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.

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 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.

scholarly journals Mutant Kinesin-2 Motor Subunits Increase Chromosome Loss

2005 ◽  
Vol 16 (8) ◽  
pp. 3810-3820 ◽  
Author(s):  
Mark S. Miller ◽  
Jessica M. Esparza ◽  
Andrew M. Lippa ◽  
Fordyce G. Lux ◽  
Douglas G. Cole ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Single Point ◽  
Intraflagellar Transport ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Dominant Effect ◽  
Flagellar Assembly ◽  
Gene Maps

The Chlamydomonas anterograde intraflagellar transport motor, kinesin-2, is isolated as a heterotrimeric complex containing two motor subunits and a nonmotor subunit known as kinesin-associated polypeptide or KAP. One of the two motor subunits is encoded by the FLA10 gene. The sequence of the second motor subunit was obtained by mass spectrometry and sequencing. It shows 46.9% identity with the Fla10 motor subunit and the gene maps to linkage group XII/XIII near RPL9. The temperature-sensitive flagellar assembly mutants fla1 and fla8 are linked to this kinesin-2 motor subunit. In each strain, a unique single point mutation gives rise to a unique single amino acid substitution within the motor domain. The fla8 strain is named fla8-1 and the fla1 strain is named fla8-2. The fla8 and fla10 alleles show a chromosome loss phenotype. To analyze this chromosome loss phenotype, intragenic revertants of fla8-1, fla8-2, and fla10-14 were generated. The analysis of the mutants and the revertants demonstrates the importance of a pocket in the amino terminus of these motor subunits for both motor activity and for a novel, dominant effect on the fidelity of chromosome segregation.

scholarly journals The short flagella 1 (SHF1) gene in Chlamydomonas encodes a Crescerin TOG-domain protein required for late stages of flagellar growth.

2021 ◽  
Author(s):  
Karina Perlaza ◽  
Mariya Mirvis ◽  
Hiroaki Ishikawa ◽  
Wallace F Marshall
Keyword(s):  
Model Organism ◽  
Size Control ◽  
Intraflagellar Transport ◽  
Loss Of Function ◽  
Wild Type ◽  
Cytoplasmic Microtubule ◽  
Flagellar Assembly ◽  
Flagellar Regeneration ◽  
Domain Protein ◽  
Late Stages

Length control of flagella represents a simple and tractable system to investigate the dynamics of organelle size. Models for flagellar length control in the model organism, Chlamydomonas reinhardtii have focused on the length-dependence of the intraflagellar transport (IFT) system which manages the delivery and removal of axonemal subunits at the tip of the flagella. One of these cargoes, tubulin, is the major axonemal subunit, and its frequency of arrival at the tip plays a central role in size control models. However, the mechanisms determining tubulin dynamics at the tip are still poorly understood. We discovered a loss-of-function mutation that leads to shortened flagella, and found that this was an allele of a previously described gene, SHF1, whose molecular identity had not previously been determined. We found that SHF1 encodes a Chlamydomonas ortholog of Crescerin, previously identified as a cilia specific TOG-domain array protein that can bind tubulin via its TOG domains and increase tubulin polymerization rates. In this mutant, flagellar regeneration occurs with the same initial kinetics as wild-type cells, but plateaus at a shorter length. Using a computational model in which the flagellar microtubules are represented by a differential equation for flagellar length combined with a stochastic model for cytoplasmic microtubule dynamics, we found that our experimental results are best described by a model in which Crescerin/SHF1 binds tubulin dimers in the cytoplasm and transports them into the flagellum. We suggest that this TOG-domain protein is necessary to efficiently and preemptively increase intra-flagella tubulin levels to offset decreasing IFT cargo at the tip as flagellar assembly progresses.

scholarly journals Retrograde Intraflagellar Transport Mutants Identify Complex A Proteins With Multiple Genetic Interactions in Chlamydomonas reinhardtii

