsZ2, whose functional relationship is poorly understood. We expressed fluorescently tagged FtsZ1 and FtsZ2 in fission yeast to investigate their intrinsic assembly and dynamic properties. FtsZ1 and FtsZ2 formed filaments with differing morphologies when expressed separately. FRAP showed that FtsZ2 filaments were less dynamic than FtsZ1 filaments and that GTPase activity was essential for FtsZ2 filament turnover but may not be solely responsible for FtsZ1 turnover. When coexpressed, the proteins colocalized, consistent with coassembly, but exhibited an FtsZ2-like morphology. However, FtsZ1 increased FtsZ2 exchange into coassembled filaments. Our findings suggest that FtsZ2 is the primary determinant of chloroplast Z-ring structure, whereas FtsZ1 facilitates Z-ring remodeling. We also demonstrate that ARC3, a regulator of chloroplast Z-ring positioning, functions as an FtsZ1 assembly inhibitor.

HIV gp41 six-helix bundle constructs induce rapid vesicle fusion at pH 3.5 and little fusion at pH 7.0: understanding pH dependence of protein aggregation, membrane binding, and electrostatics, and implications for HIV-host cell fusion.

The HIV gp41 protein catalyzes fusion between HIV and target cell membranes. The fusion states of the gp41 ectodomain include early coiled-coil (CC) structure and final six-helix bundle (SHB) structure. The ectodomain has an additional N-terminal apolar fusion peptide (FP) sequence which binds to target cell membranes and plays a critical role in fusion. One approach to understanding gp41 function is study of vesicle fusion induced by constructs that encompass various regions of gp41. There are apparent conflicting literature reports of either rapid or no fusion of negatively charged vesicles by SHB constructs. These reports motivated the present study, which particularly focused on effects of pH because the earlier high and no fusion results were at pH 3.0 and 7.2, respectively. Constructs include "Hairpin," which has SHB structure but lacks the FP, "FP-Hairpin" with FP + SHB, and "N70," which contains the FP and part of the CC but does not have SHB structure. Aqueous solubility, membrane binding, and vesicle fusion function were measured at a series of pHs and much of the pH dependences of these properties were explained by protein charge. At pH 3.5, all constructs were positively charged, bound negatively charged vesicles, and induced rapid fusion. At pH 7.0, N70 remained positively charged and induced rapid fusion, whereas Hairpin and FP-Hairpin were negatively charged and induced no fusion. Because viral entry occurs near pH 7 rather than pH 3, our results are consistent with fusogenic function of early CC gp41 and with fusion arrest by final SHB gp41.