scholarly journals A Protein Methylation Pathway in Chlamydomonas Flagella Is Active during Flagellar Resorption

2008 ◽  
Vol 19 (10) ◽  
pp. 4319-4327 ◽  
Author(s):  
Mark J. Schneider ◽  
Megan Ulland ◽  
Roger D. Sloboda
Keyword(s):  
Unit Length ◽  
Protein Composition ◽  
Intraflagellar Transport ◽  
Immunoblot Analysis ◽  
Full Length ◽  
Protein Methylation ◽  
Independent Form ◽  
Arginine Residues ◽  
Flagellar Proteins ◽  
Punctate Pattern

During intraflagellar transport (IFT), the regulation of motor proteins, the loading and unloading of cargo and the turnover of flagellar proteins all occur at the flagellar tip. To begin an analysis of the protein composition of the flagellar tip, we used difference gel electrophoresis to compare long versus short (i.e., regenerating) flagella. The concentration of tip proteins should be higher relative to that of tubulin (which is constant per unit length of the flagellum) in short compared with long flagella. One protein we have identified is the cobalamin-independent form of methionine synthase (MetE). Antibodies to MetE label flagella in a punctate pattern reminiscent of IFT particle staining, and immunoblot analysis reveals that the amount of MetE in flagella is low in full-length flagella, increased in regenerating flagella, and highest in resorbing flagella. Four methylated proteins have been identified in resorbing flagella, using antibodies specific for asymmetrically dimethylated arginine residues. These proteins are found almost exclusively in the axonemal fraction, and the methylated forms of these proteins are essentially absent in full-length and regenerating flagella. Because most cells resorb cilia/flagella before cell division, these data indicate a link between flagellar protein methylation and progression through the cell cycle.

scholarly journals Protein arginine methyltransferases interact with intraflagellar transport particles and change location during flagellar growth and resorption

2017 ◽  
Vol 28 (9) ◽  
pp. 1208-1222 ◽  
Author(s):  
Katsutoshi Mizuno ◽  
Roger D. Sloboda
Keyword(s):  
Posttranslational Modifications ◽  
Intraflagellar Transport ◽  
Temperature Sensitive ◽  
Protein Arginine Methyltransferases ◽  
Cellular Processes ◽  
Arginine Residues ◽  
Flagellar Assembly ◽  
Flagellar Regeneration ◽  
Flagellar Proteins ◽  
Punctate Pattern

Changes in protein by posttranslational modifications comprise an important mechanism for the control of many cellular processes. Several flagellar proteins are methylated on arginine residues during flagellar resorption; however, the function is not understood. To learn more about the role of protein methylation during flagellar dynamics, we focused on protein arginine methyltransferases (PRMTs) 1, 3, 5, and 10. These PRMTs localize to the tip of flagella and in a punctate pattern along the length, very similar, but not identical, to that of intraflagellar transport (IFT) components. In addition, we found that PRMT 1 and 3 are also highly enriched at the base of the flagella, and the basal localization of these PRMTs changes during flagellar regeneration and resorption. Proteins with methyl arginine residues are also enriched at the tip and base of flagella, and their localization also changes during flagellar assembly and disassembly. PRMTs are lost from the flagella of fla10-1 cells, which carry a temperature-sensitive mutation in the anterograde motor for IFT. The data define the distribution of specific PRMTs and their target proteins in flagella and demonstrate that PRMTs are cargo for translocation within flagella by the process of IFT.

Review of Progress in Predicting Protein Methylation Sites

2019 ◽  
Vol 23 (15) ◽  
pp. 1663-1670 ◽  
Author(s):  
Chunyan Ao ◽  
Shunshan Jin ◽  
Yuan Lin ◽  
Quan Zou
Keyword(s):  
Gene Expression ◽  
Signal Transduction ◽  
Transcriptional Activity ◽  
Regulation Of Gene Expression ◽  
Protein Methylation ◽  
Biological Processes ◽  
Post Translational Modification ◽  
Regulatory Enzymes ◽  
Lysine Residues ◽  
Arginine Residues

Protein methylation is an important and reversible post-translational modification that regulates many biological processes in cells. It occurs mainly on lysine and arginine residues and involves many important biological processes, including transcriptional activity, signal transduction, and the regulation of gene expression. Protein methylation and its regulatory enzymes are related to a variety of human diseases, so improved identification of methylation sites is useful for designing drugs for a variety of related diseases. In this review, we systematically summarize and analyze the tools used for the prediction of protein methylation sites on arginine and lysine residues over the last decade.

