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Acute myeloid leukemia (AML) is an aggressive blood cancer with a poor prognosis. We report a comprehensive proteogenomic analysis of bone marrow biopsies from 252 uniformly treated AML patients to elucidate the molecular pathophysiology of AML in order to inform future diagnostic and therapeutic approaches. In addition to in-depth quantitative proteomics, our analysis includes cytogenetic profiling and DNA/RNA sequencing. We identify five proteomic AML subtypes, each reflecting specific biological features spanning genomic boundaries. Two of these proteomic subtypes correlate with patient outcome, but none is exclusively associated with specific genomic aberrations. Remarkably, one subtype (Mito-AML), which is captured only in the proteome, is characterized by high expression of mitochondrial proteins and confers poor outcome, with reduced remission rate and shorter overall survival on treatment with intensive induction chemotherapy. Functional analyses reveal that Mito-AML is metabolically wired toward stronger complex I-dependent respiration and is more responsive to treatment with the BCL2 inhibitor venetoclax., Competing Interests: Declaration of interests T.O. received research funding from Gilead (related to this work) and Merck KGaA (not related to this work). T.O. is a consultant for Roche and Merck KGaA (both not related to this work). K.S. receives grant funding as part of the DFCI/Novartis Drug Discovery Program, consults for and has stock options in Auron Therapeutics, and has consulted for Kronos Bio and AstraZeneca on topics not directly related to this manuscript. F.C. is a co-founder of enGene Statistics GmbH. The Max Planck institute and the Goethe University Frankfurt are filing a patent application, on which T.O., A.K.J., S.Wolf, F.B., H.S., M.M., and H.U. are listed as inventors., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Aortic valve stenosis (AVS) is one of the most common valve diseases in the world. However, detailed biological understanding of the myocardial changes in AVS hearts on the proteome level is still lacking. Proteomic studies using high-resolution mass spectrometry of formalin-fixed and paraffin-embedded (FFPE) human myocardial tissue of AVS-patients are very rare due to methodical issues. To overcome these issues this study used high resolution mass spectrometry in combination with a stem cell-derived cardiac specific protein quantification-standard to profile the proteomes of 17 atrial and 29 left ventricular myocardial FFPE human myocardial tissue samples from AVS-patients. In our proteomic analysis we quantified a median of 1980 (range 1495-2281) proteins in every single sample and identified significant upregulation of 239 proteins in atrial and 54 proteins in ventricular myocardium. We compared the proteins with published data. Well studied proteins reflect disease-related changes in AVS, such as cardiac hypertrophy, development of fibrosis, impairment of mitochondria and downregulated blood supply. In summary, we provide both a workflow for quantitative proteomics of human FFPE heart tissue and a comprehensive proteomic resource for AVS induced changes in the human myocardium., (© 2021. The Author(s).)

Background: Yeasts of the CTG-clade lineage, which includes the human-infecting Candida albicans, Candida parapsilosis and Candida tropicalis species, are characterized by an altered genetic code. Instead of translating CUG codons as leucine, as happens in most eukaryotes, these yeasts, whose ancestors are thought to have lost the relevant leucine-tRNA gene, translate CUG codons as serine using a serine-tRNA with a mutated anticodon, [Formula: see text]. Previously reported experiments have suggested that 3-5% of the CTG-clade CUG codons are mistranslated as leucine due to mischarging of the [Formula: see text]. The mistranslation was suggested to result in variable surface proteins explaining fast host adaptation and pathogenicity., Results: In this study, we reassess this potential mistranslation by high-resolution mass spectrometry-based proteogenomics of multiple CTG-clade yeasts, including various C. albicans strains, isolated from colonized and from infected human body sites, and C. albicans grown in yeast and hyphal forms. Our data do not support a bias towards CUG codon mistranslation as leucine. Instead, our data suggest that (i) CUG codons are mistranslated at a frequency corresponding to the normal extent of ribosomal mistranslation with no preference for specific amino acids, (ii) CUG codons are as unambiguous (or ambiguous) as the related CUU leucine and UCC serine codons, (iii) tRNA anticodon loop variation across the CTG-clade yeasts does not result in any difference of the mistranslation level, and (iv) CUG codon unambiguity is independent of C. albicans' strain pathogenicity or growth form., Conclusions: Our findings imply that C. albicans does not decode CUG ambiguously. This suggests that the proposed misleucylation of the [Formula: see text] might be as prevalent as every other misacylation or mistranslation event and, if at all, be just one of many reasons causing phenotypic diversity., (© 2021. The Author(s).)

