Molecular Composition of the Chloroplast Division Apparatus

项目总结。叶绿体种群的维持对光合作用真核生物的生存至关重要，并且依赖于叶绿体的复制。然而，导致叶绿体分裂的分子机制仍未确定。近年来，在高等植物中发现了两个核基因家族，FtsZ1和FtsZ2，每个家族都编码关键的细菌细胞分裂蛋白FtsZ的同源基因。细菌FtsZ是一种与微管蛋白相关的GTP酶，在胞质分裂开始时聚合，在收缩细胞的分裂平面形成环状结构。对拟南芥特异的FtsZ1和FtsZ2家族成员AtFtsZ1-1和AtFtsZ2-1进行反义抑制，证明了这两个基因家族在高等植物中调节叶绿体分裂中的重要和功能不同的作用，并确立了叶绿体分裂机制的内生共生起源。此外，已经产生了针对AtFtsZ1-1和AtFtsZ2-1的高度特异性抗体，并将其用于免疫荧光显微镜，首次表明FtsZ1和FtsZ2共同定位于叶绿体分裂部位的膜相关环。在体外实验证明FtsZ1而不是FtsZ2被导入叶绿体的基础上，提出了一种叶绿体分裂装置的大分子组织模型，其中FtsZ1和FtsZ2分别在叶绿体被膜的内外表面组装成环，并协同作用来收缩细胞器。然而，在拟南芥中发现了第二个FtsZ2基因AtFtsZ2-2，这表明这个模型可能是不完整的。该项目的总体目标是通过更全面地探索拟南芥中三个FtsZ蛋白的功能，并通过识别和分析叶绿体分裂装置的其他组件的功能，来严格测试和扩展该模型。第一个目标是确定FtsZ2的精确定位。在这一模型的部分支持下，最近发现AtFtsZ1-1和AtFtsZ2-1以及它们在其他植物中的同源物都定位在叶绿体分裂部位的膜相关环上。尽管每个FtsZ相对于包膜的精确定位还没有解决，但有证据表明FtsZ1存在于间质隔室。免疫电子显微镜和蛋白酶保护实验将被用来确定FtsZ2位于哪个隔室：间质、胞质或膜间间隙。这些发现将对评估和扩展该模型至关重要，并将显著影响与叶绿体分裂装置的组成、组装、组织和力学相关的其他实验的设计和解释。第二个目标是测试AtFtsZ2-2与AtFtsZ2-1的功能。AtFtsZ2-2与AtFtsZ2-1有80%以上的氨基酸同源性，强烈暗示它参与了叶绿体的分裂，但表明这两个蛋白可能在功能上是多余的。相反，初步数据表明AtFtsZ2-1和AtFtsZ2-2可能在功能上是不同的。为了解决这一明显的矛盾，将产生表达AtFtsZ2-2反义和正义结构的转基因植株；它们的表型将与相应的AtFtsZ2-1转基因植株的表型进行比较。这两个基因的敲除突变体也将被分离出来，用于对突变表型的单独和组合进行遗传和细胞学分析。如果在单个突变体中观察到叶绿体分裂缺陷，将确定两个基因产物中每一个相互补充的能力。为了区分AtFtsZ2-1和AtFtsZ2-2的作用，还将包括用异构体特异性抗肽抗体进行定位研究，以及用基因特异性探针和记者进行表达模式分析。第三个目标是识别与植物FtsZ相互作用的蛋白质。豌豆叶绿体系统将与多种免疫亲和和交联策略结合使用，以确定与高等植物中的FtsZ1和FtsZ2蛋白特异相互作用的蛋白质。超出这一建议范围的研究将把从豌豆系统获得的生化洞察力应用于对拟南芥叶绿体分裂的持续遗传分析。这个项目应该会对叶绿体分裂装置的组成、组装、组织和机制提供重要的新见解。此外，由于最近发现了叶绿体和线粒体分裂过程之间的机械相似之处，这些研究将补充并可能与理解线粒体分裂有关。

PROJECT SUMMARY. The maintenance of plastid populations is essential to the viability of photosynthetic eukaryotes and dependent on plastid replication. Yet, the molecular mechanisms underlying plastid division remain undetermined. In recent years, two nuclear gene families have been identified in higher plants, FtsZ1 and FtsZ2, each encoding homologues of the key bacterial cell division protein FtsZ. Bacterial FtsZ, a GTPase related to tubulin, polymerizes at the onset of cytokinesis to form a ring structure at the division plane that constricts the cell. Antisense repression of specific FtsZ1 and FtsZ2 family members from Arabidopsis, AtFtsZ1-1 and AtFtsZ2-1, has demonstrated essential and functionally distinct roles for both gene families in mediating plastid division in higher plants, and firmly established the endosymbiotic origin of the plastid division machinery. Further, antibodies highly specific for AtFtsZ1-1 and AtFtsZ2-1 have been generated and used in immunofluorescence microscopy to show for the first time that both FtsZ1 and FtsZ2 co-localize to membrane-associated rings at the plastid division site. Based on experiments demonstrating that FtsZ1 but not FtsZ2 is imported into chloroplasts in vitro, a model has been proposed for the macromolecular organization of the plastid division apparatus wherein FtsZ1 and FtsZ2 assemble into rings on the inner and outer surfaces of the chloroplast envelope membranes, respectively, and act in concert to constrict the organelle. However, the discovery of a second FtsZ2 gene in Arabidopsis, AtFtsZ2-2, suggests this model may be incomplete. The overall goal of the