A hub for regulation of mitochondrial metabolism: Fatty acid and lipoic acid biosynthesis.

Having evolved from a prokaryotic origin, mitochondria retain pathways required for the catabolism of energy-rich molecules and for the biosynthesis of molecules that aid catabolism and/or participate in other cellular processes essential for life of the cell. Reviewed here are details of the mitochondrial fatty acid biosynthetic pathway (FAS II) and its role in building both the octanoic acid precursor for lipoic acid biosynthesis (LAS) and longer-chain fatty acids functioning in chaperoning the assembly of mitochondrial multisubunit complexes. Also covered are the details of mitochondrial lipoic acid biosynthesis, which is distinct from that of prokaryotes, and the attachment of lipoic acid to subunits of pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, and glycine cleavage system complexes.

Genetic dissection of the mitochondrial lipoylation pathway in yeast.

Background: Lipoylation of 2-ketoacid dehydrogenases is essential for mitochondrial function in eukaryotes. While the basic principles of the lipoylation processes have been worked out, we still lack a thorough understanding of the details of this important post-translational modification pathway. Here we used yeast as a model organism to characterize substrate usage by the highly conserved eukaryotic octanoyl/lipoyl transferases in vivo and queried how amenable the lipoylation system is to supplementation with exogenous substrate.

MLT1 links cytoskeletal asymmetry to organelle placement in chlamydomonas.

Asymmetric placement of the photosensory eyespot organelle in Chlamydomonas is patterned by mother-daughter differences between the two basal bodies, which template the anterior flagella. Each basal body is associated with two bundled microtubule rootlets, one with two microtubules and one with four, forming a cruciate pattern. In wild-type cells, the single eyespot is positioned at the equator in close proximity to the plus end of the daughter rootlet comprising four microtubules, the D4.

Variant non ketotic hyperglycinemia is caused by mutations in LIAS, BOLA3 and the novel gene GLRX5.

Patients with nonketotic hyperglycinemia and deficient glycine cleavage enzyme activity, but without mutations in AMT, GLDC or GCSH, the genes encoding its constituent proteins, constitute a clinical group which we call 'variant nonketotic hyperglycinemia'. We hypothesize that in some patients the aetiology involves genetic mutations that result in a deficiency of the cofactor lipoate, and sequenced genes involved in lipoate synthesis and iron-sulphur cluster biogenesis. Of 11 individuals identified with variant nonketotic hyperglycinemia, we were able to determine the genetic aetiology in eight patients and delineate the clinical and biochemical phenotypes.

Defects in mitochondrial fatty acid synthesis result in failure of multiple aspects of mitochondrial biogenesis in Saccharomyces cerevisiae.

Mitochondrial fatty acid synthesis (mtFAS) shares acetyl-CoA with the Krebs cycle as a common substrate and is required for the production of octanoic acid (C8) precursors of lipoic acid (LA) in mitochondria. MtFAS is a conserved pathway essential for respiration. In a genetic screen in Saccharomyces cerevisiae designed to further elucidate the physiological role of mtFAS, we isolated mutants with defects in mitochondrial post-translational gene expression processes, indicating a novel link to mitochondrial gene expression and respiratory chain biogenesis.

Independent localization of plasma membrane and chloroplast components during eyespot assembly.

Like many algae, Chlamydomonas reinhardtii is phototactic, using two anterior flagella to swim toward light optimal for photosynthesis. The flagella are responsive to signals initiated at the photosensory eyespot, which comprises photoreceptors in the plasma membrane and layers of pigment granules in the chloroplast. Phototaxis depends on placement of the eyespot at a specific asymmetric location relative to the flagella, basal bodies, and bundles of two or four highly acetylated microtubules, termed rootlets, which extend from the basal bodies toward the posterior of the cell.

Miniature- and Multiple-Eyespot Loci in Chlamydomonas reinhardtii Define New Modulators of Eyespot Photoreception and Assembly.

The photosensory eyespot of the green alga Chlamydomonas reinhardtii is a model system for the study of organelle biogenesis and placement. Eyespot assembly and positioning are governed by several genetic loci that have been identified in forward genetic screens for phototaxis-defective mutants. These include the previously described miniature-eyespot mutant min1, the multiple-eyespot mutant mlt1, the eyeless mutants eye2 and eye3, and two previously uncharacterized eyespot mutants, min2 and mlt2.

New insights into eyespot placement and assembly in Chlamydomonas.