Genetics ◽  
2009 ◽  
Vol 183 (3) ◽  
pp. 885-896 ◽  
Author(s):  
Carlo Iomini ◽  
Linya Li ◽  
Jessica M. Esparza ◽  
Susan K. Dutcher
Keyword(s):  
Intraflagellar Transport ◽  
Gene Interaction ◽  
Retrograde Transport ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Transport Mutants ◽  
Flagellar Assembly ◽  
Biochemical Genetic ◽  
Different Levels ◽  
Assembly Defect

The intraflagellar transport machinery is required for the assembly of cilia. It has been investigated by biochemical, genetic, and computational methods that have identified at least 21 proteins that assemble into two subcomplexes. It has been hypothesized that complex A is required for retrograde transport. Temperature-sensitive mutations in FLA15 and FLA17 show defects in retrograde intraflagellar transport (IFT) in Chlamydomonas. We show that IFT144 and IFT139, two complex A proteins, are encoded by FLA15 and FLA17, respectively. The fla15 allele is a missense mutation in a conserved cysteine and the fla17 allele is an in-frame deletion of three exons. The flagellar assembly defect of each mutant is rescued by the respective transgenes. In fla15 and fla17 mutants, bulges form in the distal one-third of the flagella at the permissive temperature and this phenotype is also rescued by the transgenes. These bulges contain the complex B component IFT74/72, but not α-tubulin or p28, a component of an inner dynein arm, which suggests specificity with respect to the proteins that accumulate in these bulges. IFT144 and IFT139 are likely to interact with each other and other proteins on the basis of three distinct genetic tests: (1) Double mutants display synthetic flagellar assembly defects at the permissive temperature, (2) heterozygous diploid strains exhibit second-site noncomplemention, and (3) transgenes confer two-copy suppression. Since these tests show different levels of phenotypic sensitivity, we propose they illustrate different gradations of gene interaction between complex A proteins themselves and with a complex B protein (IFT172).

scholarly journals WW Domains of Rsp5p Define Different Functions: Determination of Roles in Fluid Phase and Uracil Permease Endocytosis in Saccharomyces cerevisiae

Genetics ◽  
2001 ◽  
Vol 157 (1) ◽  
pp. 91-101 ◽  
Author(s):  
Beata Gajewska ◽  
Joanna Kamińska ◽  
Alicja Jesionowska ◽  
Nancy C Martin ◽  
Anita K Hopper ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Fluid Phase ◽  
Temperature Sensitive ◽  
Protein Protein Interactions ◽  
Ww Domains ◽  
Ubiquitin Protein Ligase ◽  
Cellular Processes ◽  
Fluid Phase Endocytosis ◽  
Ubiquitination Pathway ◽  
Punctate Pattern

Abstract Rsp5p, ubiquitin-protein ligase, an enzyme of the ubiquitination pathway, contains three WW domains that mediate protein-protein interactions. To determine if these domains adapt Rsp5p to a subset of substrates involved in numerous cellular processes, we generated mutations in individual or combinations of the WW domains. The rsp5-w1, rsp5-w2, and rsp5-w3 mutant alleles complement RSP5 deletions at 30°. Thus, individual WW domains are not essential. Each rsp5-w mutation caused temperature-sensitive growth. Among variants with mutations in multiple WW domains, only rsp5-w1w2 complemented the deletion. Thus, the WW3 domain is sufficient for Rsp5p essential functions. To determine whether rsp5-w mutations affect endocytosis, fluid phase and uracil permease (Fur4p) endocytosis was examined. The WW3 domain is important for both processes. WW2 appears not to be important for fluid phase endocytosis whereas it is important for Fur4p endocytosis. In contrast, the WW1 domain affects fluid phase endocytosis, but it does not appear to function in Fur4p endocytosis. Thus, various WW domains play different roles in the endocytosis of these two substrates. Rsp5p is located in the cytoplasm in a punctate pattern that does not change during the cell cycle. Altering WW domains does not change the location of Rsp5p.

scholarly journals Partially redundant actin genes in Chlamydomonas control flagellum-directed traffic and transition zone organization

