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Results for “posttranslational modification”

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PubMed1989

Genetic and pharmacological suppression of oncogenic mutations in ras genes of yeast and humans.

The activity of an oncoprotein and the secretion of a pheromone can be affected by an unusual protein modification. Specifically, posttranslational modification of yeast a-factor and Ras protein requires an intermediate of the cholesterol biosynthetic pathway. This modification is apparently essential for biological activity. Studies of yeast mutants blocked in sterol biosynthesis demonstrated that the membrane association and biological activation of the yeast Ras2 protein require mevalonate, a precursor of sterols and other isoprenes such as farnesyl pyrophosphate. Furthermore, drugs that inhibit mevalonate biosynthesis blocked the in vivo action of oncogenic derivatives of human Ras protein in the Xenopus oocyte assay. The same drugs and mutations also prevented the posttranslational processing and secretion of yeast a-factor, a peptide that is farnesylated. Thus, the mevalonate requirement for Ras activation may indicate that attachment of a mevalonate-derived (isoprenoid) moiety to Ras proteins is necessary for membrane association and biological function. These observations establish a connection between the cholesterol biosynthetic pathway and transformation by the ras oncogene and offer a novel pharmacological approach to investigating, and possibly controlling, ras-mediated malignant transformations.

Amino Acid Sequence↗

PubMed1988

Comparison of posttranslational protein modification by amino acid addition after crush injury to sciatic and optic nerves of rats.

Posttranslational protein modifications by the addition of amino acids are reactions which occur in intact sciatic and optic nerves of rats. The nerves differ, however, in that 2 h after crush injury these reactions are activated in sciatic but not in optic nerves. As sciatic nerves will eventually regenerate, whereas optic nerves will not, we have proposed that the activation of these reactions is correlated with the ability of a nerve to regenerate. The current experiments examined the posttranslational addition of amino acids to proteins at times greater than 2 h after nerve crush, during sciatic nerve regeneration and optic nerve degeneration. We also examined the optic nerve for morphologic correlates to changes in protein modification and partially characterized the proteins modified by [3H]Lys in the regenerating sciatic nerve using two-dimensional sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). In a segment of sciatic nerve taken from a region just proximal to the site of crush, protein modification by covalent addition of [3H]Arg, [3H]Lys and [3H]Leu increased during both posttraumatic (2 h postcrush) and regenerative (6 days and 14 days postcrush) stages. Two-dimensional PAGE of [3H]Lys modified sciatic nerve proteins 6 days after crush injury showed labeling of proteins having molecular masses in the 18,000- to 20,000-, 30,000- to 40,000-, and 80,000- to 100,000-Da ranges, with neutral or basic isoelectric points (pI 7.1 to 8.0). In the retinal portion of the crushed optic nerve, incorporation of the same amino acids was unchanged or depressed to 21 days postcrush, except at 6 days postcrush when the incorporation of all three amino acids into proteins was increased threefold. These increases correlated with the appearance of terminal end bulbs in the portion of nerve analyzed. Histological examination of each nerve 2 h postcrush showed marked edema in the optic but not the sciatic nerve, a condition which may be related to the ability of sciatic and inability of optic nerves to activate protein modification reactions.

Amino Acids↗

PubMed2003

Flagellin glycosylation island in Pseudomonas syringae pv. glycinea and its role in host specificity.

The deduced amino acid sequences of the flagellins of Pseudomonas syringae pv. tabaci and P. syringae pv. glycinea are identical; however, their abilities to induce a hypersensitive reaction are clearly different. The reason for the difference seems to depend on the posttranslational modification of the flagellins. To investigate the role of this posttranslational modification in the interactions between plants and bacterial pathogens, we isolated genes that are potentially involved in the posttranslational modification of flagellin in P. syringae pv. glycinea (glycosylation island); then defective mutants with mutations in these genes were generated. There are three open reading frames in the glycosylation island, designated orf1, orf2, and orf3. orf1 and orf2 encode putative glycosyltransferases, and mutants with defects in these open reading frames, deltaorf1 and deltaorf2, secreted nonglycosylated and slightly glycosylated flagellins, respectively. Inoculation tests performed with these mutants and original nonhost tobacco leaves revealed that deltaorf1 and deltaorf2 could grow on tobacco leaves and caused symptom-like changes. In contrast, these mutants failed to cause symptoms on original host soybean leaves. These data indicate that putative glycosyltransferases encoded in the flagellin glycosylation island are strongly involved in recognition by plants and could be the specific determinants of compatibility between phytopathogenic bacteria and plant species.

