scholarly journals Mitochondrial mislocalization and altered assembly of a cluster of Barth syndrome mutant tafazzins

2006 ◽  
Vol 174 (3) ◽  
pp. 379-390 ◽  
Author(s):  
Steven M. Claypool ◽  
J. Michael McCaffery ◽  
Carla M. Koehler
Keyword(s):  
Point Mutations ◽  
Mutant Gene ◽  
Barth Syndrome ◽  
Membrane Association ◽  
Intermembrane Space ◽  
Mitochondrial Matrix ◽  
Mitochondrial Membranes ◽  
Membrane Bilayer ◽  
Conserved Residues ◽  
Tm Domain

None of the 28 identified point mutations in tafazzin (Taz1p), which is the mutant gene product associated with Barth syndrome (BTHS), has a biochemical explanation. In this study, endogenous Taz1p was localized to mitochondria in association with both the inner and outer mitochondrial membranes facing the intermembrane space (IMS). Unexpectedly, Taz1p does not contain transmembrane (TM) segments. Instead, Taz1p membrane association involves a segment that integrates into, but not through, the membrane bilayer. Residues 215–232, which were predicted to be a TM domain, were identified as the interfacial membrane anchor by modeling four distinct BTHS mutations that occur at conserved residues within this segment. Each Taz1p mutant exhibits altered membrane association and is nonfunctional. However, the basis for Taz1p dysfunction falls into the following two categories: (1) mistargeting to the mitochondrial matrix or (2) correct localization associated with aberrant complex assembly. Thus, BTHS can be caused by mutations that alter Taz1p sorting and assembly within the mitochondrion, indicating that the lipid target of Taz1p is resident to IMS-facing leaflets.

scholarly journals The Taz1p Transacylase Is Imported and Sorted into the Outer Mitochondrial Membrane via a Membrane Anchor Domain

2013 ◽  
Vol 12 (12) ◽  
pp. 1600-1608 ◽  
Author(s):  
Jenny D. Herndon ◽  
Steven M. Claypool ◽  
Carla M. Koehler
Keyword(s):  
Outer Membrane ◽  
Point Mutations ◽  
Pathogenic Mutation ◽  
Barth Syndrome ◽  
Mitochondrial Outer Membrane ◽  
Intermembrane Space ◽  
Membrane Anchor ◽  
Inner Membrane ◽  
Content Type ◽  
The Matrix

ABSTRACT Mutations in the mitochondrial transacylase tafazzin, Taz1p, in Saccharomyces cerevisiae cause Barth syndrome, a disease of defective cardiolipin remodeling. Taz1p is an interfacial membrane protein that localizes to both the outer and inner membranes, lining the intermembrane space. Pathogenic point mutations in Taz1p that alter import and membrane insertion result in accumulation of monolysocardiolipin. In this study, we used yeast as a model to investigate the biogenesis of Taz1p. We show that to achieve this unique topology in mitochondria, Taz1p follows a novel import pathway in which it crosses the outer membrane via the translocase of the outer membrane and then uses the Tim9p-Tim10p complex of the intermembrane space to insert into the mitochondrial outer membrane. Taz1p is then transported to membranes of an intermediate density to reach a location in the inner membrane. Moreover, a pathogenic mutation within the membrane anchor (V224R) alters Taz1p import so that it bypasses the Tim9p-Tim10p complex and interacts with the translocase of the inner membrane, TIM23, to reach the matrix. Critical targeting information for Taz1p resides in the membrane anchor and flanking sequences, which are often mutated in Barth syndrome patients. These studies suggest that altering the mitochondrial import pathway of Taz1p may be important in understanding the molecular basis of Barth syndrome.

scholarly journals A novel general approach to eucaryotic mutagenesis functionally identifies conserved regions within the adenovirus 13S E1A polypeptide.

