Post-translational modifications of mitochondrial outer membrane proteins

The mitochondrial outer membrane surrounds the entire organelle. It is composed of a phospholipid bilayer with proteins either embedded into or anchored to the bilayer and mediates the interactions between mitochondria and the rest of the cell. Most of the proteins present in the mitochondrial outer membrane are highly hydrophobic with one or more transmembrane segments. These proteins in conjunction with proteins localized in the inner membrane catalyse energy exchange reactions, the flux of small molecules such as ions, the activation and uptake of long chain fatty acids, import of proteins into the mitochondria, and elimination of biogenic amines among others. In addition, some outer membrane proteins serve as docking sites for non-resident enzymes such as hexokinase and other kinases of signal transduction. All these processes require an intact outer membrane and are highly regulated. One level of regulation with physiological/pathophysiological relevance involves post-translational modification of outer membrane proteins, either by phosphorylation, acetylation or other type of reversible covalent modification. Post-translational modification such as nitration and carbonylation becomes significant under disease states that are associated with increased oxidative stress, i.e. inflammation and ischemia. This review examines the different post-translational modifications of mitochondrial outer membrane proteins and discusses the physiological relevance of these modifications.     

Post-translational modifications of mitochondrial outer membrane proteins

Abstract

The mitochondrial outer membrane surrounds the entire organelle. It is composed of a phospholipid bilayer with proteins either embedded into or anchored to the bilayer and mediates the interactions between mitochondria and the rest of the cell. Most of the proteins present in the mitochondrial outer membrane are highly hydrophobic with one or more transmembrane segments. These proteins in conjunction with proteins localized in the inner membrane catalyse energy exchange reactions, the flux of small molecules such as ions, the activation and uptake of long chain fatty acids, import of proteins into the mitochondria, and elimination of biogenic amines among others. In addition, some outer membrane proteins serve as docking sites for non-resident enzymes such as hexokinase and other kinases of signal transduction. All these processes require an intact outer membrane and are highly regulated. One level of regulation with physiological/pathophysiological relevance involves post-translational modification of outer membrane proteins, either by phosphorylation, acetylation or other type of reversible covalent modification. Post-translational modification such as nitration and carbonylation becomes significant under disease states that are associated with increased oxidative stress, i.e. inflammation and ischemia. This review examines the different post-translational modifications of mitochondrial outer membrane proteins and discusses the physiological relevance of these modifications.     

Novel post-translational modifications of Smad2 identified by mass spectrometry

Smad2 is a crucial component of transforming growth factor-β (TGFβ) signaling, and is involved in the regulation of cell proliferation, death and differentiation. Phosphorylation, ubiquitylation and acetylation of Smad2 have been found to regulate its activity. We used mass spectrometry to search for novel post-translational modifications (PTMs) of Smad2. Peptide mass fingerprinting (PMF) indicated that Smad2 can be acetylated, methylated, citrullinated, phosphorylated and palmitoylated. Sequencing of selected peptides validated methylation at Gly122 and hydroxylation at Trp18 of Smad2. We also observed a novel, so far unidentified modification at Tyr128 and Tyr151. Our observations open for further exploration of biological importance of the detected PTMs. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.     

Biogenesis of mitochondrial outer membrane proteins

Mitochondria are surrounded by two distinct membranes: the outer and the inner membrane. The mitochondrial outer membrane mediates numerous interactions between the mitochondrial metabolic and genetic systems and the rest of the eukaryotic cell. Proteins of this membrane are nuclear-encoded and synthesized as precursor proteins in the cytosol. They are targeted to the mitochondria and inserted into their target membrane via various pathways. This review summarizes our current knowledge of the sorting signals for this specific targeting and describes the mechanisms by which the mitochondrial import machineries recognize precursor proteins, mediate their membrane integration and facilitate assembly into functional complexes.     