Flagellin↗

PubMed2004

Pseudophosphorylation and glycation of tau protein enhance but do not trigger fibrillization in vitro.

Alzheimer's disease is defined in part by the intraneuronal aggregation of tau protein into filamentous lesions. The pathway is accompanied by posttranslational modifications including phosphorylation and glycation, each of which has been shown to promote tau fibrillization in vitro when present at high stoichiometry. To clarify the site-specific impact of posttranslational modification on tau fibrillization, the ability of recombinant full-length four repeat tau protein (htau40) and 11 pseudophosphorylation mutants to fibrillize in the presence of anionic inducer was assayed in vitro using transmission electron microscopy and laser light scattering assays. Tau glycated with d-glucose was examined as well. Both glycated tau and pseudophosphorylation mutants S199E, T212E, S214E, double mutant T212E/S214E, and triple mutant S199E/S202E/T205E yielded increased filament mass at equilibrium relative to wild-type tau. Increases in filament mass correlated strongly with decreases in critical concentration, indicating that both pseudophosphorylation and glycation promoted fibrillization by shifting equilibrium toward the fibrillized state. Analysis of reaction time courses further revealed that increases in filament mass were not associated with reduced lag times, indicating that these posttranslational modifications did not promote filament nucleation. The results suggest that site-specific posttranslational modifications can stabilize filaments once they nucleate, and thereby support their accumulation at low intracellular tau concentrations.

Alzheimer Disease↗

PubMed2006

Structural basis for histone N-terminal recognition by human peptidylarginine deiminase 4.

Histone arginine methylation is a posttranslational modification linked to the regulation of gene transcription. Unlike other posttranslational modifications, methylation has generally been regarded as stable, and enzymes that demethylate histone arginine residues have not been identified. However, it has recently been shown that human peptidylarginine deiminase 4 (PAD4), a Ca(2+)-dependent enzyme previously known to convert arginine residues to citrulline in histones, can also convert monomethylated arginine residues to citrulline both in vivo and in vitro. Citrullination of histone arginine residues by the enzyme antagonizes methylation by histone arginine methyltransferases and is thus a novel posttranslational modification that regulates the level of histone arginine methylation and gene activity. Here we present the crystal structures of a Ca(2+)-bound PAD4 mutant in complex with three histone N-terminal peptides, each consisting of 10 amino acid residues that include one target arginine residue for the enzyme (H3/Arg-8, H3/Arg-17, and H4/Arg-3). To each histone N-terminal peptide, the enzyme induces a beta-turn-like bent conformation composed of five successive residues at the molecular surface near the active site cleft. The remaining five residues are highly disordered. The enzyme recognizes each peptide through backbone atoms of the peptide with a possible consensus recognition motif. The sequence specificity of the peptide recognized by this enzyme is thought to be fairly broad. These observations provide structural insights into target protein recognition by histone modification enzymes and illustrate how PAD4 can target multiple arginine sites in the histone N-terminal tails.

Amino Acid Sequence↗

PubMed2005

Analysis of minor hemoglobins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

BACKGROUND: Hemoglobin (Hb) heterogeneity arises mainly from posttranslational modifications of the globin chains, and cation-exchange chromatography reveals falsely increased concentrations of some minor Hbs in the presence of abnormal Hbs. Here we describe a method for identification of the globin chains and their posttranslational modifications contained in the Hb fractions. METHODS: We used cation-exchange HPLC (PolyCAT A column) for separation of Hb fractions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for analysis of the separated globin chains. Globin chains were identified by their molecular masses. Posttranslational modifications of globin chains were identified by digestion of the proteins with endoproteinase V8 before MALDI-TOF MS of the resulting peptides. RESULTS: Analysis of the HbA2 fractions of patients with HbS revealed 4 different globin chains. We found, in addition to the expected alpha- and delta-chains, the carbamylated alpha- and the betaS-chains. Additionally, we analyzed HbH, Hb Barts, HbA 1b, pre-HbA 1c, HbA 1c, HbF1, HbF, HbA 1d3a, HbA 1d3b, HbA2, and HbC1 fractions from control and pathologic blood samples. We identified several posttranslational modifications of the globin chains, such as pyruvatization, glycation, acetylation, carbamylation, and acetaldehyde adduct formation. CONCLUSIONS: The native and posttranslationally modified globin chains in minor and major Hbs are unambiguously identified by MALDI-TOF MS. A minor Hb containing the carbamylated alpha- and the betaS-chain elutes at the same time as normal HbA2 (alpha2delta2) and thus leads to falsely increased HbA2 values in patients with HbS when blood is analyzed with PolyCAT A chromatography.