2017 ◽  
Author(s):  
Brittany Jack ◽  
David M. Mueller ◽  
Ann C. Fee ◽  
Ashley Tetlow ◽  
Prachee Avasthi
Keyword(s):  
Transition Zone ◽  
Mammalian Cells ◽  
Intraflagellar Transport ◽  
Actin Genes ◽  
Transmission Electron ◽  
Unicellular Green Alga ◽  
Absolute Requirement ◽  
Flagellar Assembly ◽  
Flagellar Proteins ◽  
Flagellar Biogenesis

ABSTRACTFlagella of the unicellular green alga Chlamydomonas reinhardtii are nearly identical to cilia of mammalian cells and provide an excellent model to study ciliogenesis. These biflagellated cells have two actin genes: one encoding a conventional actin (IDA5) and the other encoding a divergent novel actin-like protein (NAP1). Previously, we described a role for actin in the regulation of flagella-building intraflagellar transport machinery. Here, we probe how actin redundancy contributes to this process using a nap1 mutant Chlamydomonas strain. Disruption of a single actin allows normal or slower incorporation but complete flagellar assembly. However, when we disrupt both actins using Latrunculin B (LatB) treatment on the nap1 mutant background, we find flagellar growth from newly synthesized limiting flagellar proteins is actin-dependent. Upon total actin disruption during flagellar assembly, transmission electron microscopy identified an accumulation of Golgi-adjacent vesicles, suggesting impaired vesicular trafficking may be the mechanism by which actin supports flagellar growth from new flagellar proteins. We also find there is a mislocalization of a key transition zone gating and ciliopathy protein, NPHP-4. Extended (2 hour) treatment with LatB, a condition under which NAP1 is upregulated, restores NPHP-4 localization. This suggests NAP1 can perform the functions of conventional actin at the transition zone. Our experiments demonstrate that each stage of flagellar biogenesis requires redundant actin function to varying degrees, with an absolute requirement for these actins in transport of Golgi-adjacent vesicles and flagellar incorporation of newly synthesized proteins.

scholarly journals A conserved flagella-associated protein in Chlamydomonas, FAP234, is essential for axonemal localization of tubulin polyglutamylase TTLL9

2014 ◽  
Vol 25 (1) ◽  
pp. 107-117 ◽  
Author(s):  
Tomohiro Kubo ◽  
Haru-aki Yanagisawa ◽  
Zhongmei Liu ◽  
Rie Shibuya ◽  
Masafumi Hirono ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Intraflagellar Transport ◽  
Sucrose Density Gradient ◽  
Temperature Sensitive ◽  
Sensitive Mutant ◽  
Gene Encoding ◽  
Sucrose Density Gradient Centrifugation ◽  
Dependent Transport

Tubulin undergoes various posttranslational modifications, including polyglutamylation, which is catalyzed by enzymes belonging to the tubulin tyrosine ligase–like protein (TTLL) family. A previously isolated Chlamydomonas reinhardtii mutant, tpg1, carries a mutation in a gene encoding a homologue of mammalian TTLL9 and displays lowered motility because of decreased polyglutamylation of axonemal tubulin. Here we identify a novel tpg1-like mutant, tpg2, which carries a mutation in the gene encoding FAP234, a flagella-associated protein of unknown function. Immunoprecipitation and sucrose density gradient centrifugation experiments show that FAP234 and TTLL9 form a complex. The mutant tpg1 retains FAP234 in the cell body and flagellar matrix but lacks it in the axoneme. In contrast, tpg2 lacks both TTLL9 and FAP234 in all fractions. In fla10, a temperature-sensitive mutant deficient in intraflagellar transport (IFT), both TTLL9 and FAP234 are lost from the flagellum at nonpermissive temperatures. These and other results suggest that FAP234 functions in stabilization and IFT-dependent transport of TTLL9. Both TTLL9 and FAP234 are conserved in most ciliated organisms. We propose that they constitute a polyglutamylation complex specialized for regulation of ciliary motility.