Post-translational modifications of poliovirus proteins

The post-translational modifications of poliovirus proteins have been investigated by analysis of glycosylation, sulphation, phosphorylation and acylation of the proteins made in the infected HeLa cells. No glycosylation or sulphation of proteins specific for virus-infected cells was apparent. A number of changes in the pattern of phosphorylated proteins took place. The specific myristylation of the structural protein VP4 and its precursors was clearly apparent. Acylation of viral proteins with oleic or palmitic acid was not detected. Myristylation took place in the presence of the protease inhibitor ZnCl2, but not in the presence of inhibitors of translation, such as cycloneximide and anysomycin.     

Post-translational modifications of histone proteins by monoamine neurotransmitters

Protein monoaminylation is a biochemical process through which biogenic monoamines (e.g., serotonin, dopamine, histamine, etc.) are covalently bonded to certain protein substrates via Transglutaminase 2, an enzyme that catalyzes the transamidation of primary amines to the γ-carboxamides of glutamine residues. Since their initial discovery, these unusual post-translational modifications have been implicated in a wide variety of biological processes, ranging from protein coagulation to platelet activation and G-protein signaling. More recently, histone proteins – specifically histone H3 at glutamine 5 (H3Q5) – have been added to the growing list of monoaminyl substrates in vivo, with H3Q5 monoaminylation demonstrated to regulate permissive gene expression in cells. Such phenomena have further been shown to contribute critically to various aspects of (mal)adaptive neuronal plasticity and behavior. In this short review, we examine the evolution of our understanding of protein monoaminylation events, highlighting recent advances in the elucidation of their roles as important chromatin regulators.     

Post-translational modifications of the polycystin proteins

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited cause of kidney failure and affects up to 12 million people worldwide. Germline mutations in two genes, PKD1 or PKD2, account for almost all patients with ADPKD. The ADPKD proteins, polycystin-1 (PC1) and polycystin-2 (PC2), are regulated by post-translational modifications (PTM), with phosphorylation, glycosylation and proteolytic cleavage being the best described changes. A few PTMs have been shown to regulate polycystin trafficking, signalling, localisation or stability and thus their physiological function. A key challenge for the future will be to elucidate the functional significance of all the individual PTMs reported to date. Finally, it is possible that site-specific mutations that disrupt PTM could contribute to cystogenesis although in the majority of cases, confirmatory evidence is awaited.     

Action ofl-acetylcarnitine on age-dependent modifications of mitochondrial membrane proteins from rat cerebellum

Protein patterns of mitochondrial outer membrane, inner membrane, and matrix from nonsynaptic (free) mitochondria from rat cerebellum at different ages (4, 8, 12, 16, 20, and 24 months) were analyzed by gel electrophoresis. Acutel-acetylcarnitine treatment was per-formed by a single i.p. injection (100 mg/kg body weight) of the substance 60 min before the sacrifice of the animals. Different age-dependent changes were obtained for the proteins of the three fractions. The amount of some protein subunits increased and/or decreased after drug treatment. In particular, protein composition of the inner mitochondrial membrane showed significant age-related modifications. This result probably indicates differences in protein synthesis and/or turnover rates in the various mitochondrial compartments during aging. Acutel-acetylcarnitine treatment caused: a high increase in the amount of one inner membrane protein with Mw 16 kDa, at all the ages studied; a decrease in the amount of many other inner membrane proteins; modifications of some matrix proteins. Our results show that in vivo administration ofl-acetylcarnitine affects mainly the inner membrane protein composition of cerebellar mitochondria.     

NADH-dependent aryl hydrocarbon hydroxylase in rat liver mitochondrial outer membrane.