Chromatography, High Pressure Liquid↗

PubMed2005

Transcriptional regulation by steroid receptor coactivator phosphorylation.

The basic mechanisms underlying ligand-dependent transcriptional activation by nuclear receptors (NRs) require the sequential recruitment of various coactivators. Increasing numbers of coactivators have been identified in recent years, and both biochemical and genetic studies demonstrate that these coactivators are differentially used by transcription factors, including NRs, in a cell/tissue type- and promoter-specific manner. However, the molecular basis underlying this specificity remains largely unknown. Recently, NRs and coregulators were shown to be targets of posttranslational modifications activated by diverse cellular signaling pathways. It is argued that posttranslational modifications of these proteins provide the basis for a combinatorial code required for specific gene activation by NRs and coactivators, and that this code also enables coactivators to efficiently stimulate the activity of other classes of transcription factors. In this review, we will focus on coactivators and discuss the recent progress in understanding the role of phosphorylation of the steroid receptor coactivator family and the potential ramifications of this posttranslational modification for regulation of gene expression.

Acetyltransferases↗

PubMed1997

Reactivity of nitrogen monoxide species with NADH: implications for nitric oxide-dependent posttranslational protein modification.

Nitric oxide (NO.) and NO. donors incite NAD- [i.e., mono(ADP-ribosylation)] and NADH-dependent posttranslational protein modifications by an as yet unknown mechanism. A route of pyridine nucleotide-dependent, NO.-stimulated protein modification has recently been hypothesized [S. Dimmeler, and B. Brune, (1992) Eur. J. Biochem. 210, 305-310; J. S. Stamler (1994) Cell 78, 931-936]. An essential feature of this proposed mechanism is NADH nitrosation, for a nitroso-NADH adduct is considered to be a key reactant in the generation of pyridine nucleotide-modified protein. To evaluate at the molecular level the ability of NADH to act as a nitrosation substrate, the potential effects of NO., the nitrosothiols S-nitrosoglutathione and S-nitrosocysteine, the nitrosating agent tert-butyl-nitrite, and the NO. metabolite peroxynitrite on the molecular and functional (i.e., hydride-transfer) properties of NADH have been directly assessed at physiological pH. Exposure of NADH to NO. or nitrosothiol altered neither the hydride-transfer capability of the pyridine nucleotide nor its ultraviolet spectrum in ways suggestive of NADH nitrosation. As determined by NMR spectroscopy, NADH was refractory to the well-recognized nitrosating agent tert-butyl nitrite. Consequently, it appears that NADH is an unfavorable substrate for nitrosation under physiological conditions. These data are inconsistent with the proposal that NO. or a NO.-derived nitrosating agent interacts with NADH to generate the nitroso-NADH hypothesized to be essential to NO.-stimulated, pyridine nucleotide-dependent protein modification. Peroxynitrite, a possible source of nitrosating compounds, readily oxidized NADH to NAD, but demonstrated no potential to form a nitroso-NADH adduct. The facility with which NADH is oxidized to NAD has implications for peroxynitrite-mediated tissue damage.

Magnetic Resonance Spectroscopy↗

PubMed2006

Identification of rotenone-induced modifications in alpha-synuclein using affinity pull-down and tandem mass spectrometry.

Parkinson's disease is a movement disorder that results from a loss of dopaminergic neurons in the substantia nigra. The disease is characterized by mitochondrial dysfunction, oxidative stress, and the presence of "Lewy body" inclusions enriched with aggregated forms of alpha-synuclein, a presynaptic protein. Although alpha-synuclein is modified at various sites in Lewy bodies, it is unclear how sequence-specific posttranslational modifications modulate the aggregation of the protein in oxidatively stressed neurons. To begin to address this problem, we developed an affinity pull-down/mass spectrometry method to characterize the primary structure of histidine-tagged alpha-synuclein isolated from catecholaminergic neurons. Using this method, we mapped posttranslational modifications of alpha-synuclein from untreated neurons and neurons exposed to rotenone, an inhibitor of mitochondrial complex I. Various posttranslational modifications suggestive of oxidative damage or repair were identified in a region comprising a 20-residue stretch in the C-terminal part of the protein. The results indicate that alpha-synuclein is subject to discrete posttranslational modifications in neurons with impaired mitochondrial function. Our affinity pull-down/mass spectrometry method is a useful tool to examine how specific modifications of alpha-synuclein contribute to neurologic disorders such as Parkinson's disease.