Structure and chemical composition of insoluble filamentous components of sperm flagellar microtubules

1982 ◽  
Vol 58 (1) ◽  
pp. 1-22
Author(s):  
R.W. Linck ◽  
G.L. Langevin
Keyword(s):  
Peptide Mapping ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Resistant Material ◽  
Remarkable Feature ◽  
Electrophoretic Mobilities ◽  
Helical Content ◽  
Isoelectric Points ◽  
Flagellar Proteins ◽  
Final Fraction

By progressive solvent extraction, we have obtained a series of subfragments of flagellar microtubules. Mild treatment gives rise to ribbons that contain longitudinally arranged protofilaments. Further extraction leaves a distinctive residue containing thinner ribbons, of three and eventually two protofilaments. Finally, filaments 2–3 nm in diameter and fibrous ribbons apparently containing 6 or more 2 nm subfibrils are found. This latter solvent-resistant material is consistently enriched in a characteristic set of polypeptides, which are found in flagella of several different species, including echinoderms and a mollusc. These polypeptides appear different from alpha- and beta-tubulin on the basis of their solubilities, isoelectric points and electrophoretic mobilities in sodium dodecyl sulphate/polyacrylamide gels; these conclusions are reinforced by peptide mapping after limited proteolytic digestion, although the latter method reveals certain similarities between these unique flagellar proteins, tubulin, chicken gizzard desmin and rabbit actin. A remarkable feature of the protein in the final fraction is the high alpha-helical content: 71% as measured by circular dichroism. We consider the possible origins of these filaments in the microtubule, in particular the possibility that microtubule protofilaments are heterogeneous in protein composition, and we discuss some of the implications of our findings.

scholarly journals The Arginine-1493 Residue in QRRGRTGR1493G Motif IV of the Hepatitis C Virus NS3 Helicase Domain Is Essential for NS3 Protein Methylation by the Protein Arginine Methyltransferase 1

2001 ◽  
Vol 75 (17) ◽  
pp. 8031-8044 ◽  
Author(s):  
Jaerang Rho ◽  
Seeyoung Choi ◽  
Young Rim Seong ◽  
Joonho Choi ◽  
Dong-Soo Im
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Binding ◽  
Rna Helicase ◽  
Full Length ◽  
Arginine Residue ◽  
Protein Methylation ◽  
Helicase Domain ◽  
Rna Helicases ◽  
Ns3 Protein

ABSTRACT The NS3 protein of hepatitis C virus (HCV) contains protease and RNA helicase activities, both of which are likely to be essential for HCV propagation. An arginine residue present in the arginine-glycine (RG)-rich region of many RNA-binding proteins is posttranslationally methylated by protein arginine methyltransferases (PRMTs). Amino acid sequence analysis revealed that the NS3 protein contains seven RG motifs, including two potential RG motifs in the 1486-QRRGRTGRG-1494 motif IV of the RNA helicase domain, in which arginines are potentially methylated by PRMTs. Indeed, we found that the full-length NS3 protein is arginine methylated in vivo. The full-length NS3 protein and the NS3 RNA helicase domain were methylated by a crude human cell extract. The purified PRMT1 methylated the full-length NS3 and the RNA helicase domain, but not the NS3 protease domain. The NS3 helicase bound specifically and comigrated with PRMT1 in vitro. Mutational analyses indicate that the Arg1493 in the QRR1488GRTGR1493G region of the NS3 RNA helicase is essential for NS3 protein methylation and that Arg1488 is likely methylated. NS3 protein methylation by the PRMT1 was decreased in the presence of homoribopolymers, suggesting that the arginine-rich motif IV is involved in RNA binding. The results suggest that an arginine residue(s) in QRXGRXGR motif IV conserved in the virus-encoded RNA helicases can be posttranslationally methylated by the PRMT1.

scholarly journals Antigenic Composition of Borrelia burgdorferi during Infection of SCID Mice

2003 ◽  
Vol 71 (6) ◽  
pp. 3419-3428 ◽  
Author(s):  
Timothy R. Crother ◽  
Cheryl I. Champion ◽  
Xiao-Yang Wu ◽  
David R. Blanco ◽  
James N. Miller ◽  
...  
Keyword(s):  
Borrelia Burgdorferi ◽  
Antigenic Variation ◽  
Protein Composition ◽  
Surface Protein ◽  
Immunoblot Analysis ◽  
Scid Mice ◽  
Antigenic Composition ◽  
Mouse Tissues ◽  
Molecular Masses