Macrophage migration inhibitory factor (MIF) and its homologue D-dopachrome tautomerase (D-DT) are widely expressed pro-inflammatory cytokines with chemokine-like functions that coordinate a wide spectrum of biological activities, such as migration. Here, we biotin-tagged intracellular MIF/D-DT in vivo to identify important cytosolic interactors and found a plethora of actin cytoskeleton-associated proteins. Although the receptor complex between CD74 and CD44 (CD74/CD44) is essential for signalling transduction in fibroblasts via extracellular MIF/D-DT, our interactome data suggested direct effects. We, thus, investigated whether MIF/D-DT can modulate cell migration independently of CD74/CD44. To distinguish between receptor- and non-receptor-mediated motility, we used fibroblasts that are either deficient or that express CD74/CD44 proteins, and treated them with recombinant MIF/D-DT. Interestingly, only MIF could stimulate chemokinesis in the presence or absence of CD74/CD44. The pro-migratory effects of MIF depended on lipid raft/caveolae-mediated but not clathrin-mediated endocytosis, on its tautomerase activity and, probably, on its thiol protein oxidoreductase activity. As MIF treatment restrained actin polymerisation in vitro , our findings establish a new intracellular role for MIF/D-DT in driving cell motility through modulation of the actin cytoskeleton., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Hypoxia occurs in pathological conditions, such as cancer, as a result of the imbalance between oxygen supply and consumption by proliferating cells. HIFs are critical molecular mediators of the physiological response to hypoxia but also regulate multiple steps of carcinogenesis including tumor progression and metastasis. Recent data support that sumoylation, the covalent attachment of the Small Ubiquitin-related MOdifier (SUMO) to proteins, is involved in the activation of the hypoxic response and the ensuing signaling cascade. To gain insights into differences of the SUMO1 and SUMO2/3 proteome of HeLa cells under normoxia and cells grown for 48 h under hypoxic conditions, we employed endogenous SUMO-immunoprecipitation in combination with quantitative mass spectrometry (SILAC). The group of proteins whose abundance was increased both in the total proteome and in the SUMO IPs from hypoxic conditions was enriched in enzymes linked to the hypoxic response. In contrast, proteins whose SUMOylation status changed without concomitant change in abundance were predominantly transcriptions factors or transcription regulators. Particularly interesting was transcription factor TFAP2A (Activating enhancer binding Protein 2 alpha), whose sumoylation decreased on hypoxia. TFAP2A is known to interact with HIF-1 and we provide evidence that deSUMOylation of TFAP2A enhances the transcriptional activity of HIF-1 under hypoxic conditions. Overall, these results support the notion that SUMO-regulated signaling pathways contribute at many distinct levels to the cellular response to low oxygen., (© 2019 Chachami et al.)

Post-transcriptional gene regulation controls the amount of protein produced from a specific mRNA by altering both its decay and translation rates. Such regulation is primarily achieved by the interaction of trans-acting factors with cis-regulatory elements in the untranslated regions (UTRs) of mRNAs. These interactions are guided either by sequence- or structure-based recognition. Similar to sequence conservation, the evolutionary conservation of a UTR's structure thus reflects its functional importance. We used such structural conservation to identify previously unknown cis-regulatory elements. Using the RNA folding program Dynalign, we scanned all UTRs of humans and mice for conserved structures. Characterizing a subset of putative conserved structures revealed a binding site of the RNA-binding protein Roquin. Detailed functional characterization in vivo enabled us to redefine the binding preferences of Roquin and identify new target genes. Many of these new targets are unrelated to the established role of Roquin in inflammation and immune responses and thus highlight additional, unstudied cellular functions of this important repressor. Moreover, the expression of several Roquin targets is highly cell-type-specific. In consequence, these targets are difficult to detect using methods dependent on mRNA abundance, yet easily detectable with our unbiased strategy.

