FtsZ ring formation at the chloroplast division site in plants

Among the events that accompanied the evolution of chloroplasts from their endosymbiotic ancestors was the host cell recruitment of the prokaryotic cell division protein FtsZ to function in chloroplast division. FtsZ, a structural homologue of tubulin, mediates cell division in bacteria by assembling into a ring at the midcell division site. In higher plants, two nuclear-encoded forms of FtsZ, FtsZ1 and FtsZ2, play essential and functionally distinct roles in chloroplast division, but whether this involves ring formation at the division site has not been determined previously. Using immunofluorescence microscopy and expression of green fluorescent protein fusion proteins in Arabidopsis thaliana, we demonstrate here that FtsZ1 and FtsZ2 localize to coaligned rings at the chloroplast midpoint. Antibodies specific for recognition of FtsZ1 or FtsZ2 proteins in Arabidopsis also recognize related polypeptides and detect midplastid rings in pea and tobacco, suggesting that midplastid ring formation by FtsZ1 and FtsZ2 is universal among flowering plants. Perturbation in the level of either protein in transgenic plants is accompanied by plastid division defects and assembly of FtsZ1 and FtsZ2 into filaments and filament networks not observed in wild-type, suggesting that previously described FtsZ-containing cytoskeletal-like networks in chloroplasts may be artifacts of FtsZ overexpression.     

The WD repeat protein,Mdv1p,functions as a molecular adaptor by interacting with Dnm1p and Fis1p during mitochondrial fission

Yeast mitochondrial fission is a multistep process during which the dynamin-related GTPase, Dnm1p, assembles into punctate structures that associate with the outer mitochondrial membrane and mediate mitochondrial division. Steps in the Dnm1p-dependent process of fission are regulated by the actions of the WD repeat protein, Mdv1p, and the mitochondrial outer membrane protein, Fis1p. Our previous studies suggested a model where Mdv1p functions to regulate fission at a post-Dnm1p assembly step and Fis1p functions at two distinct steps, at an early point, to regulate Dnm1p assembly, and later, together with Mdv1p, to facilitate Dnm1p-dependent mitochondrial fission. To test this model, we have examined the physical and functional relationship between Mdv1p and Fis1p and present genetic, biochemical, and two-hybrid data indicating that a Fis1p-Mdv1p complex is required to regulate mitochondrial fission. To further define the role of Mdv1p in fission, we examined the structural features of Mdv1p required for its interactions with Dnm1p and Fis1p. Data from two-hybrid analyses and GFP-tagged domains of Mdv1p indicate that it contains two functionally distinct domains that enable it to function as a molecular adaptor to regulate sequential interactions between Dnm1p and Fis1p and catalyze a rate-limiting step in mitochondrial fission.     

Molecular chaperones and the biogenesis of mitochondria and peroxisomes

Summary— A review of the proteinaceous machinery involved in protein sorting pathways and protein folding and assembly in mitochondria and peroxisomes is presented. After considering the various sorting pathways and targeting signals of mitochondrial and peroxisomal proteins, we make a comparative dissection of the protein factors involved in: i) the stabilization of cytosolic precursor proteins in a translocation competent conformation; ii) the membrane import apparatus of mitochondria and peroxisomes; iii) the processing of mitochondrial precursor proteins, and the eventual processing of certain peroxisomal precursor, in the interior of the organelles; and iv) the requirement of molecular chaperones for appropriate folding and assembly of imported proteins in the matrix of both organelles. Those aspects of mitochondrial biogenesis that have developed rapidly during the last few years, such as the requirement of molecular chaperones, are stressed in order to stimulate further parallel investigations aimed to understand the origin, biochemistry, molecular biology and pathology of peroxisomes. In this regard, a brief review of findings from our group and others is presented in which the role of the F₁-ATPase α-subunit is pointed out as a molecular chaperone of mitochondria and chloroplasts. In addition, data are presented that could question our previous indication that the immunoreactive protein found in the rat liver peroxisomes is due to the presence of the F₁-ATPase α-subunit.     

The genetic control of plant mitochondrial morphology and dynamics

Little is known about the genetic control of mitochondrial morphology and dynamics in higher plants. We used a genetic screen involving fluorescence microscopic analysis of ethyl methane sulphonate (EMS)-mutated Arabidopsis thaliana seedlings expressing GFP targeted to mitochondria to isolate eight mutants displaying distinct perturbations of the normal mitochondrial morphology or distribution. We describe five mutants with distinct and unique mitochondrial phenotypes, which map to five different loci, not previously implicated in mitochondrial behaviour in plants. We have used a combination of forward and reverse genetics to identify one of the genes, friendly mitochondria (FMT), a homologue of the CluA gene of Dictyostelium discoideum, which is involved in the correct distribution of mitochondria in the cell. The five mutants constitute a powerful resource to aid our understanding of mitochondrial dynamics in higher plants.     