1986 ◽  
Vol 6 (5) ◽  
pp. 1487-1496 ◽  
Author(s):  
D Kimelman
Keyword(s):  
Shuttle Vector ◽  
Point Mutations ◽  
Mutant Gene ◽  
Adenovirus Type ◽  
Cellular Morphology ◽  
Morphological Alteration ◽  
Morphological Alterations ◽  
E1a Gene ◽  
Adenovirus Type 5

A new approach to the isolation of mutations in mammalian genes was developed which permits both the selection of infrequently occurring mutants that alter the cellular morphology of recipient cells and the rapid reisolation of the mutant gene. The adenovirus type 5 13S early region 1a (E1a) gene was mutagenized in vitro with sodium bisulfite and then efficiently transferred into cells with a retrovirus shuttle vector. Three classes of mutants of the 13S E1a gene product were isolated, each of which induced a distinct morphological alteration. The mutant E1a gene was reisolated from each cell line, and the precise nucleotide changes were determined. The E1a-induced morphological alterations were further examined by the construction of single and double point mutations within different regions of the polypeptides by utilizing the amino acid substitutions obtained from the original mutants. The results suggest that each of the three regions of highly conserved amino acids within the E1a 13S polypeptide has a distinct role in the alteration of cellular morphology and the activation of gene expression.

scholarly journals Barth syndrome mutations that cause tafazzin complex lability

2011 ◽  
Vol 192 (3) ◽  
pp. 447-462 ◽  
Author(s):  
Steven M. Claypool ◽  
Kevin Whited ◽  
Santi Srijumnong ◽  
Xianlin Han ◽  
Carla M. Koehler
Keyword(s):  
Mitochondrial Function ◽  
Protein Complexes ◽  
Barth Syndrome ◽  
Human Diseases ◽  
Intermembrane Space ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Low Levels ◽  
Tafazzin Gene ◽  
Mutant Polypeptides

Deficits in mitochondrial function result in many human diseases. The X-linked disease Barth syndrome (BTHS) is caused by mutations in the tafazzin gene TAZ1. Its product, Taz1p, participates in the metabolism of cardiolipin, the signature phospholipid of mitochondria. In this paper, a yeast BTHS mutant tafazzin panel is established, and 18 of the 21 tested BTHS missense mutations cannot functionally replace endogenous tafazzin. Four BTHS mutant tafazzins expressed at low levels are degraded by the intermembrane space AAA (i-AAA) protease, suggesting misfolding of the mutant polypeptides. Paradoxically, each of these mutant tafazzins assembles in normal protein complexes. Furthermore, in the absence of the i-AAA protease, increased expression and assembly of two of the BTHS mutants improve their function. However, the BTHS mutant complexes are extremely unstable and accumulate as insoluble aggregates when disassembled in the absence of the i-AAA protease. Thus, the loss of function for these BTHS mutants results from the inherent instability of the mutant tafazzin complexes.

scholarly journals Protein sorting between mitochondrial membranes specified by position of the stop-transfer domain.

1988 ◽  
Vol 106 (5) ◽  
pp. 1499-1505 ◽  
Author(s):  
M Nguyen ◽  
A W Bell ◽  
G C Shore
Keyword(s):  
G Protein ◽  
Simple Extension ◽  
Intermembrane Space ◽  
Mitochondrial Membranes ◽  
Inner Mitochondrial Membrane ◽  
The Matrix ◽  
Targeting Signal ◽  
Vesicular Stomatitis ◽  
Matrix Precursor ◽  
Identical Fashion