Patients with head-and-neck cancer can develop both lung metastasis and primary lung cancer during the course of their disease. Despite the clinical importance of discrimination, reliable diagnostic biomarkers are still lacking. Here, we have characterised a cohort of squamous cell lung (SQCLC) and head-and-neck (HNSCC) carcinomas by quantitative proteomics. In a training cohort, we quantified 4,957 proteins in 44 SQCLC and 30 HNSCC tumours. A total of 518 proteins were found to be differentially expressed between SQCLC and HNSCC, and some of these were identified as genetic dependencies in either of the two tumour types. Using supervised machine learning, we inferred a proteomic signature for the classification of squamous cell carcinomas as either SQCLC or HNSCC, with diagnostic accuracies of 90.5% and 86.8% in cross- and independent validations, respectively. Furthermore, application of this signature to a cohort of pulmonary squamous cell carcinomas of unknown origin leads to a significant prognostic separation. This study not only provides a diagnostic proteomic signature for classification of secondary lung tumours in HNSCC patients, but also represents a proteomic resource for HNSCC and SQCLC., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

Although the "universal" genetic code is now known not to be universal, and stop codons can have multiple meanings, one regularity remains, namely that for a given sense codon there is a unique translation. Examining CUG usage in yeasts that have transferred CUG away from leucine, we here report the first example of dual coding: Ascoidea asiatica stochastically encodes CUG as both serine and leucine in approximately equal proportions. This is deleterious, as evidenced by CUG codons being rare, never at conserved serine or leucine residues, and predominantly in lowly expressed genes. Related yeasts solve the problem by loss of function of one of the two tRNAs. This dual coding is consistent with the tRNA-loss-driven codon reassignment hypothesis, and provides a unique example of a proteome that cannot be deterministically predicted. VIDEO ABSTRACT., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

During microRNA (miRNA) biogenesis, two endonucleolytic reactions convert stem-loop-structured precursors into mature miRNAs. These processing steps can be posttranscriptionally regulated by RNA-binding proteins (RBPs). Here, we have used a proteomics-based pull-down approach to map and characterize the interactome of a multitude of pre-miRNAs. We identify ∼180 RBPs that interact specifically with distinct pre-miRNAs. For functional validation, we combined RNAi and CRISPR/Cas-mediated knockout experiments to analyze RBP-dependent changes in miRNA levels. Indeed, a large number of the investigated candidates, including splicing factors and other mRNA processing proteins, have effects on miRNA processing. As an example, we show that TRIM71/LIN41 is a potent regulator of miR-29a processing and its inactivation directly affects miR-29a targets. We provide an extended database of RBPs that interact with pre-miRNAs in extracts of different cell types, highlighting a widespread layer of co- and posttranscriptional regulation of miRNA biogenesis., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The genetic code is the cellular translation table for the conversion of nucleotide sequences into amino acid sequences. Changes to the meaning of sense codons would introduce errors into almost every translated message and are expected to be highly detrimental. However, reassignment of single or multiple codons in mitochondria and nuclear genomes, although extremely rare, demonstrates that the code can evolve. Several models for the mechanism of alteration of nuclear genetic codes have been proposed (including "codon capture," "genome streamlining," and "ambiguous intermediate" theories), but with little resolution. Here, we report a novel sense codon reassignment in Pachysolen tannophilus, a yeast related to the Pichiaceae. By generating proteomics data and using tRNA sequence comparisons, we show that Pachysolen translates CUG codons as alanine and not as the more usual leucine. The Pachysolen tRNACAG is an anticodon-mutated tRNA(Ala) containing all major alanine tRNA recognition sites. The polyphyly of the CUG-decoding tRNAs in yeasts is best explained by a tRNA loss driven codon reassignment mechanism. Loss of the CUG-tRNA in the ancient yeast is followed by gradual decrease of respective codons and subsequent codon capture by tRNAs whose anticodon is not part of the aminoacyl-tRNA synthetase recognition region. Our hypothesis applies to all nuclear genetic code alterations and provides several testable predictions. We anticipate more codon reassignments to be uncovered in existing and upcoming genome projects., (© 2016 Mühlhausen et al.; Published by Cold Spring Harbor Laboratory Press.)

