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1. Details of Module and its Structure
Module Detail
Subject Name <BOTANY>
Paper Name <Cell Biology>
Module Name/Title < Chloroplast and mitochondria – biogenesis of Chloroplast and mitochondria >
Module Id <10b>
Pre-requisites Basic knowledge about plant cells, their contents and basic idea about cell organelles
Objectives To make the students aware of the biogenesis, multiplication and evolution of chloroplast and mitochondria
Keywords Biogenesis, fission, fusion, evolution, chloroplast, mitochondria
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2. 2. Development Team
TABLE OF CONTENTS
1. Biogenesis / development of chloroplast 2. Multiplication of chloroplasts
3. Evolution of chloroplasts
4. Biogenesis / development of mitochondria 5. Division, fusion and fission of mitochondria
5.1 Mitochondrial division 5.2 Mitochondrial fusion 5.3 Mitochondrial fission
Role Name Affiliation
National Coordinator <NA>
Subject Coordinator <Dr. Sujata Bhargava>
Paper Coordinator <Dr. Nutan Malpathak>
Content Writer/Author (CW) <Dr. Gauri Abhyankar>
Content Reviewer (CR) <Dr. Nutan Malpathak >
Language Editor (LE) < Dr. Nutan Malpathak>
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1. Biogenesis / Development of chloroplasts –
The chloroplast is an essential organelle of photosynthetic eukaryotes. It captures and converts light energy to chemical energy, and produces various metabolites necessary for cellular activities. Each chloroplast arises from a pre-existing chloroplast by binary fission. The chloroplasts are considered to be originated from a free-living cyanobacteria-like prokaryote through engulfment over one billion years ago. Just like a prokaryotic cell, chloroplast is also surrounded by an outer envelope membrane (OEM) and an inner envelope membrane (IEM) (Inoue, 2011). In higher plants, the chloroplast gained ability to differentiate into various non-green plastids viz. chromoplasts (in yellow and orange fruits and flower petals), amyloplasts (in non-colored starch accumulating tissues) and etioplasts (in dark-grown seedlings). These non-green plastid lack photosynthetic machinery. They are usually inter-convertible.
Chloroplast biogenesis is a multistage process that ultimately directs to fully differentiated and functionally mature plastids. Chloroplasts develop from small organelles called proplastids. They are present in leaf primordia and possess small vesicles that do not contain any photosynthetic complexes. On exposure to light, proplastids develop the thylakoid network and achieve photosynthetic capacity (Rudowska et al, 2012). The process of thylakoid biogenesis requires harmonized assembly of lipids, proteins, and chlorophylls, which together account for >98% of the mass of the thylakoid membrane (Pribil et al, 2014).
De-etiolation and biogenesis of thylakoids –
During biogenesis, if light is absent, then the proplastids mature into etioplasts.
Inner membrane of this etioplast forms a semi-crystalline network of interconnected tubules called a prolamellar body. The prolamellar body produces extensions and forms prothylakoids. Because they contain lower amounts of monogalactosyldiacylglycerol (MGDG), they permit them to form a planar bilayer structure (Fig 1A). The prolamellar body is photosynthetically inactive, but it contains 64 proteins that are linked to the photosynthetic light reactions, the Calvin cycle, protein synthesis and pigment biosynthesis. By now, ATP synthase is fully assembled. Cyt b6/f complex is present as a dimer excluding mature chlorophyll a. PSII biogenesis is arrested at a pre-complex stage and PSI assembly probably takes place later in the greening process. In etioplasts, LHCs are not yet inserted into the membrane. The most abundant protein in prolamellar bodies is the NADPH:protochlorophyllide oxidoreductase (POR). NADPH, protochlorophyllide and MGDG are the major constituents of the prolamellar body (Adam et al, 2011). The high content of POR (90% of the protein content) is responsible for the semi-crystalline
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form of prolamellar bodies. Adaptation to light brings about reduction of protochlorophyllide to chlorophyllide by POR and finally it is esterified into chlorophyll. At the same time, the prolamellar body loses its semi-crystalline structure and the extruded lamellae align in parallel throughout the stroma (Fig. 1B, C). The semi-crystalline prolamellar body then becomes planar thylakoids (Fig. 1D).