NADH-dependent 3,4-benzpyrene hydroxylase activity was detected in the purified mitochondrial outer membrane fraction from the livers of rats treated with 3-methylcholanthrene. The specific activity in the outer membrane fraction is nearly equal to that of microsomes, a level too high to be accounted for only by the microsomal contamination. On the other hand, the NADPH-dependent 3,4-benzpyrene hydroxylase activity in the outer membrane fraction is about 50% of that of microsomes. The ratio of the specific activity of NADPH- to NADH-dependent 3,4-benzpyrene hydroxylase in microsomal fraction was about 3.5, while that of the outer membrane fraction was about 1.5. Moreover, it was found that NADH-dependent 3,4-benzpyrene hydroxylase activity in mitochondrial outer membrane from control rat liver was cyanide-insensitive, while that in microsomes was cyanide-sensitive. These results suggest the presence in the mitochondrial outer membrane fraction of aryl hydrocarbon hydroxylase activity which uses as electron donor NADH nearly to the same extent as NADPH. The hydroxylase system is composed of cyanide-insensitive cytochrome P-450 and is inducible markedly by 3-methylcholanthrene treatment. The probable electron transfer pathways in the mitochondrial outer membrane cytochrome P-450 oxidase system are discussed.     

Analysis of posttranslational modifications of proteins by tandem mass spectrometry

Protein activity and turnover is tightly and dynamically regulated in living cells. Whereas the three-dimensional protein structure is predominantly determined by the amino acid sequence, posttranslational modification (PTM) of proteins modulates their molecular function and the spatial-temporal distribution in cells and tissues. Most PTMs can be detected by protein and peptide analysis by mass spectrometry (MS), either as a mass increment or a mass deficit relative to the nascent unmodified protein. Tandem mass spectrometry (MS/MS) provides a series of analytical features that are highly useful for the characterization of modified proteins via amino acid sequencing and specific detection of posttranslationally modified amino acid residues. Large-scale, quantitative analysis of proteins by MS/MS is beginning to reveal novel patterns and functions of PTMs in cellular signaling networks and biomolecular structures.     

In vitro synthesis of monoamine oxidase of rat liver outer mitochondrial membrane

Monoamine oxidase, an intrinsic protein of outer mitochondrial membrane, was purified from bovine liver and rabbit antibody against the enzyme was prepared. The antibody could react with the monoamine oxidase of rat liver mitochondria. When rat liver RNA was translated $\text{in}\text{vitro}$ using rabbit reticulocyte lysate and monoamine oxidase peptide in the translation products was immunoprecipitated by the antibody, the peptide was detected in the products programmed by the messenger RNA's from total and free polysomes but not that from bound polysomes. The enzyme synthesized $\text{in}\text{vitro}$ had the same apparent molecular size as the mature protein in outer mitochondrial membrane.     

Regulating mitochondrial outer membrane proteins by ubiquitination and proteasomal degradation

Mitochondrial outer membrane proteins have been found to be ubiquitinated and degraded by the proteasome. This process shares at least one component of the ERAD pathway of ER membrane protein degradation, the AAA ATPase cdc48/p97/VCP, thought to extract integral membrane proteins from the lipid bilayer and chaperone them to the proteasome. Proteasomal degradation of the outer mitochondrial membrane (OMM) protein Mcl1 regulates apoptosis whereas Parkin-mediated ubiquitination and degradation of Mitofusins can inhibit mitochondrial fusion and promote mitophagy. The breadth of OMM ubiquitin/proteasome substrates and the physiological relevance of their turnover are only beginning to be understood.     

Sorting and assembly of mitochondrial outer membrane proteins

In the last years the picture of protein import into the mitochondria has become much more complicated in terms of new components and new sorting pathways. These novel findings have also changed views concerning the biogenesis pathway of mitochondrial outer membrane proteins. In addition to proteins anchored with transmembrane alpha-helices, the endosymbiotic origin of the mitochondria has resulted in the presence of transmembrane beta-barrels in this compartment. The sorting and assembly pathway of outer membrane proteins involves three machineries: the translocase of the outer membrane (TOM complex) the sorting and assembly machinery (SAM complex) and the MDM complex (mitochondrial distribution and morphology). Here we review recent developments on the biogenesis pathways of outer membrane proteins with a focus on Tom proteins, the most intensively studied class of these precursor proteins.     