Amino Acid Sequence↗

PubMed2004

Modification of the erythroid transcription factor GATA-1 by SUMO-1.

The activity of transcription factors is tightly modulated by posttranslational modifications affecting stability, localization, and protein-protein interactions. Conjugation to SUMO is a reversible posttranslational modification that has been shown to regulate important transcription factors involved in cell proliferation, differentiation, and tumor suppression. Here, we demonstrate that the erythroid transcription factor GATA-1 is sumoylated in vitro and in vivo and map the single lysine residue involved in SUMO-1 attachment. We show that the nuclear RING finger protein PIASy promotes sumoylation of GATA-1 and dramatically represses its transcriptional activity. We present evidence that a nonsumoylatable GATA-1 mutant is indistinguishable from the WT protein in its ability to transactivate a reporter gene in mammalian cells and in its ability to trigger endogenous globin expression in Xenopus explants. These observations open interesting questions about the biological role of this posttranslational modification of GATA-1.

Amino Acid Sequence↗

PubMed2004

Posttranslational mechanisms of peripheral sensitization.

The sensation of pain can be dramatically altered in response to injury or disease. This sensitization can occur at the level of the primary sensory neuron, and can be mediated by multiple biochemical mechanisms, including, but not limited to, changes in gene transcription, changes in translation, stability, or subcellular localization of translated proteins, and posttranslational modifications. This review focuses on posttranslational modifications to ion channels expressed in primary sensory neurons that form the machinery driving peripheral sensitization and pain hypersensitivity. Studies published to date show strong evidence for modulation of ion channels involved in transduction and transmission of nociceptive inputs coincident with biophysical and behavioral sensitization. The roles of phosphorylation and oxidation/reduction reactions of voltage-dependent sodium, potassium, and calcium channels are discussed, as well as phosphorylation-mediated modulation of sensory transduction channels.

Animals↗

PubMedDate not supplied

Redox-sensitive kinases of the nuclear factor-kappaB signaling pathway.

NF-kappaB is an inducible transcription factor that plays a role in the expression of over one hundred genes involved in immunity, inflammation, proliferation, and in defense against apoptosis. NF-kappaB has been known to be redox regulated for some time and is a direct target for oxidation that can affect its ability to bind to DNA. Reactive oxygen species (ROS) have been identified as second messengers in cells, and play a role in receptor signaling and posttranslation modification of signaling molecules. These posttranslation modifications include oxidations of critical cysteines to sulfenic acids or mixed disulfides, which can affect the activity of proteins. Many kinases involved in direct or indirect activation of NF-kappaB are affected by oxidants and therefore, have the potential to alter NF-kappaB activity. This review will provide a summary of the NF-kappaB family, their activation and regulation, followed by a summary of cytoplasmic and nuclear kinases in this pathway whose activity is affected by oxidants. Additionally, recent investigations have revealed that the JNK signaling pathway, which is known to be redox regulated, and pro-apoptotic, is inhibited by NF-kappaB signaling. The crosstalk of NF-kappaB with other signaling pathways is therefore critical for cellular fate, notably survival or cell death under oxidative conditions, and will also be reviewed.

Animals↗

PubMed1994

Alpha-A crystallin: quantitation of C-terminal modification during lens aging.

Previous studies have demonstrated that the C-terminal region of alpha-A crystallin is susceptible to age-dependent, posttranslational modification. To quantitate the amount of modification, alpha-A crystallin was purified from total proteins of the aging bovine lens, then digested with lys-C endoproteinase. Reverse phase, high pressure liquid chromatography was used to resolve and quantitate the resulting peptides, to determine the amount of C-terminal peptide relative to peptides from other regions of the protein that have not been reported to undergo modification. The results indicate that relative to alpha-A crystallin from newborn lens, posttranslational modification has occurred in approximately 45-55% of the C-terminal region from mature lens. These results demonstrate extensive modification of the C-terminal region of alpha-A crystallin from the mature lens, indicating that during the aging process, posttranslational modifications in this region may make significant contributions to the aggregated state and/or molecular chaperone properties of the molecule.