Fig 1: Schematic overview of the light-dependent de-etiolation process and the biogenesis of thylakoid membranes that focuses on PSII, PSI, and LHC. (A) The etioplast contains prothylakoids and the semi- crystalline prolamellar body. MGDG and POR together form the cubic lipid structure that makes up the prolamellar body. (B) Etio-chloroplast stage. The de-etiolation process is initiated by exposure to light and the light- and NADPH-dependent reduction of protochlorophyllide by POR. The semi-crystalline prolamellar body disassembles. Mainly monomeric proteins are incorporated into the developing thylakoids. (C) The lamellar structures align in parallel within the chloroplast. With the formation of protein complexes, the thylakoids enter a photoactive state. (D) The grana stacks characteristic of mature thylakoids form upon incorporation of mega- and super complexes (Pribil et al, J Exp Bot 65(8): 1955 – 1972, 2014).
The thylakoid membrane contains five major lipids. The non-bilayer forming MGDG accounts for 52% of total lipids by weight. Along with MGDG, 27% DGDG (Digalactosyldiacylglycerol), 15% SQDG (sulphoquinovosyldiacylglycerol), 3% PG (phosphatidylglycerol), and 3% PC (phosphatidylcholine) account for the rest (Kirchhoff et
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al, 2002). Synthesis of all of these lipids is carried out in the chloroplast envelope. So they need to be transported continuously to thylakoids. This lipid transfer may occur through one of the following 3 pathways - (i) a vesicular pathway; (ii) soluble glycerolipid transfer proteins; or (iii) invaginations that directly connect the envelope to thylakoids. Which pathway works is not clear (Fig. 1D). If you see a mutant with a defective MGDG synthase 1 (mgd1), it is unable to produce photosynthetically active membranes, but shows invaginations of the inner envelope (Kobayashi et al, 2013).
Chloroplasts are found in different plant organs like cotyledons, leaves, stems, fruits, and flowers, depending upon the species and stage of development. Due to the specialization of tissues, the biogenesis and development of the chloroplasts differs between organs as well as between species. E.g. chloroplast development proceeds differently in cotyledons and true leaves (Pogson & Albrecht, 2011). Tight assembly of proteins encoded by nuclear as well as chloroplast genome along with chlorophylls and carotenoids is very crucial for photosynthesis. This requires coordination between the two organelles at the level of transcription, translation, import, protein turnover, and metabolite flux. These we can call as plastidic and nuclear factors required for biogenesis and development of chloroplasts (Fig 2). Along with these, there are also certain non plastidic factors essential for chloroplast biogenesis and development (Fig 3).
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Fig 2: Plastidic and nuclear factors. Numbers refer to factors influencing chloroplast biogenesis in the text (Pogson & Albrecht, Plant Physiol 155: 1545 – 1551, 2011).
Factors influencing chloroplast biogenesis and development – a) Nuclear gene transcription –
When the seedlings receive light, most of the genes encoding chloroplast-targeted proteins are up regulated. This confirms that majority of the chloroplast proteins are encoded by nuclear genes. The perception of light is required for activation of the phytochromes like phyA and phyB. The light-mediated transcription requires involvement of phytochrome interacting factors (PIFs). These are transcription factors and loss of either PIF1 or PIF3 results in delayed development of the chloroplasts (Moon et al, 2008). In addition to PIFs, other transcription factors like GLKs (Golden 2- like proteins) also assist for chloroplast development.
b) Chloroplast Protein Import and Processing –
Most of chloroplast targeted proteins enter the plastids via the translocon at outer (TOC) or inner (TIC) envelope of chloroplast. The proteins are transported to translocons by their chloroplast targeting peptide HSP90 and TOC159. TOC59 is very crucial for chloroplast biogenesis. If it is knocked out, then chloroplast biogenesis is dramatically impaired. A targeting system is operational within the chloroplast also. The system functions for integration of proteins into the thylakoid membranes and/or lumen.