Aspects of cellular architecture, such as cytoskeletal asymmetry cues, play critical roles in directing the placement of organelles and establishing the sites of their formation. In the model green alga Chlamydomonas, the photosensory eyespot occupies a defined position in relation to the flagella and microtubule cytoskeleton. Investigations into the cellular mechanisms of eyespot placement and assembly have aided our understanding of the interplay between cytoskeletal and plastid components of the cell.

The daughter four-membered microtubule rootlet determines anterior-posterior positioning of the eyespot in Chlamydomonas reinhardtii.

The characteristic geometry of the unicellular chlorophyte Chlamydomonas reinhardtii has contributed to its adoption as a model system for cellular asymmetry and organelle positioning. The eyespot, a photosensitive organelle, is localized asymmetrically in the cell at a precisely defined position relative to the flagella and cytoskeletal microtubule rootlets. We have isolated a mutant, named pey1 for posterior eyespot, with variable microtubule rootlet lengths.

Asymmetric properties of the Chlamydomonas reinhardtii cytoskeleton direct rhodopsin photoreceptor localization.

The eyespot of the unicellular green alga Chlamydomonas reinhardtii is a photoreceptive organelle required for phototaxis. Relative to the anterior flagella, the eyespot is asymmetrically positioned adjacent to the daughter four-membered rootlet (D4), a unique bundle of acetylated microtubules extending from the daughter basal body toward the posterior of the cell. Here, we detail the relationship between the rhodopsin eyespot photoreceptor Channelrhodopsin 1 (ChR1) and acetylated microtubules.

Thioredoxin-family protein EYE2 and Ser/Thr kinase EYE3 play interdependent roles in eyespot assembly.

The eyespot of the biflagellate unicellular green alga Chlamydomonas reinhardtii is a complex organelle that facilitates directional responses of the cell to environmental light stimuli. The eyespot, which assembles de novo after every cell division and is associated with the daughter four-membered (D4) microtubule rootlet, comprises an elliptical patch of rhodopsin photoreceptors on the plasma membrane and stacks of carotenoid-rich pigment granule arrays in the chloroplast. Two loci, EYE2 and EYE3, define factors involved in the formation and organization of the eyespot pigment granule arrays.

Mitochondrial fatty acid synthesis and respiration.

Recent studies have revealed that mitochondria are able to synthesize fatty acids in a malonyl-CoA/acyl carrier protein (ACP)-dependent manner. This pathway resembles bacterial fatty acid synthesis (FAS) type II, which uses discrete, nuclearly encoded proteins. Experimental evidence, obtained mainly through using yeast as a model system, indicates that this pathway is essential for mitochondrial respiratory function.

Lipoic acid synthesis and attachment in yeast mitochondria.

Lipoic acid is a sulfur-containing cofactor required for the function of several multienzyme complexes involved in the oxidative decarboxylation of alpha-keto acids and glycine. Mechanistic details of lipoic acid metabolism are unclear in eukaryotes, despite two well defined pathways for synthesis and covalent attachment of lipoic acid in prokaryotes. We report here the involvement of four genes in the synthesis and attachment of lipoic acid in Saccharomyces cerevisiae.

Mitochondrial fatty acid synthesis type II: more than just fatty acids.

Eukaryotes harbor a highly conserved mitochondrial pathway for fatty acid synthesis (FAS), which is completely independent of the eukaryotic cytosolic FAS apparatus. The activities of the mitochondrial FAS system are catalyzed by soluble enzymes, and the pathway thus resembles its prokaryotic counterparts. Except for octanoic acid, which is the direct precursor for lipoic acid synthesis, other end products and functions of the mitochondrial FAS pathway are still largely enigmatic.

C2 domain protein MIN1 promotes eyespot organization in Chlamydomonas reinhardtii.

Assembly and asymmetric localization of the photosensory eyespot in the biflagellate, unicellular green alga Chlamydomonas reinhardtii requires coordinated organization of photoreceptors in the plasma membrane and pigment granule/thylakoid membrane layers in the chloroplast. min1 (mini-eyed) mutant cells contain abnormally small, disorganized eyespots in which the chloroplast envelope and plasma membrane are no longer apposed. The MIN1 gene, identified here by phenotypic rescue, encodes a protein with an N-terminal C2 domain and a C-terminal LysM domain separated by a transmembrane sequence.

Intersection of RNA processing and the type II fatty acid synthesis pathway in yeast mitochondria.

Distinct metabolic pathways can intersect in ways that allow hierarchical or reciprocal regulation. In a screen of respiration-deficient Saccharomyces cerevisiae gene deletion strains for defects in mitochondrial RNA processing, we found that lack of any enzyme in the mitochondrial fatty acid type II biosynthetic pathway (FAS II) led to inefficient 5' processing of mitochondrial precursor tRNAs by RNase P. In particular, the precursor containing both RNase P RNA (RPM1) and tRNA(Pro) accumulated dramatically.