A targeted proteomic approach for the analysis of rat liver mitochondrial outer membrane proteins with extensive sequence coverage

Membrane proteins play an important role in cellular function. However, their analysis by mass spectrometry often is hindered by their hydrophobicity and/or low abundance. In this article, we present a method for the mass spectrometric analysis of membrane proteins based on the isolation of the resident membranes, isolation of the proteins by gel electrophoresis, and electroelution followed by enzymatic digestion by both trypsin and proteinase K. With this method, we have achieved 82-99% sequence coverage for the membrane proteins carnitine palmitoyltransferase-I (CPT-I), long-chain acyl-CoA synthetase (LCAS), and voltage-dependent anion channel (VDAC), isolated from rat liver mitochondrial outer membranes, including the transmembrane domains of these integral membrane proteins. This high sequence coverage allowed the identification of the isoforms of the proteins under study. This methodology provides a targeted approach for examining membrane proteins in detail.     

Structural mass spectrometry of membrane proteins

The analysis of proteins and protein complexes by mass spectrometry (MS) has come a long way since the invention of electrospray ionization (ESI) in the mid 80s. Originally used to characterize small soluble polypeptide chains, MS has progressively evolved over the past 3 decades towards the analysis of samples of ever increasing heterogeneity and complexity, while the instruments have become more and more sensitive and resolutive. The proofs of concepts and first examples of most structural MS methods appeared in the early 90s. However, their application to membrane proteins, key targets in the biopharma industry, is more recent. Nowadays, a wealth of information can be gathered from such MS-based methods, on all aspects of membrane protein structure: sequencing (and more precisely proteoform characterization), but also stoichiometry, non-covalent ligand binding (metals, drug, lipids, carbohydrates), conformations, dynamics and distance restraints for modelling. In this review, we present the concept and some historical and more recent applications on membrane proteins, for the major structural MS methods.     

Rapid typing of Moraxella catarrhalis subpopulations based on outer membrane proteins using mass spectrometry

Moraxella catarrhalis is a major mucosal pathogen of the human respiratory tract both in children and in adults. Two subpopulations of this organism have been described that differ in 16S rRNA gene sequence and virulence traits. Three 16S rRNA types have been defined. 2-DE followed by protein identification by MS revealed significant differences in the outer membrane protein (OMP) patterns of each M. catarrhalis 16S rRNA type. Approximately 130 features were detected on the 2-DE map of each M. catarrhalis 16S rRNA type. However, only 50 features were expressed by all strains. Furthermore, direct profiling of isolated OMP using MALDI-TOF MS resulted in a characteristic spectral fingerprint for each 16S rRNA type. Fingerprints remained identical when intact cells instead of isolated OMP were analyzed. This finding suggests that the source of desorbed ions is the outer membrane. Based on the fingerprint we were able to assign 18 well-characterized clinical M. catarrhalis isolates to the correct subpopulation. Therefore, MALDI-TOF of intact M. catarrhalis provides a rapid and robust tool for M. catarrhalis strain typing that could be applied in epidemiological studies.     

Yeast mitochondrial outer membrane specifically binds cytoplasmically-synthesized precursors of mitochondrial proteins

The precursor of cytochrome b₂ (a cytoplasmically-synthesized mitochondrial protein) binds to isolated mitochondria or to isolated outer membrane vesicles. Binding does not require an energized inner membrane, is diminished by trypsin treatment of the membranes and is not observed with the partially processed (intermediate) form of the cytochrome b₂ precursor or with non-mitochondrial proteins. Upon energization of the mitochondria, the bound precursor is imported and cleaved to the mature form. Similar results were obtained with the precursor of citrate synthase. This receptor-like binding activity was present in isolated outer, but not inner membrane. It was solubilized from outer membrane with non-ionic detergent and reconstituted into liposomes.