Integration of proteins into the membrane can either be spontaneous or via the chloroplast signal recognition particle pathway. If any of the pathways is impaired, it may show lethal effect for the seedling or may affect thylakoid formation (Cline and Dabney-Smith, 2008). A thylakoidal processing peptidase, PISP1, is required for removing the thylakoid-targeting sequence from thylacoidal proteins and has been shown to be involved in processing PsbO and PsbP proteins. Loss of the protein may be seedling lethal.
c) Chloroplast gene transcription and translation –
Chloroplast biogenesis and development are dependent on chloroplast gene transcription, RNA maturation, protein translation, and modification brought about by both nuclear-encoded polymerase and plastid encoded polymerase machinery. Loss of the plastidic transcription factors - sig2 and sig6 – delays chloroplast biogenesis (Chi et al, 2010). Also, nuclear encoded RNA Polymerases (rpoT) affect chloroplast
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biogenesis (Azevedo et al, 2008). pTAC12/HEMERA is a dual targeted protein required for plastid transcription. In chloroplasts, it is essential for the regulation of PIF1 and PIF3 protein accumulation. Loss of this protein results in an albino phenotype.
Recently, a large class of nuclear-encoded proteins known as PPRs (pentatricopeptide repeats) have been shown to be critical for RNA processing, splicing, editing, stability, maturation, and translation in the chloroplast. In most cases the phenotypes of PPR mutations are seedling lethal (Chi et al, 2010).
d) Protein folding and degradation –
Chaperones like HSP70 and Cpn60 mediate chloroplast protein folding. Mutation in HSP70 leads to variegated cotyledons (Latijnhouwers et al, 2010). The rates of protein translation and degradation are important for chloroplast development. Mutations in FtsH proteases impair PSII D1 protein degradation and so causes a white-patched phenotype in true leaves, but not cotyledons (Miura et al, 2007).
e) Thylakoid formation and pigment biosynthesis –
The thylakoids are formed mainly by mono- and digalactosyldiacylglycerol. Many mutations have been reported to affect thylakoid formation. Thus, carotenoid and chlorophyll biosynthesis is tightly regulated during chloroplast development as they are essential for the assembly of photosynthetic complexes and photoprotection.
f) Retrograde signaling –
Majority of the proteins needed for chloroplast function are encoded in the nucleus.
Various factors are involved in the control of retrograde signaling. The PSII-associated proteins, EXECUTER1 and EXECUTER2, mediate singlet oxygen signalling pathways (Kim et al, 2009). The double mutant ex1ex2 exhibit white cotyledon regions that contain undifferentiated plastids.
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Fig 3: Model of environmental, cellular, and temporal factors that influence chloroplast biogenesis and development in seedlings. Environmental factors involved are, among others, light, the cellular factors include the communication between the organelles, and the embryo development is one of the temporal factors that influences chloroplast biogenesis in germinating seedlings (Pogson & Albrecht, Plant Physiol 155: 1545 – 1551, 2011).
2. Multiplication of chloroplasts –
Chloroplasts divide by binary fission (Fig 4a) but in certain cases, they show multiple fission. Binary fission supports the endosymbiotic origin of chloroplasts. Two processes were very much essential for evolution of chloroplasts –
a) establishment of host-controlled division machinery and b) protein import apparatus.
The molecular mechanism of chloroplast division has been studied extensively using Arabidopsis and the red alga Cyanidioschyzon merolae. Cytological studies have revealed the formation of constriction with plastid-dividing (PD) rings. These are electron- dense structures observed on the cytoplasmic and stromal sides of the chloroplast envelope (Miyagishima, 2011). Recently, the outer PD ring from Cyanidioschyzon chloroplasts has been studied. It is made up of a bundle of polyglucan filaments and a glycogenin-like protein. Whether this is the case in vascular plant chloroplasts remains to be determined. Such structures have not been identified from land plants yet.