Aging↗

PubMed1998

Keratin modifications and solubility properties in epithelial cells and in vitro.

The gains that have been made in characterizing some of the keratin posttranslational modifications have helped answer some questions regarding these modifications and have generated an information base for asking additional refined questions in future studies. Highlights of where we believe we currently stand with regard to keratin posttranslational modifications are as follows: 1. Keratin glycosylation, via O-GlcNAc, is a dynamic modification that has been conclusively identified in K13, K8, and K18. Three serine glycosylation sites in the head domain of K18 have been identified, and it is possible that all keratins are glycosylated. The function of this modification remains to be defined, but is likely to be different from phosphorylation, since the two modifications are generally segregated on different molecules and several examples exist whereby both modifications increase simultaneously. 2. Keratin phosphorylation occurs within the tail and/or head domains of all keratins that have been examined. Several serine phosphorylation sites and some of the relevant kinases have been characterized in K8, K6, and K18, and serine/threonine sites have been identified in K1. Functions of keratin phosphorylation that have significant experimental support include a role in filament solubility and reorganization and a role in regulating keratin binding with other cytoplasmic proteins. The significance of filament reorganization and increased solubility under a variety of physiologic conditions such as mitosis and cell stress are important areas of future and ongoing investigation. Other associations with keratin phosphorylation include protection against cell stress, cell signaling, apoptosis, and cell compartment-specific roles. At this stage, however, it is not known if these associations play direct or indirect roles. 3. Keratin transglutamination occurs in epidermal and simple epithelial keratins under physiologic and pathologic states, respectively. In the physiological context, the role of this modification is clear in terms of providing a compact protective structure, while in the pathologic context of liver disease the role remains ambiguous. 4. Proteolysis of K18 and K19 by caspases occurs during apoptosis, and generates stable keratin fragments that are highly enriched within the cytoskeletal compartment. Proteolysis of the type II keratins appears to be spared for reasons that remain to be defined. It is likely that this apoptosis-associated degradation involves all type I keratins. Keratin fragments are also noted in sera of patients in association with a variety of epithelial tumors. If a signal does exist for the apoptosis-associated fragmentation, aside from caspase activation, then it appears that the overall increase in keratin phosphorylation during apoptosis does not account for this signal. 5. Keratins undergo several other posttranslational modifications including disulfide bond formation (not found in K8/18 due to lack of cystienes) and acetylation of their N-terminal serines. Modification by lipids is also possible, but this modification requires further confirmation. 6. Keratin solublility is highly dynamic and varies profoundly depending on the keratin pair and the physiologic state of the cell. Within the keratin family, simple epithelial keratins are among the most soluble (approximately 5% of K8/18 is soluble at basal conditions). Phosphorylation plays an important role in modulating keratin solubility, and distinct differences occur in site-specific phosphorylation depending on the soluble versus cytoskeletal partitioning of the keratin. Keratin solubility (at least for K8/18) also appears to be regulated by 14-3-3 proteins via K18 Ser33 phosphorylation.

Acetylation↗

PubMed2002

Shotgun identification of protein modifications from protein complexes and lens tissue.

Large-scale genomics has enabled proteomics by creating sequence infrastructures that can be used with mass spectrometry data to identify proteins. Although protein sequences can be deduced from nucleotide sequences, posttranslational modifications to proteins, in general, cannot. We describe a process for the analysis of posttranslational modifications that is simple, robust, general, and can be applied to complicated protein mixtures. A protein or protein mixture is digested by using three different enzymes: one that cleaves in a site-specific manner and two others that cleave nonspecifically. The mixture of peptides is separated by multidimensional liquid chromatography and analyzed by a tandem mass spectrometer. This approach has been applied to modification analyses of proteins in a simple protein mixture, Cdc2p protein complexes isolated through the use of an affinity tag, and lens tissue from a patient with congenital cataracts. Phosphorylation sites have been detected with known stoichiometry of as low as 10%. Eighteen sites of four different types of modification have been detected on three of the five proteins in a simple mixture, three of which were previously unreported. Three proteins from Cdc2p isolated complexes yielded eight sites containing three different types of modifications. In the lens tissue, 270 proteins were identified, and 11 different crystallins were found to contain a total of 73 sites of modification. Modifications identified in the crystallin proteins included Ser, Thr, and Tyr phosphorylation, Arg and Lys methylation, Lys acetylation, and Met, Tyr, and Trp oxidations. The method presented will be useful in discovering co- and posttranslational modifications of proteins.