ABSTRACT The general concept that during infection of mice the Borrelia burgdorferi surface protein composition differs profoundly from that of tick-borne or in vitro-cultivated spirochetes is well established. Specific knowledge concerning the differences is limited because the small numbers of spirochetes present in tissue have not been amenable to direct compositional analysis. In this report we describe novel means for studying the antigenic composition of host-adapted Borrelia (HAB). The detergent Triton X-114 was used to extract the detergent-phase HAB proteins from mouse ears, ankles, knees, and hearts. Immunoblot analysis revealed a profile distinct from that of in vitro-cultivated Borrelia (IVCB). OspA and OspB were not found in the tissues of SCID mice 17 days after infection. The amounts of antigenic variation protein VlsE and the relative amounts of its transcripts were markedly increased in ear, ankle, and knee tissues but not in heart tissue. VlsE existed as isoforms having both different unit sizes and discrete lower molecular masses. The hydrophobic smaller forms of VlsE were also found in IVCB. The amounts of the surface protein (OspC) and the decorin binding protein (DbpA) were increased in ear, ankle, knee, and heart tissues, as were the relative amounts of their transcripts. Along with these findings regarding VlsE, OspC, and DbpA, two-dimensional immunoblot analysis with immune sera also revealed additional details of the antigenic composition of HAB extracted from ear, heart, and joint tissues. A variety of novel antigens, including antigens with molecular masses of 65 and 30 kDa, were found to be upregulated in mouse tissues. Extraction of hydrophobic B. burgdorferi antigens from tissue provides a powerful tool for determining the antigenic composition of HAB.

scholarly journals The role of retrograde intraflagellar transport in flagellar assembly, maintenance, and function

2012 ◽  
Vol 199 (1) ◽  
pp. 151-167 ◽  
Author(s):  
Benjamin D. Engel ◽  
Hiroaki Ishikawa ◽  
Kimberly A. Wemmer ◽  
Stefan Geimer ◽  
Ken-ichi Wakabayashi ◽  
...  
Keyword(s):  
Intraflagellar Transport ◽  
Retrograde Transport ◽  
Full Length ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Flagellar Beat ◽  
Flagellar Assembly ◽  
Ph Shock ◽  
And Function

The maintenance of flagellar length is believed to require both anterograde and retrograde intraflagellar transport (IFT). However, it is difficult to uncouple the functions of retrograde transport from anterograde, as null mutants in dynein heavy chain 1b (DHC1b) have stumpy flagella, demonstrating solely that retrograde IFT is required for flagellar assembly. We isolated a Chlamydomonas reinhardtii mutant (dhc1b-3) with a temperature-sensitive defect in DHC1b, enabling inducible inhibition of retrograde IFT in full-length flagella. Although dhc1b-3 flagella at the nonpermissive temperature (34°C) showed a dramatic reduction of retrograde IFT, they remained nearly full-length for many hours. However, dhc1b-3 cells at 34°C had strong defects in flagellar assembly after cell division or pH shock. Furthermore, dhc1b-3 cells displayed altered phototaxis and flagellar beat. Thus, robust retrograde IFT is required for flagellar assembly and function but is dispensable for the maintenance of flagellar length. Proteomic analysis of dhc1b-3 flagella revealed distinct classes of proteins that change in abundance when retrograde IFT is inhibited.

Subcellular distribution of 3 functional platelet SNARE proteins: human cellubrevin, SNAP-23, and syntaxin 2

Blood ◽  
2002 ◽  
Vol 99 (11) ◽  
pp. 4006-4014 ◽  
Author(s):  
Dian Feng ◽  
Katharine Crane ◽  
Nataliya Rozenvayn ◽  
Ann M. Dvorak ◽  
Robert Flaumenhaft
Keyword(s):  
Plasma Membrane ◽  
Subcellular Distribution ◽  
Molecular Mechanisms ◽  
Protein Composition ◽  
Immunoblot Analysis ◽  
Snare Proteins ◽  
Ultrastructural Studies ◽  
Streptolysin O ◽  
Membrane Compartments ◽  
Granule Secretion