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The plastid division apparatus is basically evolved from the cell division apparatus of cyanobacteria. This was postulated when homologs of the bacterial cell division protein FtsZ (Filamentous temperature-sensitive) was observed during chloroplast division (Fig 4b) (Osteryoung & Vierling, 1995).
a b
Fig 4: a) Dividing chloroplasts in the moss P. patens and b) localization of the FtsZ protein at the chloroplast division site in Arabidopsis (Miyagishima, Plant Physiol. 155, 1533–1544, 2011).
Bacterial FtsZ is a tubulin-like cytoskeletal GTPase and is able to self-assemble spontaneously into Z-ring at the division site. Once Z-ring is formed, assembly of the cell division complex starts. The assembly helps for the constriction. Similarly, Z-ring is formed at the equator in chloroplast division. This may initiate the assembly of the plastid division complex (Fig 5a) (Miyagishima et al, 2003). Almost all prokaryotes, including cyanobacteria, have a single FtsZ gene whereas, plants and green algae have two nuclear-encoded forms of FtsZ viz. FtsZ1 and FtsZ2. Both are components of the plastidic Z-ring and function in plastid division (Vitha et al, 2001). FtsZ2 is more similar to its bacterial orthologs. Also it is able to assemble in absence of FtsZ1.
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FtsZ dynamics in bacteria is governed by several positive and negative regulators.
Combined activities of both these factors control the positioning, assembly, and disassembly of the Z-ring. Two key candidate FtsZ regulators in Arabidopsis are ARC3 (Accumulation and Replication of Chloroplasts) and ARC6.
Fig 5: A mechanistic working model of chloroplast division in higher plants. (a) Pathway of division complex assembly (steps 1–6). Only known components are shown. The FtsZ filaments are shown as heteropolymers but the stoichiometry between FtsZ1 and FtsZ2 in the Z-ring is unknown. The formation of spirals of FtsZ
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filaments at step 5 is proposed based on cytological observations. The patch of PDV1 and ARC5 at step 6 was observed in cytological studies but the organization and function of the patch is unknown. Symbols are as labeled in (b). (b) Localizations and interaction of known plastid division proteins. (c) A transverse view of the chloroplast constriction site at step 4 of panel (a), showing the hypothesized arrangement and connections between different components. Interactions between PDV1, PDV2, and ARC5 have not been experimentally demonstrated. In order to show the protein interactions, ARC6 molecules are shown as separated from one another, although the ARC6 ring appears to be continuous and ARC6 has been shown to interact with itself.
OEM: outer envelope membrane. IEM: inner envelope membrane. Z1: FtsZ1. Z2: FtsZ2. MSL: MSL2 and MSL3 (Yang et al, Current Opin in Plant Biol 11:577–584, 2008).
ARC6 is a regulator of Z-ring assembly and dynamics. It is homologous to the cyanobacterial cell division protein Ftn2 (filamentation transposon 2). The Arabidopsis arc6 mutant has 1–2 enlarged chloroplasts per leaf mesophyll cell, whereas, normal wild type cells have about 100 chloroplasts (Vitha et al, 2003). ARC6 is localized at the chloroplast division site (Fig 6) and it spans the inner envelope membrane (IEM).
Fig 6: Ring patterns formed by GFP-tagged or YFP-tagged ARC6, PDV2, PDV1, and ARC5 in dividing plastids of Arabidopsis as observed by fluorescence microscopy. ARC6 and PDV2 fusion proteins appear as continuous rings , whereas the rings formed by PDV1 and ARC5 fusion proteins are punctate (small arrows in the lower panels). The functional significance of these distinct localization patterns is not yet clear. Scale bars, 2 mM. (Yang et al, Current Opin in Plant Biol 11:577–584, 2008).