scholarly journals Distinct Mutants of Retrograde Intraflagellar Transport (IFT) Share Similar Morphological and Molecular Defects

1998 ◽  
Vol 143 (6) ◽  
pp. 1591-1601 ◽  
Author(s):  
Gianni Piperno ◽  
Edward Siuda ◽  
Scott Henderson ◽  
Margarethe Segil ◽  
Heikki Vaananen ◽  
...  
Keyword(s):  
Molecular Motors ◽  
Protein Complexes ◽  
Intraflagellar Transport ◽  
Temperature Sensitive ◽  
Single Particles ◽  
Molecular Defects ◽  
Video Images ◽  
Bidirectional Transport ◽  
Flagellar Assembly ◽  
Novel Method

A microtubule-based transport of protein complexes, which is bidirectional and occurs between the space surrounding the basal bodies and the distal part of Chlamydomonas flagella, is referred to as intraflagellar transport (IFT). The IFT involves molecular motors and particles that consist of 17S protein complexes. To identify the function of different components of the IFT machinery, we isolated and characterized four temperature-sensitive (ts) mutants of flagellar assembly that represent the loci FLA15, FLA16, and FLA17. These mutants were selected among other ts mutants of flagellar assembly because they displayed a characteristic bulge of the flagellar membrane as a nonconditional phenotype. Each of these mutants was significantly defective for the retrograde velocity of particles and the frequency of bidirectional transport but not for the anterograde velocity of particles, as revealed by a novel method of analysis of IFT that allows tracking of single particles in a sequence of video images. Furthermore, each mutant was defective for the same four subunits of a 17S complex that was identified earlier as the IFT complex A. The occurrence of the same set of phenotypes, as the result of a mutation in any one of three loci, suggests the hypothesis that complex A is a portion of the IFT particles specifically involved in retrograde intraflagellar movement.

scholarly journals The short flagella 1 (SHF1) gene in Chlamydomonas encodes a Crescerin TOG-domain protein required for late stages of flagellar growth

2021 ◽  
Author(s):  
Karina Perlaza ◽  
Mary Mirvis ◽  
Hiroaki Ishikawa ◽  
Wallace Marshall
Keyword(s):  
Model Organism ◽  
Size Control ◽  
Intraflagellar Transport ◽  
Loss Of Function ◽  
Wild Type ◽  
Cytoplasmic Microtubule ◽  
Flagellar Assembly ◽  
Flagellar Regeneration ◽  
Domain Protein ◽  
Late Stages

Length control of flagella represents a simple and tractable system to investigate the dynamics of organelle size. Models for flagellar length control in the model organism, Chlamydomonas reinhardtii have focused on the length-dependence of the intraflagellar transport (IFT) system which manages the delivery and removal of axonemal subunits at the tip of the flagella. One of these cargoes, tubulin, is the major axonemal subunit, and its frequency of arrival at the tip plays a central role in size control models. However, the mechanisms determining tubulin dynamics at the tip are still poorly understood. We discovered a loss-of-function mutation that leads to shortened flagella, and found that this was an allele of a previously described gene, SHF1, whose molecular identity had not previously been determined.  We found that SHF1 encodes a Chlamydomonas ortholog of Crescerin, previously identified as a cilia-specific TOG-domain array protein that can bind tubulin via its TOG domains and increase tubulin polymerization rates. In this mutant, flagellar regeneration occurs with the same initial kinetics as wild-type cells, but plateaus at a shorter length. Using a computational model in which the flagellar microtubules are represented by a differential equation for flagellar length combined with a stochastic model for cytoplasmic microtubule dynamics, we found that our experimental results are best described by a model in which Crescerin/SHF1 binds tubulin dimers in the cytoplasm and transports them into the flagellum. We suggest that this TOG-domain protein is necessary to efficiently and preemptively increase intra-flagella tubulin levels to offset decreasing IFT cargo at the tip as flagellar assembly progresses.

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