Gluing the Respiratory Chain Together

Abstract

Cytochrome bc 1 complex (complex III) and cytochrome c oxidase complex (complex IV) are multisubunit homodimers that are essential components of the mitochondrial respiratory chain. Complexes III and IV associate to form a supercomplex that can be displayed using blue native polyacrylamide gel electrophoresis. Both homodimeric complexes contain tightly associated cardiolipin (CL) required for function. We report here that in a crd1⌬ strain of yeast (null in expression of CL synthase) ϳ90% of complexes III and IV were observed as individual homodimers; only the supercomplex was observed with CRD1 wild type cells. Introduction of a plasmid born copy of the CRD1 gene under exogenous regulation by doxycycline made possible controlled variation in the in vivo CL levels. At an intermediate level of CL, a mixture of individual homodimers (30%) and supercomplex (70%) was observed. These results strongly indicate that CL plays a central role in higher order organization of components of the respiratory chain of mitochondria.Broader understanding of how lipid-protein interactions determine protein structure and function is increasing. Recent crystallographic results of multimeric membrane-associated complexes have revealed lipids inserted between the protein subunits (1, 2). Even monomeric proteins such as OmpF of Escherichia coli show outer membrane-specific lipid A molecules in the crystal structure (3). The highly ordered state of these lipids necessary for their resolution supports a role for specific and tight association of defined lipid species with membrane proteins. Considerable evidence also supports the organization of the respiratory chain in both prokaryotes and eukaryotes into "respirasomes" or supercomplexes made up of smaller multimeric complexes (4). For oxidative phosphorylation, such supercomplexes may provide added efficiency by eliminating the need for diffusion of substrates and products between individual complexes and provide a means for channeling intermediates between individual complexes. What role does the lipid environment play in supporting supercomplex formation? Are there specific protein-lipid interactions involved or is the collective property of the membrane responsible for higher order complex formation?In 1974, Skulachev (5) suggested that cardiolipin (CL) 1 might participate in linking together components of the respiratory chain. CL is a minor phospholipid with distribution limited to bacterial cytoplasmic membranes and to the inner membrane of mitochondria where energy transducing oxidative processes of cells are centered. Structured CL is associated with the b subunit of the cytochrome bc 1 complex (complex III) in yeast (6). Complex III is a homodimer of 10 different subunits in yeast and 11 subunits in mammalian cells (7). When CL was removed from mammalian complex III, structure was perturbed and the complex lost all function (8). Only CL and its immediate metabolic precursor phosphatidylglycerol (PG) restored activity to the resolved complex. Although PG restored full activity,...

C-terminal Lysines Determine Phospholipid Interaction of Sarcomeric Mitochondrial Creatine Kinase

“…8) in which MtCK C termini and ANT are brought into close vicinity by binding to a common CL patch in the inner mitochondrial membrane (3). In fact, CL can act as a glue for supercomplex formation between proteins in the inner mitochondrial membrane (33,34). Since four C termini are exposed at one binding face of the cuboidal MtCK octamer, such a 2 Available online at genome.jgi-psf.org/ciona4/ciona4.home.html.…”

“…In support of this, mutations that reduce MtCK membrane binding through destabilization of its octameric structure also reduce creatinestimulated respiration in cardiomyocyte mitochondria (30). In fact, it is well known that CL-protein interactions are important not only for activity and structural integrity of mitochondrial inner membrane proteins but also for subunit assembly and supercomplex formation (31)(32)(33)(34). CL (diphosphatidyl glycerol) is unique in having a large dianionic headgroup that consists of three glycerol molecules connected by two phosphodiester linkages with the terminal glycerols bound to four acyl chains via their primary and secondary hydroxyl groups (31).…”

Taz1, an Outer Mitochondrial Membrane Protein, Affects Stability and Assembly of Inner Membrane Protein Complexes: Implications for Barth Syndrome

“…Thus it appears that dissociation of respiratory supercomplexes can decrease the efficiency of respiration. In a S. cerevisiae cardiolipin synthase mutant (crd1⌬), which lacks cardiolipin, oligomerization of AAC was affected (Jiang et al, 2000) and the respiratory chain supercomplexes dissociated in organello and on BN-PAGE (Zhang et al, 2002(Zhang et al, , 2005, suggesting a role for cardiolipin in the oligomerization of inner membrane protein complexes. We find that AAC oligomerization is affected in taz1⌬ mitochondria, similar to that in crd1⌬; however, supercomplexes only partially dissociate on BN-PAGE.…”

“…The formation of supercomplexes is thought to increase the efficiency of respiration through both substrate channeling and sequestration of reactive intermediates and is thus of advantage for efficient respiration (Cruciat et al, 2000;Schägger and Pfeiffer, 2000;Zhang et al, 2005). When yeast cells are deleted for the CRD1 gene (cardiolipin synthase) cardiolipin is not synthesized and dissociation of the respiratory chain supercomplexes and oligomers of AAC is observed (Jiang et al, 2000;Zhang et al, 2002Zhang et al, , 2005. The reduced stability of these protein complex oligomers reflects the complete loss of cardiolipin from the mitochondrial membranes (Pfeiffer et al, 2003).…”

Peer Reviewed: The Chemistry of Thought: Neurotransmitters in the Brain

“…Unfortunately, improvements are needed to enhance the selectivity of these reagents because cross-reaction with other cellular constituents complicates the signals. These interferences include divalent cations and ascorbic acid compounds, which are often naturally present in concentrations that are orders of magnitude greater than NO itself in the same brain tissues (27,28). However, for rapid spatial analysis of large tissues, such methods remain an excellent choice.…”