Acetylation↗

PubMed1998

Serine-70 is one of the critical sites for drug-induced Bcl2 phosphorylation in cancer cells.

Taxoids and other microtubule-damaging drugs are known to induce Bcl2 phosphorylation at the G2-M phase of the cell cycle, with concomitant apoptosis in malignant cells derived from a variety of human malignancies, including leukemia, lymphoma, and breast and prostate cancer. We have investigated the ability of another antineoplastic drug, dolastatin 10, in inducing Bcl2 phosphorylation and apoptosis. We also investigated the effects of a phosphatase inhibitor okadaic acid in the regulation of Bcl2 phosphorylation, cell cycle arrest, and programmed cell death. Moreover, site-directed mutagenesis studies were performed to determine the specific serine residue(s) responsible for drug-induced Bcl2 phosphorylation. Our results indicate that these antimicrotubule agents or okadaic acid can induce posttranslational modification (phosphorylation) of Bcl2 protein at multiple serine residues. Interestingly, mutation of a serine residue at position 70 to alanine can significantly decrease drug-induced posttranslational modification (phosphorylation) of Bcl2 protein. Apparently, Ser70 seems to be a critical site for drug-induced posttranslational modification (phosphorylation) of the Bcl2 protein.

Alanine↗

PubMed2003

Proteomic profiling of epididymis and vas deferens: identification of proteins regulated during rat genital tract development.

Epididymis and vas deferens form part of the male internal genital tract and are dependent on androgens for their growth and development. To better understand the molecular action of androgens during male genital tract development, protein expression profiles were generated using two-dimensional gels, for rat epididymides and vasa deferentia isolated on embryonic days (E) 17-21. Proteins that were differentially expressed between E17 and E21 were cut from the gels, digested into tryptic peptides and analyzed on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer. Using this approach, 20 proteins could be identified that were regulated in time and were categorized into cytoskeletal proteins, nuclear proteins, transport proteins, chaperones, and enzymes (mainly glycolytic). Furthermore, epididymides and vasa deferentia isolated on E19 were cultured in vitro in the absence or presence of 10 nm of the synthetic androgen R1881, for 9, 24, and 48 h. Under these conditions, regulation and posttranslational modification were observed for glyceraldehyde 3-phosphate dehydrogenase, triosephosphate isomerase, heterogeneous nuclear ribonucleoprotein A2/B1 and heterogeneous nuclear ribonucleoprotein A3, similar to the observed changes in vivo. In addition, posttranslational modification of RhoGDI1 (also named RhoGDIalpha) was found in response to androgen. Androgen-induced posttranslational modification of RhoGDI1 and glycolytic enzymes may be an important functional link between signaling pathways and cytoskeletal rearrangements in control of growth and development of the male internal genital tract.

Androgens↗

PubMed1985

Chlamydomonas alpha-tubulin is posttranslationally modified by acetylation on the epsilon-amino group of a lysine.

Previous work has shown that the principal alpha-tubulin within Chlamydomonas reinhardtii flagellar axonemes differs from the major alpha-tubulin in the cell body. These two variants of alpha-tubulin are related to one another since posttranslational modification of the cell body form converts it to the axonemal form. When flagella are induced to assemble in the absence of de novo protein synthesis, tritiated acetate can be used to posttranslationally label alpha-tubulin in vivo, and under these conditions, no other flagellar polypeptides exhibit detectable labeling [L'Hernault, S. W., & Rosenbaum, J. L. (1983) J. Cell Biol. 97, 258-263]. In the present report, this labeling method has been used to provide material for chemical analysis of the tritiated moiety that is posttranslationally added to flagellar alpha-tubulin. This radioactivity was volatile after acid hydrolysis, suggesting that the posttranslational modification is the addition of neither an amino acid nor carbohydrate. Treatment of posttranslationally 3H-labeled alpha-tubulin with hydrazine yields radioactive acetylhydrazine, indicating that the moiety involved in posttranslational modification is an acetyl group. Analysis of complete proteolytic digests by thin-layer chromatography has revealed that this acetyl group is located on the epsilon-amino group of a flagellar alpha-tubulin lysine residue.

Acetylation↗