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Symmetric division in both plastids and bacteria is achieved by placing the Z-ring in the centre. Such placement of ring is dynamically regulated by the Min (Minicell) system in E. coli. Min system consists of MinC, MinD, and MinE (Margolin, 2005). MinC is a direct inhibitor of FtsZ polymerization. Distribution of MinC in a cell in unequal showing highest concentration near the cell poles and lowest at the centre of the cell. This distribution allows construction of Z-ring only at the centre of the cell. The MinC concentration gradient is maintained by the pole-to-pole oscillations of MinD and MinE, which continually sweep MinC away from the centre of the cell via a complex set of transient interactions. Maple et al. (2007) have suggested that in higher plants MinC was replaced by ARC3. The ARC3 is chloroplast division protein. It is composed of an incomplete FtsZ-like domain, a middle domain with no recognizable sequence motifs and a domain with partial similarity to phosphatidylinositol-4-phosphate 5-kinase (PIP5K). ARC3 functions like MinC. It interacts with MinD, MinE, and FtsZ1. ARC3 exhibits both polar (like AtMinD and AtMinE) and medial (like FtsZ) localization in chloroplasts (Margolin, 2005) (Fig 5b).
ARC5 (also called DRP5B), is a dynamin-related protein and it mediates division from outside the plastid. It is a member of the dynamin superfamily of eukaryotic membrane-remodeling GTPases. They form spiral-like structures that twist to pinch membranes. In Arabidopsis, GFP-tagged ARC5 forms a punctate ring-like structure around the mid-plastid division site (Fig 6).
PDV1 and PDV2 share approximately 55% sequence similarity and have a similar domain organization. They both are present at medial plastidic rings. Both span outer envelope membrane (OEM) with their N-termini exposed to the cytosol. PDV1 and PDV2 have partially redundant functions in recruiting dynamin (ARC5) to the division site (Fig 1a step 4).
3. Evolution of chloroplasts –
Chloroplasts share common ancestors with existing cyanobacteria. Chloroplasts in green algae and mosses usually do not develop from or into non-green plastids. Sequence analyses suggest that non-vascular plants do not have the extensive diversity of Toc159 homologs seen in vascular plants. In addition, the N-terminal regions of Toc159 homologs from some non-vascular plants are less acidic than those from vascular plants. Therefore, the gene duplication producing diverse A-domains of Toc159 homologs might have driven the evolution of non-green plastids (Inoue, 2011).
The original endosymbiosis of the cyanobacterium gave rise to plastids (primary plastids) in the Archaeplastida, This contains Glaucophya, Rhodophyta (red algae), and
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Viridiplantae (green algae and land plants). All other plastids, such as those found in kelps, dinoflagellates, malaria parasites (these three belong to the Chromalveolata), euglenids (Excavata), and chlorarachniophytes (Rhizaria), were established by subsequent endosymbiotic events in which eukaryotic algae had become integrated into other eukaryotes.(Miyagishima, 2011).
4. Biogenesis / development of mitochondria –
Plant mitochondria are complex organelles. They carry out various metabolic processes related with energy generation required for cellular functions as well as synthesis and degradation of several compounds. Mitochondria are semi-autonomous and dynamic organelles that can change their shape, number and composition depending on tissue or developmental stage (Welchen et al, 2014).
In general, mitochondrial biogenesis is the process by which new mitochondria are formed in the cell. As new mitochondria arise only from pre-existing ones, mitochondrial biogenesis involves different mechanisms related to synthesis and assembly of new mitochondrial components. The synthesis and assembly of mitochondrial components is quite complex process. Owing to the complexity of the process and functionalities of various mitochondrial components, biogenesis of mitochondria is considered to be a highly coordinated process. The coordination is observed in two different ways. One way of coordination is for synthesis of different components of mitochondria to form new mitochondria. The second way refers to coordination of mitochondrial biogenesis with other cellular activities.
