Posttranslational Modifications in the C-terminal Tail of Axonemal Tubulin from Sea Urchin Sperm (original) (raw)
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Posttranslational Modifications of Axonemal Tubulin
1997
Axonemal tubulin exhibits a high degree of heterogeneity mostly due to several posttranslational modifications (PTM). The aim of this work was to chemically characterize the different PTM occurring in the C-terminal tail of axonemal tubulin purified from sea urchin, Paracentrotus lividus, spermatozoa. After its purification, tubulin was enzymatically cleaved. The C-terminal peptides were chromatographically isolated, first by anion exchange and then by reverse-phase HPLC. Peptides were characterized by their sequence, determined by Edman degradation, and by their mass, determined by MALDI-TOF/MS. The two major conclusions arc that the majority of the isolated C-terminal peptides were unmodified and that polyglycylation and polyglutamylation can occur simultaneously on one molecule of a-tubulin.
Biochemistry, 1996
The R -tubulin heterodimer, the structural unit of microtubules, comes in many variants. There are different R and isotypes encoded by multigene families. Additional heterogeneity is generated by a set of posttranslational modifications. Detyrosination of R-tubulin, removal of the carboxy-terminal Glu-Tyr dipeptide of R-tubulin, phosphorylation of some tubulins, polyglutamylation, and polyglycylation of Rand -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 R -tubulin. In B tubules, R-tubulin is ∼65% detyrosinated and both Rand -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.
Sperm axoneme: A tale of tubulin posttranslation diversity
Molecular Reproduction and Development, 2002
Two microtubule-containing structures are assembled during spermiogenesis: a transient manchette and a stable axoneme. Both structures contain microtubules enriched in posttranslationally modified tubulins. Despite the existence of a spectrum of tubulin isotypes postulated by the multi-tubulin hypothesis, further extended by an elaborated array of posttranslational modifications, it is unknown how this diversity influences microtubule function. There is increasing evidence that different ab-tubulin isotypes can affect the structure and function of microtubules. It is also becoming increasingly clear that eukaryotic cells encode other tubulin proteins expressed by the tubulin superfamily: g, d e, z h, and FtsZ have been identified so far. Although the role of g-tubulin in the nucleation of microtubule assembly is well established, the function of d-, e-, z-, h-, and FtsZ-tubulins is less understood. The members of the tubulin superfamilies found in spermatids include the ab-tubulin dimer, in addition to g-tubulin in the centrosome, and d-tubulin in the perinuclear ring region of the mouse spermatid manchette, the centrosome region, and flagellum. Posttranslational modifications in tubulin isotypes are predominant in the C-terminus exposed on the outside surface of the microtubule. This target site may influence the interaction of microtubule-associated proteins, including motor proteins, and therefore determine the functional specificity of tubulin isotypes. It remains to be determined whether other newcomers to the superfamily of tubulins contain sites prone to posttranslational modification. Mol. Reprod. Dev. 62: 1± 3,
Proceedings of the National …, 1973
Two kinds of tubulin (a and ,3) have been described in microtubules from many different systems. In this study a discontinuous acrylamide-gel system containing sodium dodecyl sulfate was used to separate milligram quantities of a-and fl-tubulin from microtubules of chick-embryo brain and from outer doublets of sea-urchin sperm. The isolated tubulins were characterized by peptide mapping and automated sequencing of the first 25 NH2-terminal amino acids. Our results show that a-and #-tubulin are related but distinctly different proteins and that each one has been highly conserved in the course of evolution.
Axoneme-dependent tubulin modifications in singlet microtubules of the Drosophila sperm tail
Cell motility and the cytoskeleton, 2008
Drosophila melanogaster sperm tubulins are posttranslationally glutamylated and glycylated. We show here that axonemes are the substrate for these tubulin C-terminal modifications. Axoneme architecture is required, but full length, motile axonemes are not necessary. Tubulin glutamylation occurs during or shortly after assembly into the axoneme; only glutamylated tubulins are glycylated. Tubulins in other testis microtubules are not modified. Only a small subset of total Drosophila sperm axoneme tubulins have these modifications. Biochemical fractionation of Drosophila sperm showed that central pair and accessory microtubules have the majority of poly-modified tubulins, whereas doublet microtubules have only small amounts of mono- and oligo-modified tubulins. Glutamylation patterns for different beta-tubulins experimentally assembled into axonemes were consistent with utilization of modification sites corresponding to those identified in other organisms, but surrounding sequence cont...