Claudins are integral transmembrane components of the tight junctions forming trans-epithelial barriers in many organs, such as the nervous system, lung, and epidermis. In Drosophila three claudins have been identified that are required for forming the tight junctions analogous structure, the septate junctions (SJs). The lack of claudins results in a disruption of SJ integrity leading to a breakdown of the trans-epithelial barrier and to disturbed epithelial morphogenesis. However, little is known about claudin partners for transport mechanisms and membrane organization. Here we present a comprehensive analysis of the claudin proteome in Drosophila by combining biochemical and physiological approaches. Using specific antibodies against the claudin Megatrachea for immunoprecipitation and mass spectrometry, we identified 142 proteins associated with Megatrachea in embryos. The Megatrachea interacting proteins were analyzed in vivo by tissue-specific knockdown of the corresponding genes using RNA interference. We identified known and novel putative SJ components, such as the gene product of CG3921. Furthermore, our data suggest that the control of secretion processes specific to SJs and dependent on Sec61p may involve Megatrachea interaction with Sec61 subunits. Also, our findings suggest that clathrin-coated vesicles may regulate Megatrachea turnover at the plasma membrane similar to human claudins. As claudins are conserved both in structure and function, our findings offer novel candidate proteins involved in the claudin interactome of vertebrates and invertebrates.

Photoswitchable fluorescent proteins have enabled new approaches for imaging cells, but their utility has been limited either because they cannot be switched repeatedly or because the wavelengths for switching and fluorescence imaging are strictly coupled. We report a bright, monomeric, reversibly photoswitchable variant of GFP, Dreiklang, whose fluorescence excitation spectrum is decoupled from that for optical switching. Reversible on-and-off switching in living cells is accomplished at illumination wavelengths of ∼365 nm and ∼405 nm, respectively, whereas fluorescence is elicited at ∼515 nm. Mass spectrometry and high-resolution crystallographic analysis of the same protein crystal in the photoswitched on- and off-states demonstrate that switching is based on a reversible hydration/dehydration reaction that modifies the chromophore. The switching properties of Dreiklang enable far-field fluorescence nanoscopy in living mammalian cells using both a coordinate-targeted and a stochastic single molecule switching approach.

This unit provides a robust, reliable, and easy-to-use kit-based method for extraction of intact, non-degraded proteins from formalin-fixed, paraffin-embedded (FFPE) tissue, and their subsequent use for analysis by liquid chromatography/mass spectrometry (LC/MS). After deparaffinization, proteins are extracted from unstained sections of FFPE rat liver tissue. After a simple cleanup step using organic extraction, the sample is transferred into a buffer optimized for trypsin digestion of the extracted proteins. Subsequently, LC/MS is used to identify the proteins that gave rise to the tryptic peptides. Comparing formalin-fixed and frozen tissues, good correlation is observed in the mass spectrometric pattern attributable to the tryptic peptides and number of identified proteins. Since FFPE tissues are generally available in clinical practice, this method can be used to analyze biomarkers in different pathological situations (e.g., healthy vs. diseased). The method can also be used for protein extraction from fresh-frozen tissue., ((c) 2010 by John Wiley & Sons, Inc.)

Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) are members of the MAPK family and participate in the transduction of stimuli in cellular responses. Their long-term actions are accomplished by promoting the expression of specific genes whereas faster responses are achieved by direct phosphorylation of downstream effectors located throughout the cell. In this study we determined that hERK1 translocates to the mitochondria of HeLa cells upon a proliferative stimulus. In the mitochondrial environment, hERK1 physically associates with (i) at least 5 mitochondrial proteins with functions related to transport (i.e. VDAC1), signalling, and metabolism; (ii) histones H2A and H4; and (iii) other cytosolic proteins. This work indicates for the first time the presence of diverse ERK-complexes in mitochondria and thus provides a new perspective for assessing the functions of ERK1 in the regulation of cellular signalling and trafficking in HeLa cells.