Recently, we fused a matrix-targeting signal to a large fragment of vesicular stomatitis virus G protein, which contains near its COOH-terminus a well-characterized endoplasmic reticulum (ER) stop-transfer sequence; the hybrid G protein was sorted to the inner mitochondrial membrane (Nguyen, M., and G. C. Shore. 1987. J. Biol. Chem. 262:3929-3931). Here, we show that the 19 amino acid G stop-transfer domain functions in an identical fashion when inserted toward the COOH-terminus of an otherwise normal matrix precursor protein, pre-ornithine carbamyl transferase; after import, the mutant protein was found anchored in the inner membrane via the stop-transfer sequence, with its NH2 terminus facing the matrix and its short COOH-terminal tail located in the intermembrane space. However, when the G stop-transfer sequence was placed near the NH2 terminus, the protein was inserted into the outer membrane, in the reverse orientation (NH2 terminus facing out, with a large COOH-terminal fragment located in the intermembrane space). These observations for mitochondrial topogenesis can be explained by a simple extension of existing models for ER sorting.

scholarly journals Coassembly of Mgm1 isoforms requires cardiolipin and mediates mitochondrial inner membrane fusion

2009 ◽  
Vol 186 (6) ◽  
pp. 793-803 ◽  
Author(s):  
Rachel M. DeVay ◽  
Lenin Dominguez-Ramirez ◽  
Laura L. Lackner ◽  
Suzanne Hoppins ◽  
Henning Stahlberg ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Coiled Coil ◽  
Intermembrane Space ◽  
Dependent Manner ◽  
Related Protein ◽  
Mitochondrial Membranes ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
N Terminus ◽  
In Trans

Two dynamin-related protein (DRP) families are essential for fusion of the outer and inner mitochondrial membranes, Fzo1 (yeast)/Mfn1/Mfn2 (mammals) and Mgm1 (yeast)/Opa1 (mammals), respectively. Fzo1/Mfns possess two medial transmembrane domains, which place their critical GTPase and coiled-coil domains in the cytosol. In contrast, Mgm1/Opa1 are present in cells as long (l) isoforms that are anchored via the N terminus to the inner membrane, and short (s) isoforms were predicted to be soluble in the intermembrane space. We addressed the roles of Mgm1 isoforms and how DRPs function in membrane fusion. Our analysis indicates that in the absence of a membrane, l- and s-Mgm1 both exist as inactive GTPase monomers, but that together in trans they form a functional dimer in a cardiolipin-dependent manner that is the building block for higher-order assemblies.

scholarly journals MEMBRANE JUNCTIONS IN THE INTERMEMBRANE SPACE OF MITOCHONDRIA FROM MAMMALIAN TISSUES

1974 ◽  
Vol 60 (3) ◽  
pp. 653-663 ◽  
Author(s):  
Akitsugu Saito ◽  
Murray Smigel ◽  
Sidney Fleischer
Keyword(s):  
Skeletal Muscle ◽  
Cross Sections ◽  
Rat Kidney ◽  
Respiratory Control ◽  
Heart Tissue ◽  
Intermembrane Space ◽  
Mitochondrial Membranes ◽  
Fresh Tissue ◽  
Isolated Mitochondria ◽  
Mammalian Tissues

There have been several reports describing paracrystalline arrays in the intermembrane space of mitochondria. On closer inspection these structures appear to be junctions of two adjoining membranes. There are two types. They can be formed between the outer and inner mitochondrial membranes (designated outer-inner membrane junctions) or between two cristal membranes (intercristal membrane junctions). In rat heart, adjoining membranes appeared associated via a central dense midline approximately 30 Å wide. In rat kidney, the junction had a ladder-like appearance with electron-dense "bridges" approximately 80 Å wide, spaced 130 Å apart, connecting the adjoining membranes. We have investigated the conditions which favor the visualization of such structures in mitochondria. Heart mitochondria isolated rapidly from fresh tissue (within 30 min of death) contain membrane junctions in approximately 10–15% of the cross sections. This would indicate that the percentage of membrane junctions in the entire mitochondrion is far greater. Mitochondria isolated from heart tissue which was stored for 1 h at 0°–4°C showed an increased number of membrane junctions, so that 80% of the mitochondrial cross sections show membrane junctions. No membrane junctions are observed in mitochondria in rapidly fixed fresh tissue or in mitochondria isolated from tissue disrupted in fixative. Thus, the visualization of junctions in the intermembrane space of mitochondria appears to be dependent upon the storage of tissue after death. Membrane junctions can also be observed in mitochondria from other stored tissues such as skeletal muscle, kidney, and interstitial cells from large and small intestine. In each case, no such junctions are observed in these tissues when they are fixed immediately after removal from the animal. It would appear that most studies in the literature in which isolated mitochondria from tissues such as heart or kidney were used were carried out on mitochondria which contained membrane junctions. The presence of such structures does not significantly affect normal mitochondrial function in terms of respiratory control and oxidative phosphorylation.

