Tubulin isoforms in the brine shrimp, Artemia: primary gene products and their posttranslational modification

The brine shrimp, Artemia, contains 3 alpha- and 2 beta-tubulins as shown by Coomassie Blue staining of two-dimensional gels. In order to study the biosynthetic origins of the isotubulins, we hybridized cloned Drosophila tubulin genes, under stringent conditions, to blots of Artemia DNA and RNA. Southern blot analyses indicate a tubulin gene family of limited complexity. One size class of alpha- and beta-tubulin mRNA at 1800 bases was observed on Northern blots. Fluorograms of Artemia tubulin synthesized in vitro, revealed one alpha- and one beta-tubulin on two-dimensional gels, indicating that each mRNA is translated into one polypeptide and that additional tubulin spots observed on Coomassie-stained two-dimensional gels may arise posttranslationally. Artemia tubulin, which was either purified to homogeneity, or in crude cell-free extracts, was analyzed with a panel of tubulin-specific antibodies. The presence of acetylated tubulin, restricted to one of the three major alpha-tubulin spots on two-dimensional gels, demonstrated that Artemia tubulin diversity is partially generated by posttranslational mechanisms. Artemia tubulin reacted very well with an antibody to tyrosinated tubulin, but there was no, or very little, detectable detyrosinated tubulin unless the purified Artemia tubulin was exposed to carboxypeptidase. The results suggest that all microtubule-dependent events in Artemia, a complex metazoan animal, are accomplished with microtubules composed from a limited repertoire of tubulins and that none of these events require appreciable amounts of detyrosinated tubulin.     

Microtubule cold stability in supporting cells of the gerbil auditory sensory epithelium: correlation with tubulin post-translational modifications

Sensory cells in the organ of Corti exhibit loose microtubule networks enriched in tyrosinated tubulin, whereas supporting cells have bundled microtubules containing post-translationally modified tubulin. The tubulin isoform distribution suggests that the microtubules in sensory cells are dynamic and those in supporting cells are stable. To test this, microtubule resistance to cold-induced depolymerization was examined by using immunocytochemical methods and antibodies to post-translationally modified tubulins. Microtubule labelling in cochleas perfused/immersed at room temperature was identical to that in previous studies of untreated cochleas. However, the microtubule patterns of perfused/immersed specimens were changed in cold-treated cochleas. Microtubules were no longer detected with antibodies to alpha- and tyrosinated tubulin in sensory cells from specimens exposed to cold, indicating their disassembly. Supporting cells in the same specimens showed almost total loss of detyrosinated and polyglutamylated tubulin in the middle and apical cochlear turns, and reduced labelling in the basal-most turn. Probing for alpha-, nontyrosinatable, acetylated and glycylated tubulin yielded decreased and sometimes patchy staining but these isoforms were observed even when detyrosinated and polyglutamylated tubulins were absent. The results indicate that sensory cells in the gerbil auditory sensory epithelium contain only cold-sensitive microtubules. In contrast, supporting cells possess a substantial subset of cold-stable microtubules, providing structural support to the vibratory sensory organ required for hearing.     

Posttranslationally modified tubulins and microtubule organization in hemocytes of the brine shrimp, Artemia franciscana

Crustaceans possess blood cells (hemocytes) that mediate organismal defense and are analogous to vertebrate leukocytes. In order to more fully characterize these types of cells, hemocytes of the branchiopod crustacean, Artemia franciscana, were analyzed. The data indicate that Artemia have one type of hemocyte, ranging in morphology from compact and spherical to flat and spreading when examined in vitro. Electron microscopy revealed many cytoplasmic granules in the hemocytes and only a limited number of other membrane-bound organelles. Centrioles and microtubules were also visible in thin sections of chemically fixed samples. The cytoplasm of spherical hemocytes was completely labeled by general antitubulin antibodies, but in flattened hemocytes packing of cytoskeletal elements was less tight and individual microtubules were observed. Probing of Western blots disclosed acetylated, tyrosinated, and detyrosinated tubulin isoforms in hemocyte homogenates, the first characterization of posttranslationally modified tubulins in this cell type. Acetylated tubulin was restricted to a subset of microtubules, whereas tyrosinated microtubules were displayed more abundantly. Staining obtained with antibody to detyrosinated tubulin was unusual because it was limited to the perinuclear region of hemocytes. Incubation of blood cells with a monoclonal antibody to gamma-tubulin yielded fluorescent dots sometimes in pairs, a pattern characteristic of centrosomes. The findings support the conclusion that Artemia hemocytes undergo rapid morphogenesis in vitro accompanied by extensive rearrangement of their microtubules, the latter probably indicative of cytoskeletal changes that occur during cell movement and phagocytosis. Additionally, the hemocytes contain posttranslationally modified alpha-tubulins and centrosome-associated gamma-tubulin, both with the potential to influence microtubule organization and function.     