UV crosslinking is an appropriate method to identify proteins that directly contact nucleic acid, e.g., RNA. In combination with modern mass spectrometric (MS) analysis such an approach provides the opportunity to reveal not only the nature of the crosslinked proteins but also to identify the actual crosslinking sites between the protein and the nucleic acid. However, the relatively low yield in UV-induced crosslinking makes it difficult to identify in particular those species by MS that represent peptide-nucleic acid conjugates, as the great excess of noncrosslinked material interferes with their detection in MS. Here, we present an automated enrichment strategy of crosslinked peptide-RNA oligonucleotides derived from crude mixtures of UV-irradiated ribonucleoprotein (RNP) particles that uses TiO(2) columns integrated within a two-dimensional (2D) nanoliquid chromatography (LC) system. The setup combines two C18 precolumns, a TiO(2) enrichment column and a nanoanalytical column. It allows the removal of the noncrosslinked RNA and protein moiety and the specific enrichment of crosslinked peptide-RNA conjugates so that UV-irradiated and subsequently completely hydrolyzed RNP complexes can directly be loaded and analyzed by MS. In this feasibility study, we demonstrate the specific enrichment of peptide-RNA oligonucleotides derived from UV-irradiated native spliceosomal U1 snRNPs and spliceosomal [15.5K-61K-U4atac snRNA] complex reconstituted in vitro., (2008 Wiley Periodicals, Inc)

A new atmospheric pressure (AP)-MALDI-type interface has been developed based on a free liquid (FL) microbeam/microdroplets and a mid-infrared optical parametric oscillator (mid-IR OPO). The device is integrated into a standard on-line nanoESI interface. The generation of molecular ions in the gas phase is believed to be the result of a fast (explosive) laser-induced evaporative dispersion(not desorption) of the microbeam into statistically charged nanodroplets. Only the lowest charge states appear insignificant abundance in this type of experiment. Mass spectra of some common peptides have been acquired in positive ion mode, and the limit-of-detection of this first prototype (liquid microbeam setup) was evaluated to be 17 fmol per second. To improve the duty cycle and to reduce the sample consumption, a droplet-on-demand system was implemented (generating 100 pL droplets).With this setup, about 20 attomole of bradykinin were sufficient to achieve a signal-to-noise ratio better than five.This setup can be operated at flow rates down to 100 nL/min and represents a liquid MALDI alternative to the nanoESI. Our particular interest was the application of the developed ion source for on-line coupling of liquid chromatography with mass spectrometry. The flow rates(>100 microL/min), required for stable operation of the ion source in continuous liquid microbeam mode, matches perfectly the flow rate range of micro HPLC. Therefore, online LC/MS experiments have been realized, employing a microbore C18 reversed-phase column to separate an artificial peptide mixture and tryptic peptides of bovine serum albumin (performing a peptide mass fingerprint). In the latter case, sequence coverage of more than 90%has been achieved.

RNA editing in flowering plant mitochondria is investigated by in vitro assays. These cauliflower mitochondrial lysates require added NTP or dNTP. We have now resolved the reason for this requirement to be the inhibition of the RNA binding activity of the glutamate dehydrogenases (GDH). Both GDH1 and GDH2 were identified in RNA-protein cross-links. The inhibition of in vitro RNA editing by GDH is confirmed by the ability of the GDH-specific herbicide phosphinothricin to substitute for NTP. NADH and NADPH, but not NAD or NADP, can also replace NTP, suggesting that the NAD(P)H-binding-pocket configuration of the GDH contacts the RNA. RNA editing in plant mitochondria is thus intrinsically independent of added energy in the form of NTP.

The posttranslational modifications of alpha-tubulin of Toxoplasma gondii were characterized by antibodies and biochemical analysis of the carboxy-terminal peptide. Alpha-Tubulin is acetylated and glutamylated. Side chains with up to three glutamate residues are linked to Glu445 of T. gondii alpha-tubulin. The data suggest that the site of glutamylation on alpha-tubulin is conserved over a broad range of species.