scholarly journals The highly conserved U small nuclear RNA 3'-end formation signal is quite tolerant to mutation.

1987 ◽  
Vol 7 (6) ◽  
pp. 2070-2079 ◽  
Author(s):  
R A Ach ◽  
A M Weiner
Keyword(s):  
Point Mutation ◽  
Hela Cells ◽  
Double Point ◽  
Xenopus Oocytes ◽  
Point Mutations ◽  
Small Nuclear Rna ◽  
Conserved Residues ◽  
Conserved Sequence ◽  
Nuclear Rna

Formation of the 3' end of U1 and U2 small nuclear RNA (snRNA) precursors is directed by a conserved sequence called the 3' box located 9 to 28 nucleotides downstream of all metazoan U1 to U4 snRNA genes sequenced so far. Deletion of part or all of the 3' box from human U1 and U2 genes drastically reduces 3'-end formation. To define the essential nucleotides within this box that direct 3'-end formation, we constructed a set of point mutations in the conserved residues of the human U1 3' box. The ability of the various mutations to direct 3'-end formation was tested by microinjection into Xenopus oocytes and transfection into HeLa cells. We found that the point mutations had diverse effects on 3'-end formation, ranging from no effect at all to severe inhibition; however, no single or double point mutation we tested completely eliminated 3'-end formation. We also showed that a rat U3 3' flank can effectively substitute for the human U1 3' flank, indicating that the 3' boxes of the different U snRNA genes are functionally equivalent.

scholarly journals The Dynamin-related GTPase, Dnm1p, Controls Mitochondrial Morphology in Yeast

1998 ◽  
Vol 143 (2) ◽  
pp. 333-349 ◽  
Author(s):  
Denichiro Otsuga ◽  
Brian R. Keegan ◽  
Ellen Brisch ◽  
John W. Thatcher ◽  
Greg J. Hermann ◽  
...  
Keyword(s):  
Point Mutations ◽  
Binding Domain ◽  
Cell Cortex ◽  
Mitochondrial Morphology ◽  
Wild Type ◽  
Mitochondrial Membranes ◽  
Gtp Binding ◽  
Differential Sedimentation ◽  
Gradient Fractionation ◽  
Cortical Distribution

The Saccharomyces cerevisiae Dnm1 protein is structurally related to dynamin, a GTPase required for membrane scission during endocytosis. Here we show that Dnm1p is essential for the maintenance of mitochondrial morphology. Disruption of the DNM1 gene causes the wild-type network of tubular mitochondrial membranes to collapse to one side of the cell but does not affect the morphology or distribution of other cytoplasmic organelles. Dnm1 proteins containing point mutations in the predicted GTP-binding domain or completely lacking the GTP-binding domain fail to rescue mitochondrial morphology defects in a dnm1 mutant and induce dominant mitochondrial morphology defects in wild-type cells. Indirect immunofluorescence reveals that Dnm1p is distributed in punctate structures at the cell cortex that colocalize with the mitochondrial compartment. These Dnm1p-containing structures remain associated with the spherical mitochondria found in an mdm10 mutant strain. In addition, a portion of Dnm1p cofractionates with mitochondrial membranes during differential sedimentation and sucrose gradient fractionation of wild-type cells. Our results demonstrate that Dnm1p is required for the cortical distribution of the mitochondrial network in yeast, a novel function for a dynamin-related protein.

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