首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到10条相似文献,搜索用时 93 毫秒
1.
Polyglycylation occurs through the post-translational addition of a polyglycine peptide to the gamma-carboxyl group of glutamic acids near the C terminus of alpha- and beta-tubulin, and has been found only in cells with axonemes, from protists to humans. In Tetrahymena thermophila, multiple sites of polyglycylation on alpha-tubulin are dispensable. By contrast, mutating similar sites on beta-tubulin has site-specific effects, affecting cell motility and cytokinesis, or resulting in cell death. Here, we address the lethality of a polyglycylation deficiency in T. thermophila using heterokaryons. Cells with a lethal mutation in the polyglycylation domain of beta-tubulin assembled axonemes that lack the central pair, B-subfibres and the transitional zone of outer microtubules (MTs). Furthermore, an arrest in cytokinesis occurred, and was associated with incomplete severing of cortical MTs positioned near the cleavage furrow. Thus, tubulin polyglycylation is required for the maintenance of some stable microtubular organelles that are all known to be polyglycylated in vivo, but its effects on MTs appear to be organelle-specific.  相似文献   

2.
Tubulin glycylation is a posttranslational modification found in cells with cilia or flagella. The ciliate Tetrahymena has glycylation on ciliary and cortical microtubules. We showed previously that mutating three glycylation sites on beta-tubulin produces immotile 9 + 0 axonemes and inhibits cytokinesis. Here, we use an inducible glycylation domain mutation and epitope tagging to evaluate the potential of glycylation-deficient tubulin for assembly and maintenance of microtubular systems. In axonemes, the major defects, including lack of the central pair, occurred during assembly, and newly made cilia were abnormally short. The glycylation domain also was required for maintenance of the length of already assembled cilia. In contrast to the aberrant assembly of cilia, several types of cortical organelles showed an abnormally high number of microtubules in the same mutant cells. Thus, the consequences of deficiency in tubulin glycylation are organelle type specific and lead to either insufficient assembly (cilia) or excessive assembly (basal bodies and cortical microtubules). We suggest that the diverse functions of the beta-tubulin glycylation domain are executed by spatially restricted microtubule-associated proteins.  相似文献   

3.
In the mutant BEN210 of Physarum polycephalum several beta-tubulins are detectable. beta 1-tubulin is unique to the myxamoeba, beta 2-tubulin is unique to the plasmodium, and the mutant beta 1-210 tubulin encoded by the benD210 allele is present in both cell types. In order to analyse the subcellular distribution of the beta 1-210 polypeptide, we prepared cytoskeletons from myxamoebae and mitotic spindles from plasmodia, and examined the tubulin polypeptide composition of these microtubular organelles by two-dimensional gel electrophoresis and immunoblotting. The results show that the beta 1-210 tubulin is present in microtubules of both the cytoskeleton and the intranuclear mitotic spindle. Thus a single beta-tubulin gene product can participate in multiple microtubular organelles in distinct cellular compartments.  相似文献   

4.
The occurrence of the tubulin post-translational modification, polyglycylation, in stable microtubular structures was investigated during morphogenesis in two ciliates, Paramecium and Frontonia atra, belonging to the Epiplasmata group. This analysis was carried out by means of immunofluorescence and post-embedding immunoelectron microscopy using two monoclonal antibodies, TAP 952 and AXO 49, respectively recognizing mono- and polyglycylated sites in alpha- and beta-tubulin. In the course of cell division, the TAP 952 epitope is detected in all microtubular structures including the newly assembled ones, such as cortical and oral basal bodies and cilia. In contrast, the AXO 49 epitope is only present in 'old' microtubular structures such as parental cortical and oral basal bodies and cilia. Our observations show that, in ciliates: 1) this tubulin post-translational modification takes place early in the course of morphogenesis; and 2) the lengthening of the polyglycine chains occurs after a great delay following addition of the first glycine residues on the tubulin glycylation sites, and following microtubule assembly. Thus, a sequential mechanism of polyglycylation is shown to take place in the tubulin molecule and during morphogenesis in Paramecium and Frontonia atra. Accordingly, polyglycylation, through a time-dependent polyglycine chain elongation process, appears to be a morphogenetic marker in ciliates.  相似文献   

5.
Polyglycylation is a polymeric post-translational modification of tubulin that is ubiquitous and widely present in cilia and flagella. It consists of the addition of highly variable numbers of glycyl residues as side chains onto the gamma carboxyl group of specific glutamyl residues at the C-termini of alpha- and beta-tubulin. The function of polyglycylation is poorly understood, however, studies in Tetrahymena have shown that the mutation of polyglycylation sites in beta-tubulin resulted in axonemal abnormality or lethality. This suggests that polyglycylation is functionally essential in protists. We hypothesize that polyglycylation is also essential in mammalian cilia and that the extent of polyglycylation has functional significance. In this study, we examined polyglycylation states in ciliated tissues and in mouse tracheal epithelial cell cultures. We utilized two antibodies, TAP 952 and AXO 49, which recognize glutamyl sites possessing monomeric glycylation sites and glutamyl sites possessing polymeric glycylation sites, respectively. Monomeric glycylation sites were observed in cilia of all the ciliated tissues examined but were invariably excluded from the distal tips. In contrast, polymeric glycylation sites were rare, but when observed, they were localized at the bases of cilia. During ciliogenesis, in epithelial cell cultures, monomeric glycylation sites were observed, but the extent of polymeric glycylation sites were variable and were only observed during the early stages of the cultures. Our observations suggest that while monomeric glycylation sites are universal and likely essential in mammalian cilia, polymeric glycylation sites are not required for ciliary beating. Rather, our observations suggest that the number of added glycyl residues increases progressively from the tips of cilia toward their bases.  相似文献   