Morphologic studies have demonstrated a process by which α-granule contents are released from platelets. Studies aimed at defining the molecular mechanisms of this release have demonstrated that SNARE proteins are required for α-granule secretion. These observations raise the possibility that morphologic features of α-granule secretion may be influenced by the subcellular distribution of SNARE proteins in the platelet. To evaluate this possibility, we analyzed the subcellular distribution of 3 functional platelet SNARE proteins—human cellubrevin, SNAP-23, and syntaxin 2. Exposure of streptolysin O-permeabilized platelets to antihuman cellubrevin antibody inhibited Ca++-induced α-granule secretion by approximately 50%. Inhibition of α-granule secretion by antihuman cellubrevin was reversed by a blocking peptide. Syntaxin 2 and SNAP-23 have previously been demonstrated to mediate platelet granule secretion. The subcellular localization of the 3 SNARE proteins was determined by ultrastructural studies, using a pre-embedding immunonanogold method, and by immunoblot analysis of subcellular fractions. Immunonanogold localization demonstrated that approximately 80% of human cellubrevin in resting platelets was localized to platelet granule membranes. In contrast, SNAP-23 localized predominantly to plasma membrane, whereas syntaxin 2 was more evenly distributed among membranes of α-granules, the open canalicular system, and plasma membrane. Thus, each of these SNARE proteins has a distinct subcellular distribution in platelets, and each of these membrane compartments demonstrates a unique SNARE protein composition. This distribution provides a basis for several characteristics of α-granule secretion that include homotypic α-granule fusion and the fusion of α-granules with the open canalicular system and plasma membrane.

scholarly journals Surfactant protein composition of lamellar bodies isolated from rat lung

1991 ◽  
Vol 274 (1) ◽  
pp. 115-119 ◽  
Author(s):  
M A Oosterlaken-Dijksterhuis ◽  
M van Eijk ◽  
B L M van Buel ◽  
L M G van Golde ◽  
H P Haagsman
Keyword(s):  
Protein Composition ◽  
Immunoblot Analysis ◽  
Surfactant Protein ◽  
Surfactant Proteins ◽  
Rat Lung ◽  
Lamellar Bodies

Lamellar bodies isolated from rat lung contain all three classes of surfactant proteins, SP-A, SP-B and SP-C, as determined by immunoblot analysis. The amounts of the surfactant proteins present in lamellar bodies, determined by sandwich e.l.i.s.a. (SP-A) and fluorescamine assay (SP-B and SP-C) show that these organelles are highly enriched in the hydrophobic surfactant proteins SP-B and SP-C.

scholarly journals Functional modulation of IFT kinesins extends the sensory repertoire of ciliated neurons in Caenorhabditis elegans

2006 ◽  
Vol 172 (5) ◽  
pp. 663-669 ◽  
Author(s):  
James E. Evans ◽  
Joshua J. Snow ◽  
Amy L. Gunnarson ◽  
Guangshuo Ou ◽  
Henning Stahlberg ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Intraflagellar Transport ◽  
Full Length ◽  
Sensory Stimuli ◽  
Sensory Cilia ◽  
Section Electron ◽  
Functional Modulation ◽  
Serial Section Electron Microscopy ◽  
Section Electron Microscopy ◽  
Ciliated Neurons

The diversity of sensory cilia on Caenorhabditis elegans neurons allows the animal to detect a variety of sensory stimuli. Sensory cilia are assembled by intraflagellar transport (IFT) kinesins, which transport ciliary precursors, bound to IFT particles, along the ciliary axoneme for incorporation into ciliary structures. Using fluorescence microscopy of living animals and serial section electron microscopy of high pressure–frozen, freeze-substituted IFT motor mutants, we found that two IFT kinesins, homodimeric OSM-3 kinesin and heterotrimeric kinesin II, function in a partially redundant manner to build full-length amphid channel cilia but are completely redundant for building full-length amphid wing (AWC) cilia. This difference reflects cilia-specific differences in OSM-3 activity, which serves to extend distal singlets in channel cilia but not in AWC cilia, which lack such singlets. Moreover, AWC-specific chemotaxis assays reveal novel sensory functions for kinesin II in these wing cilia. We propose that kinesin II is a “canonical” IFT motor, whereas OSM-3 is an “accessory” IFT motor, and that subtle changes in the deployment or actions of these IFT kinesins can contribute to differences in cilia morphology, cilia function, and sensory perception.

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