Dynamin-Related Protein 1-Dependent Mitochondrial Fission Changes in the Dorsal Vagal Complex Regulate Insulin Action

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Dynamin-related Protein 1 Oligomerization in Solution Impairs Functional Interactions with Membrane-anchored Mitochondrial Fission Factor

Mitochondrial fission is a crucial cellular process mediated by the mechanoenzymatic GTPase, dynamin-related protein 1 (Drp1). During mitochondrial division, Drp1 is recruited from the cytosol to the outer mitochondrial membrane by one, or several, integral membrane proteins. One such Drp1 partner protein, mitochondrial fission factor (Mff), is essential for mitochondrial division, but its mechanism of action remains unexplored. Previous studies have been limited by a weak interaction between Drp1 and Mff in vitro. Through refined in vitro reconstitution approaches and multiple independent assays, we show that removal of the regulatory variable domain (VD) in Drp1 enhances formation of a functional Drp1-Mff copolymer. This protein assembly exhibits greatly stimulated cooperative GTPase activity in solution. Moreover, when Mff was anchored to a lipid template, to mimic a more physiologic environment, significant stimulation of GTPase activity was observed with both WT and ΔVD Drp1. Contrary to recent findings, we show that premature Drp1 self-assembly in solution impairs functional interactions with membrane-anchored Mff. Instead, dimeric Drp1 species are selectively recruited by Mff to initiate assembly of a functional fission complex. Correspondingly, we also found that the coiled-coil motif in Mff is not essential for Drp1 interactions, but rather serves to augment cooperative self-assembly of Drp1 proximal to the membrane. Taken together, our findings provide a mechanism wherein the multimeric states of both Mff and Drp1 regulate their collaborative interaction.     

The mitochondrial fission regulator DRP3B does not regulate cell death in plants

BACKGROUND AND AIMS: Recent reports have described dramatic alterations in mitochondrial morphology during metazoan apoptosis. A dynamin-related protein (DRP) associated with mitochondrial outer membrane fission is known to be involved in the regulation of apoptosis. This study analysed the relationship between mitochondrial fission and regulation of plant cell death. METHODS: Transgenic plants were generated possessing Arabidopsis DRP3B (K56A), the dominant-negative form of Arabidopsis DRP, mitochondrial-targeted green fluorescent protein and mouse Bax. KEY RESULTS: Arabidopsis plants over-expressing DRP3B (K56A) exhibited long tubular mitochondria. In these plants, mitochondria appeared as a string-of-beads during cell death. This indicates that DRP3B (K56A) prevented mitochondrial fission during plant cell death. However, in contrast to results for mammalian cells and yeast, Bax-induced cell death was not inhibited in DRP3B (K56A)-expressing plant cells. Similarly, hydrogen peroxide-, menadione-, darkness- and salicylic acid-induced cell death was not inhibited by DRP3B (K56A) expression. CONCLUSIONS: These results indicate that the systems controlling cell death in animals and plants are not common in terms of mitochondrial fission.     

Recent developments in chloroplast protein transport

Most proteins located in chloroplasts are encoded by nuclear genes, synthesized in the cytoplasm, and transported into the organelle. The study of protein uptake by chloroplasts has greatly expanded over the past few years. The increased activity in this field is due, in part, to the application of recombinant DNA methodology to the analysis of protein translocation. Added interest has also been gained by the realization that the transport mechanisms that mediate protein uptake by chloroplasts, mitochondria and the endoplasmic reticulum display certain characteristics in common. These include amino terminal sequences that target proteins to particular organelles, a transport process that is mechanistically independent from the events of translation, and an ATP-requiring transport step that is thought to involve partial unfolding of the protein to be translocated. In this review we examine recent studies on the binding of precursors to the chloroplast surface, the energy-dependent uptake of proteins into the stroma, and the targeting of proteins to the thylakoid lumen. These aspects of protein transport into chloroplasts are discussed in the context of recent studies on protein uptake by mitochondria.Abbrevlations CAT chloramphenicol acetyl transferase - CCCP carbonylcyanide m-chlorophenylhydrazone - DHFR dihydrofolate reductase - EPSP 5-enol-pyruvylshikimate-3-phosphate - ER endoplasmic reticulum - LHCP light harvesting chlorophyll a/b apoprotein - NPT neomycin phosphotransferase - oATP adenosine-2,3-dialdehyde-5-triphosphate - P-inorganic phosphate Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - SSU small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase - SRP signal recognition particle