Tubulin-tyrosine ligase (TTL, EC 6.3.2.25) from porcine brain, which catalyses the readdition of tyrosine to the C-terminus of detyrosinated alpha-tubulin, was cloned and expressed in Escherichia coli as a glutathione S-transferase-fusion protein. Upon cleavage of the immobilised fusion protein, an electrophoretically homogeneous enzyme was obtained. Recombinant TTL, which exhibited similar catalytic properties as the mammalian enzyme purified from brain tissue, was capable of using nitrotyrosine as an alternative substrate in vitro. Incorporation of tyrosine into tubulin was competitively inhibited by nitrotyrosine with an apparent K(i) of 0.24 mM. The TTL-catalysed incorporation of nitrotyrosine as sole substrate into alpha-tubulin was clearly detectable at concentrations of 10 microM by immunological methods using nitrotyrosine specific antibodies. However, in competition with tyrosine 20-fold higher concentrations of nitrotyrosine were necessary before its incorporation became evident. Analysis of the C-terminal peptides of in vitro modified alpha-tubulin by MALDI-MS confirmed the covalent incorporation of nitrotyrosine into tubulin by TTL. In contrast to the C-terminal tyrosine, pancreatic carboxypeptidase A was incapable of cleaving nitrotyrosine from the modified alpha-tubulin.

The minimal sequence requirement of Crithidia tubulin polyglutamylase is already fulfilled by tubulin-related peptides carrying a free alpha-carboxylate on a glutamic acid residue. Since the product of each glutamylation step fulfills the substrate requirements necessary for the next cycle, very long side chains are generated with brain tubulin as a substrate. Up to 70 mol of glutamic acid was incorporated per alphabeta-heterodimer. We speculate that the strict choice of a particular glutamate residue for the formation of the isopeptide bond initiating a novel side chain is made by a tubulin monoglutamylase which requires the entire tubulin as substrate.

Reverse-transcriptase polymerase chain reaction cloning of the 3'-ends of the alpha-tubulin cDNAs of Tritrichomonas mobilensis in combination with Southern-blot analysis identified seven to eight distinct alpha-tubulin genes. All seven lack a carboxy-terminal tyrosine and the corresponding sequence compatible with posttranslational tyrosination. This indicates that whereas tyrosination of alpha-tubulin has been found in most species, including humans and trypanosomes, it is absent in trichomonads.

Tubulin of Giardia lamblia, a representative of the oldest eukaryotes, was screened for posttranslational modifications. Mass spectrometry of the carboxy-terminal peptides documents a large number of variants. Both alpha- and beta-tubulin show polyglycylation with up to 20 and 15 extra glycyl residues respectively. Minor variants show a low level of glutamylation without or with glycylation. The glutamylation-specific antibody GT335 detects alpha- and beta-tubulin in immunoblots. The terminal tyrosine is fully retained in alpha-tubulin, which is completely acetylated at Lys-40. Thus except for the detyrosination/tyrosination cycle all posttranslational modifications known for higher eukaryotes are already present in Giardia.

Extraction of demembranated bull sperm flagella by SDS was used to maximize tubulin solubilization. The alpha- and beta-tubulin separated by SDS-PAGE were treated with endoproteinases LysC and AspN, respectively. Carboxy-terminal fragments were isolated by Mono Q chromatography and reversed-phase HPLC. Automated sequencing and mass spectrometry revealed an astonishingly high number of tubulin variants. Many variants were due to polyglutamylation and in particular to polyglycylation. The number of side-chain glycyl residues ranged from 0 to 28 in alpha and 0 to 15 in beta. Corresponding values for side-chain glutamyl residues were 0-6 in alpha and 0-3 in beta. Additional alpha variability was based on carboxy-terminal detyrosination and partial loss of the penultimate glutamate. A major glycylation site in alpha- and beta-tubulin was mapped. Some variants seem to display both glycyl and glutamyl side chains.

