ScienceWeek

CELL BIOLOGY: ON MITOCHONDRION DOUBLE MEMBRANE FUSION

The following points are made by N. Pfanner et al (Science 2004 305:1723):

1) Mitochondria are cytoplasmic organelles that are crucial players in numerous cellular processes. These include bioenergetics, metabolism of amino acids, lipids, and iron, as well as programmed cell death (apoptosis), differentiation, and aging (1-4). Mitochondria comprise two membranes, the outer membrane and the folded inner membrane, and two aqueous compartments, the intermembrane space and the matrix.

2) From yeast to humans, mitochondria form a dynamic network in the cell that is maintained by balancing mitochondrial fusion and fission. The dynamic morphology of mitochondria is critical for the inheritance of mitochondrial DNA, for the transmission of energy, and for cellular differentiation, such as spermatogenesis. During apoptosis, enhanced mitochondrial fission and diminished mitochondrial fusion lead to mitochondrial fragmentation, potentiating the cell death response. Defects in mitochondrial fusion have been linked to neurodegenerative diseases like dominant optic atrophy and Charcot-Marie-Tooth type 2A disease, an inherited peripheral neuropathy.

3) In vivo studies in yeast, Drosophila, and mammalian cells have led to the identification of many proteins that are involved directly or indirectly in the maintenance of mitochondrial morphology. However, lack of an in vitro system for studying mitochondrial fission and fusion has hampered analysis at the molecular level. However, Meeusen et al (5) have recently described an in vitro assay that follows the fusion of isolated mitochondria, providing important insights into the ordered events of outer- and inner-membrane fusion.

4) Mitochondrial fusion has been considered a complicated process that requires essential cytosolic elements. Yet the assay established by Meeusen et al(5) is remarkably straightforward and does not require the addition of cytosolic factors. This implies that isolated mitochondria contain all of the essential ingredients required for fusion. Meeusen et al(5) used mitochondria labeled with matrix-targeted molecules: either green fluorescent protein (GFP) or red fluorescent protein (dsRED). Differently colored mitochondria were mixed, concentrated by centrifugation, and incubated in the presence of guanosine triphosphate (GTP) and energy substrates to generate an inner-membrane electrical potential. Mitochondrial fusion could be monitored directly through mixing of GFP and dsRED such that the matrix of fused mitochondria contained both fluorescently labeled proteins. The centrifugation step is a crucial element of the assay because it brings mitochondria into close proximity. In vivo, this step is probably accomplished by the cellular cytoskeleton (5).

5) With their assay, Meeusen et al(5) dissected mitochondrial fusion into sequential outer- and inner-membrane fusion events. Outer-membrane fusion required low levels of GTP and a mitochondrial proton gradient. Inner-membrane fusion depended on the hydrolysis of large amounts of GTP and the presence of an inner-membrane electrical potential. The GTP dependence of mitochondrial fusion concurs with previous in vivo studies that revealed a requirement for two large dynamin-related guanosine triphosphatases (GTPases): Fzo1 (fuzzy onions or mitofusin) of the outer membrane, and Mgm1 (mitochondrial genome maintenance) of the intermembrane space (1,2,4). In yeast cells, Fzo1 and Mgm1 are linked together in a complex by the outer-membrane protein Ugo1 (derived from the Japanese word for fusion).

References (abridged):

1. M. P. Yaffe, Science 283, 1493 (1999)

2. A. D. Mozdy, J. M. Shaw, Nature Rev. Mol. Cell Biol. 4, 468 (2003)

3. A. Sickmann et al., Proc. Natl. Acad. Sci. U.S.A. 100, 13207 (2003)

4. B. Westermann, Biochim. Biophys. Acta 1641, 195 (2003)

5. S. Meeusen, J. M. McCaffery, J. Nunnari, Science 305, 1747 (2004)

Science http://www.sciencemag.org

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Related Material:

MEDICAL BIOLOGY: MITOCHONDRIA IN HUMAN DISEASE

Notes by ScienceWeek:

Mitochondria are double-membrane enclosed organelles of cells that are involved with several important biochemical pathways, including *electron transport and *oxidative metabolism. Various types of eukaryotic cells (i.e., cells containing membrane-bound organelles) may contain from a few to several thousand mitochondria in each cell type. The mitochondria are relatively large cylindrical structures up to 10 microns long and up to 2 microns in diameter, and they are believed to have originated as organisms that became *symbiotic with eukaryotic cells.

In general, the term "apoptosis" refers to programmed cell death, whether as a part of normal tissue differentiation and development, or as a program activated in a defective cell. In the molecular biology of cancer, apoptosis is the name given to the programmed cell death provoked by certain proteins expressed by *tumor suppressor genes. Thus, malignant cells are defective cells with a deactivated apoptosis program, and this allows malignant cells to survive and replicate. In most tissues within the body, a delicate balance is maintained between cell proliferation and cell death. Aging or damaged cells are replaced with new cells, ensuring that tissues are maintained in prime condition. If this balance is disturbed in either direction, the consequences can be severe. Tissue degeneration will occur if cell death predominates over cell proliferation, and when cell proliferation outstrips cell death, the result is the development of tumors.

In recent years, it has been discovered that mitochondria play a central role in the apoptosis process, this in addition to their well-established role of providing *adenosine triphosphate (ATP) to drive the energy-requiring processes within the cell.