Factors regulating mitochondrial biogenesis (Fig 7) – a) External factors –
The changes in form, volume, number and composition of mitochondria are not random but usually they are in response to different factors like developmental stage, environmental stimuli, cell energy and metabolic demands (Howell et al, 2009). It is observed that biogenesis of mitochondria is highly regulated. E.g. in sunflower, nuclear encoded cytochrome c gene and mitochondrial genome encoded genes showed similar pattern of transcript accumulation during flower development (Ribichich et al, 2001). Secondly, pollen maturation requires large amount of energy. So, majority of the components of oxidative phosphorylation mechanism showed very high expression in flowers – particularly in anthers (Peters et al, 2012).
These higher expressions plausibly are in line to accommodate need of the specific organs.
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Mitochondria also are very crucial during seed germination. Changes occur in mitochondrial number, size, and morphology after 12h of imbibitions in continuous light (Law et al, 2012). These changes were encouraged by an increase in mitochondrial protein expression during germination.
Thus the external factors influence mitochondrial biogenesis either globally or specifically, to cope up with the cellular demands. The expression of nuclear respiratory genes is also regulated by numerous external factors like nutrient availability, hormones, light / dark conditions, the diurnal cycle, growth conditions generating oxidative stress and abiotic or biotic stress conditions.
b) Molecular mechanisms of nuclear gene expression –
The expression of many nuclear genes encoding mitochondrial proteins is controlled by elements known as site II. They are either responsible for basal gene expression or modifying the amount of response under different growth conditions.
More than 80% of genes encoding constituents of Complexes I, III, IV and V in Arabidopsis and rice have site II elements in their promoter regions (Welchen and Gonzalez, 2006). Genes encoding ribosomal proteins often show site II elements.
These elements accumulate closer to the transcription start site preferentially in genes encoding mitochondrial proteins. Some other transcription factors like bZip, AP2/ERF, bHLH, AREB2/ABF4 and trihelix families are also involved in expression of nuclear genes encoding mitochondrial components.
Recently, involvement of microRNAs (miRNAs) for regulation of mitochondrial biogenesis has been proposed (Li et al, 2012). miRNAs are a class of 20-24 nucleotide, small, non-coding, endogenous RNAs. They can bind to mRNA and so inhibit translation or promote RNA degradation. This miRNA-mediated regulation is sequence-specific and occurs at the post-transcriptional level. E.g.
miR398 – it binds to the 5’ UTR of Cox5b-1 transcript. It functions in regulation of gene expression encoding copper binding proteins. These proteins are present in different cell compartments according to copper availability, thus may work to coordinate the biogenesis of Complex IV with other cellular processes.
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Fig 7: Integrative view of different regulatory pathways involved in mitochondrial biogenesis. 1) Mitochondrial biogenesis is regulated by numerous external factors. 2) It is also internally regulated by different stages of development, organ/tissue type, hormones, and cell energy and metabolic demands; 3) The expression of a majority of nuclear genes for mitochondrial proteins is controlled by site II elements.
They are either responsible for basal gene expression or modify the magnitude of the response under different growth conditions. 4) & 5) Evidences show presence of proteins that are dual targeted to different organelles, which may act to coordinate the activities of these organelles; 6) Physical and functional interactions between the inner membrane (IM) import machinery and complexes I, III, and IV help to adjust gene expression and protein assembly & 7) Mitochondria generate signals to modify nuclear gene expression according to organelle demands (Welchen et al, Frontiers in Plant Sci, 2014).
5. Division, fusion and fission of mitochondria –
Basically, mitochondrial division in all types of cells maintains the proper cellular distribution of mitochondria. Disruption of mitochondrial division produces an extensively interconnected and collapsed mitochondrial network in both yeast and higher eukaryotes.