Basal apparatuses consisting of two basal bodies and several attached fibers were isolated from the naked green flagellate Spermatozopsis similis by detergent extraction and mechanical disintegration. Sucrose density centrifugation yielded highly enriched basal apparatuses as shown by electron microscopy. SDS-PAGE revealed the absence of histones, indicating the removal of nuclear contaminations from the isolated basal apparatuses. A mass spectrometric analysis of the carboxyterminal peptides of alpha tubulin documented detyrosination and glutamylation as posttranslational modifications and showed that some 5% of the alpha tubulin carries a polyglutamyl side chain which can reach at least 17 residues in length. Monoclonal antibodies raised against the purified basal apparatuses were used to characterize novel components in the basal apparatus. A 210-kD component identified by mAB BAS (basal apparatus of Spermatozopsis) 1.4 was localized in the flagellar transitional region by immunogold electron microscopy. Antibody BAS 16.4 reacted with two high molecular weight bands (approximately 265 and 240 kD) in Western blotting and decorated a fiber attached to the proximal end of the basal bodies. Immunofluorescence staining of isolated cytoskeletons with these mABs demonstrated that the antigens are also present in the basal apparatuses of Chlamydomonas reinhardtii and Dunahella bioculata. These antibodies are useful tools for the molecular cloning of components from the basal apparatus.

We have searched for post-translational modifications in tubulin of the diplomonad Giardia lamblia, which is a representative of the earliest branches in eukaryotic evolution. The carboxyterminal peptide of alpha-tubulin was isolated and characterized by automated sequencing and mass spectrometry. Some 60% of the peptide is unmodified, while the remainder shows various degrees of polyglycylation. The number of glycyl residues in the lateral side chain ranges from 2 to 23. All peptide species encountered end with alanine-tyrosine, indicating the absence of a detyrosination/tyrosination cycle. We conclude that tubulin-specific polyglycylation could be as old as tubulin and axonemal structures.

The alpha beta-tubulin heterodimer, the structural unit of microtubules, comes in many variants. There are different alpha and beta isotypes encoded by multigene families. Additional heterogeneity is generated by a set of posttranslational modifications. Detyrosination of alpha-tubulin, removal of the carboxy-terminal Glu-Tyr dipeptide of alpha-tubulin, phosphorylation of some tubulins, polyglutamylation, and polyglycylation of alpha- and beta-tubulins all involve the acidic carboxy-terminal region. We have investigated the distribution of tubulin variants in the axonemal microtubules of sea urchin sperm flagella by immunological procedures and by direct sequence and mass spectrometric analysis of the carboxy-terminal peptides. The A and B tubules that comprise the nine outer doublets differ strongly in tubulin variants. A tubules contain over 95% unmodified, tyrosinated alpha beta-tubulin. In B tubules, alpha-tubulin is approximately 65% detyrosinated and both alpha- and beta-tubulin are 40-45% polyglycylated. These results show a segregation of tubulin variants between two different axonemal structures and raise the possibility that posttranslational modifications of tubulins reflect or specify structurally and functionally distinct microtubules.

We report a method for isolating homogeneous myomesin from mammalian skeletal muscle. The identity of the purified bovine protein was confirmed by its reactivity with myomesin-specific monoclonal antibodies and with polyclonal antibodies raised against peptides derived from the amino-terminal and carboxy-terminal ends of the sequence predicted by the human myomesin cDNA. All partial sequences obtained from bovine myomesin can be aligned along the human sequence predicted by its cloned cDNA. Electron microscopy of myomesin revealed short flexible rods with a molecular length of about 50 nm. Circular dichroism spectra showed a high degree of beta structure as expected for a member of the immunoglobulin superfamily of proteins. Alignment of the sequences of the class I and II domains of myomesin with the sequences of domains of known three-dimensional structure provides a more detailed model of myomesin. In agreement with this view, the cleavage sites observed by limited proteolysis locate primarily between individual domains. In a solid-phase overlay assay myomesin specifically bound to the myosin rod and to light meromyosin (LMM), but not to the carboxy-terminal 30-kDa fragment of LMM. The myosin-binding site seemed to be confined to the amino-terminal 240 residues of the molecule. The cross-reactivity of myomesin with the phosphorylation-dependent monoclonal neurofilament antibody NE14 [Shaw, G.E., Debus, E. & Weber, K. (1984) Eur. J. Cell Biol. 34, 130-136] was analyzed. NE14 reactivity of myomesin was abolished by alkaline phosphatase. Reactivity of the antibody on stable proteolytic fragments of myomesin showed that the phosphorylation site must reside within the carboxy-terminal 60 residues.

