The Saccharomyces cerevisiae TCM62 Gene Encodes a Chaperone Necessary for the Assembly of the Mitochondrial Succinate Dehydrogenase (Complex II)

Abstract

The assembly of the mitochondrial respiratory chain is mediated by a large number of helper proteins. To better understand the biogenesis of the yeast succinate dehydrogenase (SDH), we searched for assembly-defective mutants. SDH is encoded by the SDH1, SDH2, SDH3, and SDH4 genes. The holoenzyme is composed of two domains. The membrane extrinsic domain, consisting of Sdh1p and Sdh2p, contains a covalent FAD cofactor and three iron-sulfur clusters. The membrane intrinsic domain, consisting of Sdh3p and Sdh4p, is proposed to bind two molecules of ubiquinone and one heme. We isolated one mutant that is respiration-deficient with a specific loss of SDH oxidase activity. SDH is not assembled in this mutant. The complementing gene, TCM62 (also known as SCYBR044C), does not encode an SDH subunit and is not essential for cell viability. It encodes a mitochondrial membrane protein of 64,211 Da. The Tcm62p sequence is 17.3% identical to yeast hsp60, a molecular chaperone. The Tcm62p amino terminus is in the mitochondrial matrix, whereas the carboxyl terminus is accessible from the intermembrane space. Tcm62p forms a complex containing at least three SDH subunits. We propose that Tcm62p functions as a chaperone in the assembly of yeast SDH.The respiratory chain of the mitochondrial inner membrane is typically composed of four multisubunit enzymes (complexes I-IV) and the ATP synthase (complex V). In Saccharomyces cerevisiae, the succinate dehydrogenase (SDH) 1 or complex II subunits are encoded by the SDH1, SDH2, SDH3, and SDH4 genes (1, 2). The four SDH subunits are as follows: a large flavoprotein (Sdh1p) subunit of 67 kDa to which is covalently attached an FAD cofactor (3), an iron-sulfur subunit (Sdh2p) of 28 kDa that contains three iron-sulfur clusters (4), and two small hydrophobic membrane subunits of 16.7 (Sdh3p) and 16.6 (Sdh4p) kDa (5, 6). The two small subunits serve as membrane anchors and are thought to contain two ubiquinonebinding sites and a b-type heme (7,8).A large number of gene products are necessary for the biogenesis of the respiratory chain. It is a complex process involving the expression of two genomes and the transport of hundreds of proteins into the organelle (9, 10). In addition to the many nuclear and to the seven (in yeast) mitochondrially encoded subunits of the five complexes, many gene products are necessary to replicate and express the mitochondrial DNA. Perhaps more surprising are the long lists of genes required for the post-translational assembly of the respiratory chain and the ATP synthase (9, 11-13). Some of these, like the COX10 and COX11 gene products, are involved in heme a synthesis for complex IV biogenesis (14, 15). The COX17 gene encodes a mitochondrial copper shuttle necessary for metal delivery to complex IV (16). The ABC1 gene is essential for the correct assembly of complexes II-IV, and a loss of Abc1p impairs electron transport in complex III (8, 17). The BCS1 gene product may be necessary for the formation or insertion of the activesite iron-sulfur cluster of the Rieske...

Mitochondrial respiratory chain complexes: apoptosis sensors mutated in cancer?

“…It can be hypothesized that these proteins render complex II dissociation reversible and reassemble the subcomplexes, in contrast to the irreversible NDUFS1 cleavage observed in complex I inhibition (Ricci et al, 2004). In line with this, Tcm62, another complex II assembly factor discovered in yeast (Dibrov et al, 1998), and prohibitin (PHB), its putative mammalian homologue, have been implicated in cell death inhibition following growth factor withdrawal (Vander Heiden et al, 2002). Interestingly, both Tcm62 and PHB were shown to act as mitochondrial chaperones and to be required for mitochondrial respiratory function (Dibrov et al, 1998;Klanner et al, 2000;Nijtmans et al, 2000Nijtmans et al, , 2002.…”

“…In line with this, Tcm62, another complex II assembly factor discovered in yeast (Dibrov et al, 1998), and prohibitin (PHB), its putative mammalian homologue, have been implicated in cell death inhibition following growth factor withdrawal (Vander Heiden et al, 2002). Interestingly, both Tcm62 and PHB were shown to act as mitochondrial chaperones and to be required for mitochondrial respiratory function (Dibrov et al, 1998;Klanner et al, 2000;Nijtmans et al, 2000Nijtmans et al, , 2002. This could indicate that members of this chaperone family can re-assemble complex II during apoptosis and thereby decrease ROS formation and cell death, making this process subject to additional levels of regulations.…”

Endoplasmic Reticulum-Associated Secretory Proteins Sec20p, Sec39p, and Dsl1p Are Involved in Peroxisome Biogenesis

“…Polyclonal antibodies to Sec20p and Dsl1p were raised in guinea pig. Antibodies to thiolase and Sdh2p have been previously described (10,13). Rabbit polyclonal antibodies to Act1p, GFP, Sec39p, Dsl1p, and Sec61p were kind gifts from Gary Eitzen (University of Alberta, Canada), Luc Berthiaume (University of Alberta, Canada), Daniel Unger (University of York, United Kingdom), Hans Dieter Schmitt (Max Planck Institute, Germany), and Randy Schekman (University of California, Berkeley), respectively.…”

J-domain Protein, Jac1p, of Yeast Mitochondria Required for Iron Homeostasis and Activity of Fe-S Cluster Proteins

“…Iron accumulation within mitochondria could be correlated with abrogation of the small amount of residual aconitase activity, suggesting a toxic effect. Succinate dehydrogenase (SDH), a protein complex of the inner membrane containing Fe-S and heme cofactors, was also evaluated (39). Like aconitase, SDH activity was markedly decreased in the mutants under all conditions, and the small amount of residual activity (6% or less) appeared to be further compromised by growth in high iron media (Fig.…”