In vivo quantitative relationship between plastid division proteins FtsZ1 and FtsZ2 and identification of ARC6 and ARC3 in a native FtsZ complex

Abstract

FtsZ1 and FtsZ2 are phylogenetically distinct homologues of the tubulin-like bacterial cell division protein FtsZ that play major roles in the initiation and progression of plastid division in plant cells. Both proteins are components of a mid-plastid ring, the Z-ring, which functions as a contractile ring on the stromal surface of the chloroplast IEM (inner envelope membrane). FtsZ1 and FtsZ2 have been shown to interact, but their in vivo biochemical properties are largely unknown. To gain insight into the in vivo biochemical relationship between FtsZ1 and FtsZ2, in the present study we investigated their molecular levels in wild-type Arabidopsis thaliana plants and endogenous interactions in Arabidopsis and pea. Quantitative immunoblotting and morphometric analysis showed that the average total FtsZ concentration in chloroplasts of 3-week-old Arabidopsis plants is comparable with that in Escherichia coli. FtsZ levels declined as plants matured, but the molar ratio between FtsZ1 and FtsZ2 remained constant at approx. 1:2, suggesting that this stoichiometry is regulated and functionally important. Density-gradient centrifugation, native gel electrophoresis, gel filtration and co-immunoprecipitation experiments showed that a portion of the FtsZ1 and FtsZ2 in Arabidopsis and pea chloroplasts is stably associated in a complex of approximately 200-245 kDa. This complex also contains the FtsZ2-interacting protein ARC6 (accumulation and replicatioin of chloroplasts 6), an IEM protein, and analysis of density-gradient fractions suggests the presence of the FtsZ1-interacting protein ARC3. Based on the mid-plastid localization of ARC6 and ARC3 and their postulated roles in promoting and inhibiting chloroplast FtsZ polymer formation respectively, we hypothesize that the FtsZ1-FtsZ2-ARC3-ARC6 complex represents an unpolymerized IEM-associated pool of FtsZ that contributes to the dynamic regulation of Z-ring assembly and remodelling at the plastid division site in vivo.

GTP-dependent Heteropolymer Formation and Bundling of Chloroplast FtsZ1 and FtsZ2

“…A typical preparation was Ͼ95% pure. Quantitative amino acid analysis also demonstrated that the BCA assay underestimates recombinant FtsZ1 and FtsZ2 by ϳ20%, similar to EcFtsZ (30) and consistent with our previous quantitative analyses (20).…”

“…Depletion or overexpression of either protein impairs division and results in enlarged chloroplasts, which are the phenotypic equivalent of the bacterial filamentation phenotype (17)(18)(19). These findings, along with data showing that the molar ratio between Arabidopsis FtsZ1 and FtsZ2 remains constant throughout leaf development (20), suggest that their stoichiometry is important for chloroplast division activity. In vitro, FtsZ1 and FtsZ2 have been reported to exhibit GTPase activity and to undergo GTPdependent assembly into protofilaments (21)(22)(23).…”

“…However, the previous analyses have been limited in scope, and whether FtsZ1 and FtsZ2 form heteropolymers has not been investigated. Heteropolymerization is suggested by the observation that FtsZ1 and FtsZ2 are always tightly colocalized in vivo even when FtsZ filament morphology is perturbed (24,25) and by studies showing that FtsZ1 and FtsZ2 interact in two-hybrid assays (26) and in a native complex (20).Here, we present a detailed study of the in vitro biochemical behavior of FtsZ1 and FtsZ2 individually and in combination. We show that, similar to bacterial FtsZ, FtsZ1 and FtsZ2 both bind and hydrolyze GTP and individually undergo GTP-dependent assembly into thin protofilaments, although only when stabilized by calcium.…”

“…However, the previous analyses have been limited in scope, and whether FtsZ1 and FtsZ2 form heteropolymers has not been investigated. Heteropolymerization is suggested by the observation that FtsZ1 and FtsZ2 are always tightly colocalized in vivo even when FtsZ filament morphology is perturbed (24,25) and by studies showing that FtsZ1 and FtsZ2 interact in two-hybrid assays (26) and in a native complex (20).…”

Proteomic Analysis of Chloroplast-to-Chromoplast Transition in Tomato Reveals Metabolic Shifts Coupled with Disrupted Thylakoid Biogenesis Machinery and Elevated Energy-Production Components

“…11C; Supplemental Table S4). Two nuclear-encoded forms of filamenting temperature-sensitive Z mutants (FtsZ1, Solyc07g065050 and FtsZ2, Solyc09g009430) play a major role in the initiation and progression of plastid division in plant cells (McAndrew et al, 2008). They are both present at MG and B stages, but FtsZ1 was absent at the R stage while FtsZ2 remained stable at all three stages.…”