6.
By means of immunofluorescence, immunoelectron microscopy and immunoblotting, we show that polyglycylation, a posttranslational modification of tubulin widely spread among eukaryotes, is present in the diplomonad, Giardia lamblia, a putative ancestral cell possessing a highly developed microtubular cytoskeleton. This modification was recently discovered in the ciliated protist, Paramecium, and was not found in the Euglenozoa, a lineage considered as ancient. We used two monoclonal antibodies (mAbs), TAP 952 and AXO 49, specifically recognizing mono- and polyglycylated tubulin isoforms, to detect this modification in Giardia extracts and to localize it in the different classes of microtubules within the cell. The alpha- and beta-tubulin subunits were recognized by the two mAbs, indicating that both tubulin subunits are glycylated, in agreement with lately reported mass spectrometry results. Noticeably, Giardia tubulin was much more reactive with AXO 49 than with TAP 952. In situ, AXO 49 intensely labeled the microtubules present in the four pairs of flagella and the median body, and lightly decorated the microtubules from the adhesive disc. In contrast, TAP 952 intensely labeled only the microtubules of the median body. The results indicate a differential expression of glycylated isoforms within various microtubular structures of Giardia lamblia. They also suggest that the complete set of enzymes required for polyglycylation is expressed in very divergent eukaryotes.  相似文献   

7.
《The Journal of cell biology》1995,129(5):1301-1310
In Tetrahymena, at least 17 distinct microtubule structures are assembled from a single primary sequence type of alpha- and beta- tubulin heterodimer, precluding distinctions among microtubular systems based on tubulin primary sequence isotypes. Tetrahymena tubulins also are modified by several types of posttranslational reactions including acetylation of alpha-tubulin at lysine 40, a modification found in most eukaryotes. In Tetrahymena, axonemal alpha-tubulin and numerous other microtubules are acetylated. We completely replaced the single type of alpha-tubulin gene in the macronucleus with a version encoding arginine instead of lysine 40 and therefore cannot be acetylated at this position. No acetylated tubulin was detectable in these transformants using a monoclonal antibody specific for acetylated lysine 40. Surprisingly, mutants lacking detectable acetylated tubulin are indistinguishable from wild-type cells. Thus, acetylation of alpha- tubulin at lysine 40 is non-essential in Tetrahymena. In addition, isoelectric focusing gel analysis of axonemal tubulin from cells unable to acetylate alpha-tubulin leads us to conclude that: (a) most or all ciliary alpha-tubulin is acetylated, (b) other lysines cannot be acetylated to compensate for loss of acetylation at lysine 40, and (c) acetylated alpha-tubulin molecules in wild-type cells contain one or more additional charge-altering modifications.  相似文献   

8.
9.
We analyzed the role of tubulin polyglycylation in Tetrahymena thermophila using in vivo mutagenesis and immunochemical analysis with modification-specific antibodies. Three and five polyglycylation sites were identified at glutamic acids near the COOH termini of alpha- and beta-tubulin, respectively. Mutants lacking all polyglycylation sites on alpha-tubulin have normal phenotype, whereas similar sites on beta-tubulin are essential. A viable mutant with three mutated sites in beta-tubulin showed reduced tubulin glycylation, slow growth and motility, and defects in cytokinesis. Cells in which all five polyglycylation sites on beta-tubulin were mutated were viable if they were cotransformed with an alpha-tubulin gene whose COOH terminus was replaced by the wild-type COOH terminus of beta-tubulin. In this double mutant, beta-tubulin lacked detectable polyglycylation, while the alpha-beta tubulin chimera was hyperglycylated compared with alpha-tubulin in wild-type cells. Thus, the essential function of polyglycylation of the COOH terminus of beta-tubulin can be transferred to alpha-tubulin, indicating it is the total amount of polyglycylation on both alpha- and beta-tubulin that is essential for survival.  相似文献   

10.
Polyglycylation is a posttranslational modification specific to tubulin. This modification was originally identified in highly stable microtubules from Paramecium cilia. As many as 34 posttranslationally added glycine residues have been located in the C-terminal domains of Paramecium alpha- and beta-tubulin. In this study, post source decay matrix-assisted laser desorption/ionization mass spectrometry (PSD MALDI MS) and electrospray ionization on a hybrid quadrupole orthogonal time-of-flight tandem mass spectrometer (ESI Q-TOF MS/MS) were both used to demonstrate that a single molecule of beta-tubulin, from either dynamic cytoplasmic microtubules or stable axonemal microtubules, can be glycylated on each of the last four C-terminal glutamate residues Glu437, Glu438, Glu439, and Glu441 in the sequence 427DATAEEEGEFEEEGEQ442. In both dynamic and stable microtubules the most abundant beta-tubulin isoform contains six posttranslationally added glycine residues: two glycine residues on both Glu437 and Glu438 and one glycine residue on both Glu439 and Glu441. The number and relative abundance of glycylated isoforms of beta-tubulin in both cytoplasmic and axonemal microtubules were compared by MALDI MS.1 The abundance of the major glycylated isoforms in axonemal tubulin decreases regularly with glycylation levels from 6 to 19 whereas it drops abruptly in cytoplasmic tubulin with glycylation levels from 6 to 9. However, the polyglycine chains are similarly distributed on the four C-terminal glutamate residues of cytoplasmic and axonemal tubulin. The polyglycylation results in bulky C-terminal domains with negatively charged surfaces, all surrounding the microtubular structure.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号