Differential Roles of Arabidopsis Dynamin‐Related Proteins DRP3A, DRP3B, and DRP5B in Organelle DivisionF
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Abstract Dynamin‐related proteins (DRPs) are key components of the organelle division machineries, functioning as molecular scissors during the fission process. In Arabidopsis , DRP3A and DRP3B are shared by peroxisomal and mitochondrial division, whereas the structurally‐distinct DRP5B (ARC5) protein is involved in the division of chloroplasts and peroxisomes. Here, we further investigated the roles of DRP3A, DRP3B, and DRP5B in organelle division and plant development. Despite DRP5B's lack of stable association with mitochondria, drp5B mutants show defects in mitochondrial division. The drp3A‐2 drp3B‐2 drp5B‐2 triple mutant exhibits enhanced mitochondrial division phenotypes over drp3A‐2 drp3B‐2 , but its peroxisomal morphology and plant growth phenotypes resemble those of the double mutant. We further demonstrated that DRP3A and DRP3B form a supercomplex in vivo , in which DRP3A is the major component, yet DRP5B is not a constituent of this complex. We thus conclude that DRP5B participates in the division of three types of organelles in Arabidopsis , acting independently of the DRP3 complex. Our findings will help elucidate the precise composition of the DRP3 complex at organelle division sites, and will be instrumental to studies aimed at understanding how the same protein mediates the morphogenesis of distinct organelles that are linked by metabolism.Keywords:
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Cell organelles are compartments that occur in eukaryotic cells (for instance plant, animal or fungal cells). Recent studies indicated that cell organelles do not function in isolation, but extensively communicate and collaborate with each other. For these processes membrane contact sites are very important. Membrane contact sites are regions where two membranes form tight physical associations. These contact sites play roles in multiple important cellular processes, such as transport of various molecules like lipids, organelle formation and fission, organelle positioning and degradation. Peroxisomes are cell organelles that occur in almost all eukaryotic cells. Peroxisomes have been implicated in multiple functions including the detoxification of hydrogen peroxide and cellular lipid metabolism. In human, defects in peroxisome formation or function cause various symptoms and often are lethal.The research described in this thesis focuses on peroxisomal contact sites, using yeast as model organism. The research resulted in the identification of two novel contact sites, namely peroxisome-vacuole and peroxisome-plasma membrane contact sites. These contacts are important for peroxisomal membrane expansion and peroxisomal anchoring, respectively. The formation of both contacts require the peroxisomal membrane protein Pex3.
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This is the detailed protocol for the measurement of the number of peroxisomes described by Shibata et al. (2013). It is difficult to count the number of organelles in a cell because of the thickness of plant leaves. To overcome this challenge, protoplasts were isolated from leaves, and the number of peroxisomes per protoplast was counted. This method can be applied to other organelles such as mitochondria that are labeled with GFP or its derivatives.
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Cellular compartmentalization of central metabolic pathways as lipid metabolism to mitochondria and peroxisomes enables high efficient control processes. The basis to understand mitochondrial or peroxisomal function is exactly to determine proteins physically present. For proteomic investigations of mouse liver organelles, we developed 2-DE reference maps covering the range pH 4–9, available under (www.diabesityprot.org). MALDI-TOF-MS/MS analyses identified a total of 799 (mitochondria) and 681 (peroxisome) protein spots resembling 323 and 293 unique proteins, respectively. Direct comparison of mitochondrial and peroxisomal proteins indicated an approximate overlap of 2/3 of identified proteins. Gene Ontologies (GO) of the identified proteins in respect to physical presence confirmed functional specifications within the organelles. The 2-DE organelle reference maps will aid to point out functional differences and similarities. Our observations suggest that for functional analyses metabolic alterations focusing on one organelle are not sufficient and parallel comparison of both organelles is to be preferred.
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Peroxisomes are subcellular organelles and are present in virtually all eukaryotic cells.Characteristic features of these organelles are their inducibility and their functional versatility.Their importance in the intermediary metabolism of cells is exemplified by the discovery of several inborn, fatal peroxisomal errors in man, the so-called peroxisomal disorders.Recent findings in research on peroxisome biogenesis and function have demonstrated that peroxisomal matrix proteins and peroxisomal membrane proteins (PMPs) follow separate pathways to reach their target organelle.This paper addresses the principles of PMP sorting and summarizes the current knowledge of the role of these proteins in organelle biogenesis and function.
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The formation of membrane contact sites between cellular organelles is required for proper organelle communication and maintenance in the compartmentalized eukaryotic cell. We recently identified a tether that links peroxisomes to the cortical ER in the yeast, Saccharomyces cerevisiae. The tether is made up of the peroxisome biogenic protein Pex3p and the peroxisome inheritance factor Inp1p, and is formed by Inp1p-mediated linkage of ER-bound Pex3p and peroxisomal Pex3p. Here we discuss how this tether is fine-tuned to ensure that peroxisomes are stably maintained over generations of yeast cells.
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Peroxisomes are subcellular organelles and are present in virtually all eukaryotic cells. Characteristic features of these organelles are their inducibility and their functional versatility. Their importance in the intermediary metabolism of cells is exemplified by the discovery of several inborn, fatal peroxisomal errors in man, the so-called peroxisomal disorders. Recent findings in research on peroxisome biogenesis and function have demonstrated that peroxisomal matrix proteins and peroxisomal membrane proteins (PMPs) follow separate pathways to reach their target organelle. This paper addresses the principles of PMP sorting and summarizes the current knowledge of the role of these proteins in organelle biogenesis and function.
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Organelle biogenesis
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Peroxisomes are ubiquitous subcellular organelles of eukaryotic cells. As with all organelles, peroxisomes can be purified from cell lysates using a combination of differential centrifugation and density gradient centrifugation. Here, we describe a method for purifying peroxisomes from the yeast Saccharomyces cerevisiae. The method involves gentle lysis of yeast spheroplasts, followed by differential centrifugation to obtain a crude organelle pellet enriched for peroxisomes and mitochondria. To separate peroxisomes from mitochondria, the organelle pellet is resuspended and spun through a sucrose density gradient. Peroxisomes purified in this way can be used to explore whether a protein of interest might be associated with the organelle.
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From today's perspective, it seems surprising that peroxisomes were regarded as uninteresting organelles for almost two decades after their discovery in 1954 (Rhodin 1954). Only after the elucidation of the peroxisomal β-oxidation pathway in rat liver (Lazarow and De Duve 1976; Lazarow 1978) and the recognition of peroxisomal disorders in humans (for review, see Van den Bosch et al. 1992; Moser 1993) did it become clear that these ubiquitous eukaryotic cell structures fulfill important metabolic functions. The delay in acceptance of peroxisomes as more than “fossil organelles” was due in part to the fact that, in contrast to most other organelles, peroxisomes lack a general unifying metabolic function. This apparent shortcoming is actually an indication of their specific metabolic role. Because a multiplicity of biochemical functions are now known to reside in these organelles, peroxisomes can be appropriately described as “ulti-purpose” organelles (Opperdoes 1988). The large number of biochemical reactions...
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