project is to rigorously test and expand upon the model by probing more fully the functions of the three FtsZ proteins in Arabidopsis and by identifying and analyzing the functions of additional components of the plastid division apparatus. The first objective is to determine the precise localization of FtsZ2. In partial support of this model, it was recently determined that both AtFtsZ1-1 and AtFtsZ2-1 and their orthologues in other plants localize to membrane-associated rings at the plastid division site. Although the precise localization of each FtsZ in relation to the envelope membranes has not been resolved, evidence that FtsZ1 resides in the stromal compartment is compelling. Immunoelectron microscopy and protease protection experiments will be employed to determine in which compartment FtsZ2 is localized: stromal, cytosolic, or intermembrane space. The findings will be critical in evaluating and expanding upon the model and will significantly influence the design and interpretation of other experiments related to the composition, assembly, organization and mechanics of the plastid division apparatus. The second objective is to test the function of AtFtsZ2-2 in relation to that of AtFtsZ2-1. AtFtsZ2-2 shares over 80% amino acid identity with AtFtsZ2-1, strongly implicating it in plastid division but suggesting the two proteins may be functionally redundant. In contrast, preliminary data suggest that AtFtsZ2-1and AtFtsZ2-2 may be functionally distinct. To resolve this apparent contradiction, transgenic plants expressing antisense and sense constructs of AtFtsZ2-2 will be generated; their phenotypes will be compared to those of the corresponding AtFtsZ2-1 transgenic plants. Knockout mutants for both genes will also be isolated for genetic and cytological analyses of the mutant phenotypes singly and in combination. If plastid division defects are observed in the single mutants, the capacity of each of the two gene products to complement the other will be determined. Attempts to distinguish the roles of AtFtsZ2-1 and -2 will also include localization studies with isoform-specific anti-peptide antibodies and expression pattern analyses with gene-specific probes and reporters. The third objective is to identify proteins that interact with plant FtsZs. The well characterized and biochemically tractable pea chloroplast system will be used in conjunction with several immunoaffinity and crosslinking strategies to identify proteins that interact specifically with FtsZ1 and FtsZ2 proteins in higher plants. Studies beyond the scope of this proposal will apply the biochemical insight gained from the pea system to continued genetic analysis of chloroplast division in Arabidopsis. This project should provide significant new insights into the composition, assembly, organization and mechanics of the plastid division apparatus. In addition, because of recently revealed mechanistic parallels between the processes of chloroplast and mitochondrial division, these studies will complement and may have relevance for understanding mitochondrial division.