The following points are made by Anne Murphy (The Biochemist April 2000):

1) Mitochondrial dysfunction is an apparent underlying contributor to many diseases. It has long been known that the lack of mitochondrial ATP production can lead to necrotic death in many pathologies, including *ischemia/reperfusion injury and *toxin exposure. More recently, it has become apparent that apoptosis can be induced by a wide variety of events, many of which involve the release of "apoptogens" (e.g., *cytochrome c and *caspases) from mitochondria. Often these stimuli initiate a caspase-dependent cascade of *proteolysis from which cells generally do not recover.

2) Forms of apoptosis exist that may not be dependent on mitochondrial apoptogen release. These forms of apoptosis appear to involve responses to certain *inflammatory mediators that bind specific death receptors. The extent to which mitochondria play a role in these varieties of cell death may be dependent upon cell type and the level of expression of specific caspases.

3) There are numerous diseases with components of apoptosis, but notable among these are certain neurodegenerative diseases, ischemic/reperfusion injury, autoimmune disorders and inflammatory diseases (including arthritis), and viral infection (including human immunodeficiency virus [HIV])). Also, the development of certain cancers is apparently associated with the loss of appropriate levels of apoptosis. Furthermore, in cancer, resistance of malignant cells to chemotherapeutic agents is associated with the expression of proteins, localized to mitochondria, that inhibit apoptosis.

4) Clinical studies have implicated mitochondrial dysfunction in certain chronic diseases, including *Alzheimer's, *Huntington's, and *Parkinson's diseases. There is evidence of metabolic abnormalities in the rates of *glucose utilization and *lactate production in the affected brain regions of patients with these diseases. There is also evidence of a decrease in the maximal activity of *electron-transport-chain complexes in the brains or peripheral tissues of patients with these diseases. The question is whether these defects are sufficient to compromise normal neuronal function, and whether they are the cause or the result of the respective disease.

The Biochemist http://www.portlandpress.com/biochemist/biochemist.htm

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Notes:

electron transport: This refers to a sequence of steps in the final stage of the aerobic respiration biochemical pathway in which high energy electrons are effectively passed through a series of membrane-bound carrier molecules to support a proton gradient involved in energy storage. The term "transport" here refers essentially to a chemical flow diagram and not necessarily to an actual spatial translocation of electrons.

oxidative metabolism: In general, a set of biochemical pathways dependent on the utilization of supplied oxygen.

symbiotic: In biology, "symbiosis" is an intimate and protracted association of individuals of different species.

tumor suppressor genes: In general, cancer genes have been divided into 2 classes, proto-oncogenes and tumor suppressor genes. Proto-oncogenes are genes that sustain activating changes in human cancer. These changes may take the form of point mutations or gene rearrangements that lead to increased or uncontrolled activity of the encoded protein, or they make take the form of gene amplification, which results in increased levels of protein expression. In contrast, tumor suppressor genes are characterized by inactivating changes in human cancer, typically point mutations that result in truncation or functional inactivation of the encoded protein, or gross deletions of chromosomal fragments carrying these genes.

adenosine triphosphate (ATP): ATP is the most important chemical energy source in all living cells, intimately involved in various cell functions and cell metabolism, and an entity in numerous cyclic chemical pathways involved in the synthesis of various cell components.

ischemia/reperfusion injury: In general, "ischemia" is a sudden loss of blood supply to a tissue caused by blockage of a blood vessel. The term "ischemia/reperfusion injury" refers to the damage that can occur to a tissue when it is reperfused after a prolonged period of ischemia. The phenomenon is of considerable clinical importance, especially in connection with heart attacks and strokes.

toxin: In general, any noxious or poisonous substance, especially substances produced by living systems.

cytochrome c: The cytochromes, categorized as hemoproteins with differing porphyrin groups, are widely distributed respiratory (oxygen-utilizing) catalysts involved in the electron transport chain of living cells. They do not combine with substrates, but alternate between Fe(2+) and Fe(3+) states. There are various cytochromes, with cytochrome c present in the greatest amounts, and most importantly in the mitochondria of eukaryotic cells.

caspases: Proteases are a class of enzymes that hydrolyze proteins, splitting them into various groups of subunits, with the sites of hydrolysis dependent on the particular enzyme and the protein substrate, and a caspase is a type of protease implicated in apoptosis.

proteolysis: In general, hydrolysis (breakdown) of proteins.

inflammatory mediators: In general, an "inflammatory change" is a response of tissues to irritation or injury. The response involves a dynamic complex of cellular and chemical reactions that occur in the affected blood vessels and adjacent tissues.

Alzheimer's, *Huntington's, and *Parkinson's diseases: All three of these neurodegenerative diseases involve considerable loss of nerve cells in certain areas of the brain.

glucose utilization: In biological systems, glycolysis (also known as Embden-Meyerhof pathway), involving the breakdown of glucose, is one of the main energy producing pathways in the cell.

lactate production: Lactate is a salt or ester of lactic acid, and lactic acid is a common end product of glycolysis in biological systems.

electron-transport-chain complexes: The term "electron-transport chain" (respiratory chain) refers to the sequence of reactions involving enzymes and other proteins within the mitochondrion by which substrates are oxidized.

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