Due to this, many areas of cell do not have mitochondria at all. Such altered distribution of mitochondria has some adverse effects. In sporulating yeast cells that do not have
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essential mitochondrial division proteins show defects in mtDNA inheritance (Gorsich &
Shaw, 2004). In higher eukaryotes, more adverse effects are seen when mitochondrial distribution is disrupted. Defective C. elegans shows embryonic lethality partly due to improper mitochondrial segregation (Labrousse et al, 1999).
5.1 Mitochondrial division –
In yeast and in higher eukaryotes, mitochondrial division occurs through Dnm1 and its homolog Drp1, respectively. Both Dnm1 and Drp1 are involved in the scission of mitochondrial membranes. They belong to a family of dynamin related proteins (DRPs).
DRPs are large, self assembling GTPases possessing a highly conserved GTPase domain. However, they differ from regulatory GTPases by their large size as well as kinetic and structural properties (Song & Schmid, 2003). DRPs show two functionally unique and essential features - a) GTP-driven self-assembly and b) assembly stimulated GTP-hydrolysis. DRPs exhibit lower affinities for GTP and GDP and faster rates of GTP hydrolysis, so they do not require nucleotide exchange factors and GTPase activating proteins. In yeast, Dnm1 is localized to the mitochondrial surface, whereas, in higher eukaryotes it is diffusely dispersed throughout the cytosol, and a small fraction of the protein is found assembled on mitochondria (Fig 8) (Lackner & Nunnari, 2009).
Fig 8: The cellular distribution of Dnm1 and Drp1. (A) In yeast, Dnm1 is predominantly found in self- assembled structures, a majority of which are associated with mitochondria. (B) In higher eukaryotes, the bulk of Drp1 in COS cells is diffusely dispersed in the cytosol with only a small fraction of the protein found assembled on mitochondria. Scale bars: (A) 1 μm, (B) 10 μm (Lackner & Nunnari, Biochim Biophys Acta, 1792:1138 – 1144, 2009).
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The current working model of mitochondrial division involves Dnm1 / Drp1. The process can be broadly classified into 3 stages: a) targeting, b) assembly-driven constriction, and c) hydrolysis-mediated constriction/scission. The process (Fig 9) can be summarized as follows:
1) Dnm1/Drp1 is targeted to surface of mitochondria.
2) It undergoes GTP driven assembly into helical structures.
3) This self-assembly drives constriction of membrane / mitochondrial tubule.
4) GTP driven assembly also stimulates GTP hydrolysis.
5) GTP hydrolysis induces conformational changes within the Dnm1/Drp1 helix required for further constriction.
6) Membrane division completes.
Fig 9: A model of Dnm1/Drp1-driven mitochondrial division. Dnm1/Drp1 is shown in red and a portion of a mitochondrial tubule is shown in green (Lackner & Nunnari, Biochim Biophys Acta, 1792:1138 – 1144, 2009).
5.2 Mitochondrial fusion –
We know that mitochondria are dynamic organelles, constantly undergoing both division and fusion. Mitochondrial fusion is a highly conserved process from yeast to human cells. Two members of DRP family viz. Fzo1 and Mgm1, are highly conserved mitochondrial DRPs. They play a vital role in outer and inner mitochondrial fusion respectively. Amongst the eukaryotic DRPs, the mitochondrial outer membrane fusion DRPs – Fzo1, Mfn1 and Mfn2 are the most closely related to prokaryotic dynamins (Hoppins & Nunnari, 2009). Fzo1 has coiled-coil regions that match to eukaryotic middle and GED (GTPase effector domain) regions. These regions are important for
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intermolecular interactions. Mgm1 is more closely related to eukaryotic like dynamins. It contains canonical GTPase, middle and GED regions. The N-terminus of Mgm1 shows a mitochondrial targeting signal and two regions of hydrophobicity. All the regions are necessary for targeting Mgm1 to the mitochondrial inner membrane and intermembrane space (Fig 10).