Pet127 governs a 5' -> 3'-exonuclease important in maturation of apocytochrome b mRNA in Saccharomyces cerevisiae.

The details of mRNA maturation in Saccharomyces mitochondria are not well understood. All seven mRNAs are transcribed as part of multigenic units. The mRNAs are processed at a common 3'-dodecamer sequence, but the 5'-ends have seven different sequences.

The Chlamydomonas genome reveals the evolution of key animal and plant functions.

Chlamydomonas reinhardtii is a unicellular green alga whose lineage diverged from land plants over 1 billion years ago. It is a model system for studying chloroplast-based photosynthesis, as well as the structure, assembly, and function of eukaryotic flagella (cilia), which were inherited from the common ancestor of plants and animals, but lost in land plants. We sequenced the approximately 120-megabase nuclear genome of Chlamydomonas and performed comparative phylogenomic analyses, identifying genes encoding uncharacterized proteins that are likely associated with the function and biogenesis of chloroplasts or eukaryotic flagella.

An ancient genetic link between vertebrate mitochondrial fatty acid synthesis and RNA processing.

In bacteria, functionally related gene products are often encoded by a common transcript. Such polycistronic transcripts are rare in eukaryotes. Here we isolated several clones from human cDNA libraries, which rescued the respiratory-deficient phenotype of a yeast mitochondrial 3-hydroxyacyl thioester dehydratase 2 (htd2) mutant strain.

CBT1 interacts genetically with CBP1 and the mitochondrially encoded cytochrome b gene and is required to stabilize the mature cytochrome b mRNA of Saccharomyces cerevisiae.

Mutation of a CCG sequence in the 5'-untranslated region of the mitochondrially encoded cytochrome b mRNA in Saccharomyces cerevisiae results in destabilization of the message and respiratory deficiency of the mutant strain. This phenotype mimics that of a mutation in the nuclear CBP1 gene. Here it is shown that overexpression of the nuclear CBT1 gene, due to a transposon insertion in the 5'-untranslated region, rescues the respiratory defects resulting from mutating the CCG sequence to ACG.

Analysis of transcription asymmetries along the tRNAE-COB operon: evidence for transcription attenuation and rapid RNA degradation between coding sequences.

Mitochondrial gene expression in yeast is believed to be regulated predominantly at the post-transcriptional level. However, the contribution of mitochondrial transcription and RNA-turnover rates to differential gene regulation is still largely unknown. Mitochondrial run-on transcription and hybrid selection assays showed that some of the multigenic transcription units of the mitochondrial genome are transcribed evenly, whereas others are transcribed asymmetrically, with higher transcription rates for promoter-proximal genes, than for promoter-distal genes.

The mitochondrial message-specific mRNA protectors Cbp1 and Pet309 are associated in a high-molecular weight complex.

In Saccharomyces cerevisiae, the nuclear-encoded protein Cbp1 promotes stability and translation of mitochondrial cytochrome b transcripts through interaction with the 5' untranslated region. Fusion of a biotin binding peptide tag to the C terminus of Cbp1 has now allowed detection in mitochondrial extracts by using peroxidase-coupled avidin. Cbp1 is associated with the mitochondrial membranes when high ionic strength extraction conditions are used.

Aep3p stabilizes the mitochondrial bicistronic mRNA encoding subunits 6 and 8 of the H+-translocating ATP synthase of Saccharomyces cerevisiae.

Subunits 6 and 8 of the mitochondrial ATPase in Saccharomyces cerevisiae are encoded by the mitochondrial genome and translated from bicistronic mRNAs containing both reading frames. The stability of the two major species of ATP8/6 mRNA, which differ in the length of the 5'-untranslated region, depends on the expression of several nuclear-encoded factors. In the present study, the product of the gene designated AEP3 (open reading frame YPL005W) is shown to be required for stabilization and/or processing of both ATP8/6 mRNA species.

Suppressor mutations define two regions in the Cbp1 protein important for mitochondrial cytochrome b mRNA stability in Saccharomyces cerevisiae.

Nuclear-encoded Cbp1 stabilizes and promotes translation of mitochondrial cytochrome b (COB) mRNA. A CCG triplet within the 5'UTR of COB mRNA is essential for Cbp1-dependent stability. Like cbp1 mutations, mutation of any nucleotide in CCG results in degradation of COB transcripts.