Carboxy-terminal fragments of alpha and beta tubulin from bull sperm were isolated and characterized by automated sequencing and mass spectrometry. About 60% of sperm alpha tubulin is polyglycylated. The lateral chain, which can reach 13 residues in length, is covalently attached via an isopeptide bond. The fully detyrosinated sperm alpha tubulin lacks polyglycylation. Thus mammalian sperm microtubules differ from the ciliary axonemal microtubules of the protozoan Paramecium for which others have documented a complete polyglycylation of both alpha and beta tubulin.

The protein auxilin is a coat component of brain clathrin-coated vesicles. It interacts directly with the heavy chain of clathrin and supports its assembly into regular cages [Ahle, S. & Ungewickell, E. (1990) J. Cell Biol. 111, 19-29]. The combined open reading frames of three cow brain cDNA clones with a total of 4531 nucleotides predict a molecular mass of 99,504 Da for auxilin. The coding region is followed by a very long untranslated region of at least 1670 nucleotides. By Northern analysis, auxilin transcripts are found only in brain tissue. Auxilin is not related to any of the previously sequenced clathrin-binding proteins, but the region of positions 50-350 is 29% identical (similarity 56%) to the corresponding region of the actin-binding protein tensin from chicken fibroblasts. Recombinant auxilin expressed in and purified from bacteria by affinity chromatography is functional with respect to clathrin binding.

A method for the purification of the Golgi adaptor protein 1 from bovine adrenal gland tissue was devised to investigate the relationship of its beta 1 (formerly referred to as beta') subunit to known beta-type sequences. Adrenal gland tissue was chosen for this study because it yielded 2-3 times more adaptor protein 1 than a comparable preparation from bovine brain. Like its neuronal isoform, the beta 1 subunit from adrenal gland adaptor protein 1 is readily cleaved by trypsin into a 63-kDa N-terminal fragment and a 40-kDa C-terminal fragment, while the gamma subunit is largely refractory to digestion. Based on microsequencing of 167 residues from the 63-kDa fragment, we noted 11 differences to the corresponding region of the beta 2 (formerly beta) subunit of the plasma membrane adaptor protein 2, but only one difference to the corresponding region of a beta-type protein encoded by the rat cDNA clone AP105a which is supposed to be a variant of the beta 2 subunit of the plasma membrane adaptor protein 2 [Kirchhausen, T., Nathanson, K. L., Matsui, W., Vaisberg, A., Chow, E. P., Burne, C., Keen, J. H. & Davis, A. E. (1989) Proc. Natl Acad. Sci. USA 84, 8805-8809]. Alignment of 187 residues from the 40-kDa beta 1 C-terminal fragment revealed differences in 77 positions to the corresponding region of the beta 2 subunit and differences in 23 positions compared to the supposed beta 2-like protein. These findings suggest that the protein encoded by the rat cDNA clone AP105a is more closely related to the beta 1 subunit of the bovine adrenal Golgi adaptor protein 1 than to the beta 2 subunit of the rat plasma membrane adaptor protein 2.

Recombinant vimentin expressed in E. coli JM 101 cells is cleaved after cell lysis between arginines 11 and 12. The truncated vimentin is assembly incompetent. Expression of the same cDNA construct in BL21 cells, which lack the protease ompT, provides intact and polymerization-competent vimentin. The ompT cleavage site is contained in a short sequence motif (YRRMF) shared by the head domains of type III and IV intermediate filament (IF) proteins. We propose that a related motif present in the N-terminal 32 residues of lambda CII accounts for the known IF formation of a fusion protein formed with a truncated GFAP.

Lamin B purified from murine EAT cells was characterized by partial protein sequences. Contrary to the current view that mammals express only a single lamin B polypeptide corresponding to a characterized murine cDNA clone, our analysis documents two distinct B lamins. One protein follows the established cDNA sequence while the other identifies a novel murine lamin B. Comparison with the two chicken lamin B sequences established by cDNA cloning identifies the first murine lamin B sequence as a B1 type and the second as a B2 type. We conclude that mammals express two distinct lamin B forms as established by others for chicken.