Fig 10: Domain structure of dynamin and the mitochondrial fusion dynamin-related proteins. The GTPase domain found in all dynamin-related proteins binds and hydrolyzes GTP. The middle, GTPase effector domains (GED) and heptad repeats (HR) are involved in DRP oligomerization. The pleckstrin homology (PH) domain and the proline rich domain (PRD) are not conserved in the fusion DRPs. Both mitochondrial fusion DRPs, Fzo1/Mfn1,2 and Mgm1/OPA1, contain transmembrane domains (T) to anchor the proteins to the outer and inner mitochondrial membrane respectively. In addition, Mgm1/OPA1 has an N-terminal mitochondrial targeting sequence (MTS) required for its targeting and import into mitochondria (Hoppins &
Nunnari, Biochim Biophys Acta 1793:20 – 26, 2009).
Outer and inner membrane fusion events can occur separately in vitro, but they are temporally linked in vivo. This linking suggests that a there is some mechanism which synchronizes the outer and inner membrane fusion. Ugo1is a member of the mitochondrial transport family and it serves as an essential fusion component. It is a multi-spanning membrane protein with 3 or 5 transmembrane domains (Sesaki & Jensen, 2001) and has energy transfer motifs each containing one positively and one negatively charged residue.
Ugo1 functions as an adaptor, creating a two membrane spanning complex with Fzo1 and Mgm1. There are two possible functions for Ugo1 mitochondrial fusion. First, the fusion DRP complex may promote a specific conformation or oligomeric structure of Fzo1 required to initiate fusion events in the outer membrane and Ugo1 would for the formation of this initiation complex. Secondly, Ugo1 may have additional post-initiation roles in both mitochondrial outer and inner membrane fusion events besides forming the complex.
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5.2 Mitochondrial fission –
Mitochondrial fission contributes to the proper distribution of mitochondria in response to the local demand for ATP. It also helps for the elimination of damaged mitochondrial fragments through mitophagy (autophagy for mitochondria). Mitochondrial fusion and fission are controlled by four high molecular weight GTPases conserved from yeast to mammals. In yeast there are three GTPases viz. Fzo1 (in mitochondrial outer membrane fusion, Mgm1 (in mitochondrial inner membrane fusion and cristae organization and Dnm1 (in mitochondrial fission).
Dnm1 is mainly found in the cytosol. During mitochondrial fission, it is translocated from the cytosol to fission sites on the mitochondria (Fig 11) (Otera & Mihara, 2011).
Fig 11: Drp1/Dmn1 assembly on the OMM in yeast and mammals. Mitochondrial fission in yeast involves the interaction of Dnm1 with Fis1 via the soluble adaptor proteins Mdv1/Caf4. Mitochondrial fission in mammals requires the tail-anchored protein Mff. Cytosolic Drp1 is recruited to the OMM by Mff. Although homologues of yeast Mdv1/Caf4 have not been identified in higher eukaryotes, Mff seems to function simultaneously as an adaptor and a receptor for the mitochondrial recruitment of Drp1 (Otera & Mihara, J Biochem 149:241 – 251, 2011).
Dnm1 or Drp1 forms self-assembled higher-order structures that wrap around the mitochondrial tubule. These spiral structures squeeze and eventually cut off the
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mitochondrial membrane. The process is dependent on GTP hydrolysis. Mitochondrial tubules are observed to divide at these perforated sites.
Fis1 is an outer mitochondrial membrane protein with its N-terminal multiple TPR (tetra tricopeptide repeat) motif exposed to the cytoplasm. In yeast, during mitochondrial fission, TPR motif of Fis1 transiently interacts with Dnm1 via cytosolic adaptor proteins Mdv1/Caf4. So it functions as Dnm1 receptor and Fis1 is required for its recruitment.
Different stressors induce mitochondrial fission for modifying mitochondria and cellular function. During apoptosis, cytoplasmic Drp1 is transported to mitochondria and it induces mitochondrial fragmentation. Such increased fission events are required for breakdown of dysfunctional mitochondria or even for proper segregation of mitochondria into daughter cells during mitosis.
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