Posttranslational modifications of tubulin in teleost photoreceptor cytoskeletons

1. Posttranslational modifications of tubulin by acetylation and detyrosination have been correlated previously with microtubule stability in numerous cell types. 2. In this study, posttranslational modifications of tubulin and their regional distribution within teleost photoreceptor cones and rods are demonstrated immunohistochemically using antibodies specific for acetylated, detyrosinated, or tyrosinated tubulin. 3. Immunolocalization was carried out on isolated whole cones and mechanically detached rod and cone inner/outer segments. 4. Acetylated tubulin within rods and cones is found only in microtubules of the ciliary axoneme of the outer segment. Detyrosinated tubulin is also enriched in axonemes of both rod and cone outer segments. 5. Distributions of tyrosinated and detyrosinated cytoplasmic microtubules differ within cones and rods. In cones, detyrosinated and tyrosinated tubulins are both abundant throughout the cell body. In rods, the ellipsoid and myoid contain much more tyrosinated tubulin than detyrosinated tubulin. Comparisons between whole cones and cone fragments suggest that detyrosinated microtubules are more stable than tyrosinated microtubules in teleost photoreceptors. 6. Our findings provide further evidence that microtubules of teleost cones differ from rod microtubules in their stabilities and rapidity of turnover within the photoreceptor inner segment.     

Age-related changes in microtubules in the guinea pig organ of Corti

Biochemical and immunocytochemical analyses have been used to provide new insights into age-related changes in the sensory and supporting cells of the guinea pig organ of Corti. Quantitative densitometry of immunoblots showed that, while levels of alpha-tubulin remained relatively constant in guinea pigs from 3 weeks to 18 months old, there were progressive shifts in some tubulin isoforms. Levels of tyrosinated tubulin increased with age, nontyrosinatable tubulin (delta2-tubulin) showed a compensatory decrease, but detyrosinated tubulin did not change; acetylated, polyglutamylated, and glycylated tubulin levels also decreased. Immunolabeled tissue sections showed that cell type-specific distribution of tubulin seen in young guinea pigs (tyrosinated in the microtubules of the sensory cells, and post-translationally modified isoforms in the supporting cells) did not change as animals aged. However, there were age-related decreases in labeling for alpha-tubulin and all post-translationally modified isoforms. Biochemical and immunocytochemical results both support an age-related decrease in the number and/or length of microtubules as well as an increase in the pool of soluble tyrosinated and detyrosinated tubulin. They further suggest that microtubules containing nontyrosinatable tubulin from older animals are the sites for further modification of tubulin by acetylation, polyglutamylation, and glycylation. Changes in tubulin isoform levels and stability of microtubules in the organ of Corti may alter its micromechanical properties; the resulting changes in conduction of sound-induced vibration would provide one mechanism for age-related hearing loss.     

Rapid and reversible tubulin tyrosination in human neutrophils stimulated by the chemotactic peptide,fMet-Leu-Phe

Neutrophil activation by specific stimuli, such as the oligopeptide chemotactic factor fMet-Leu-(fMLF), is associated with an increased enzymatic addition of tyrosine to tubulin α -subunits, as measured by ¹⁴C tyrosine uptake. In studies using immunoblots we have found that this increased tyrosine uptake into tubulin in activated neutrophils reflects an increase in the proportion of cellular tubulin that is tyrosinated rather than simply an increase in the turnover of tyrosinated subunits. However, the increased accumulation of tyrosinated tubulin was also found to follow an initial depletion of tyrosinated tubulin and concomitant increase in detyrosinated tubulin between 0 and 60 sec following stimulation of neutrophils with fMLF. Immunogold electron microscopy studies of intact micro tubules recovered from activated neutrophils demonstrated that these rapid changes in the relative content of tubulin isoforms in the cells were not associated with the formation or disappearance of microtubule microdomains composed of only one form of tubulin. Previously, we have shown that under conditions of fMLF-stimulated exocytosis there is an increased binding of neutrophil granules to endogenous microtubules. Since neutrophil activation by fMLF is associated with increased tyrosination of α -tubulin subunits, we speculated that rapid changes in the levels of tyrosinated tubulin in the microtubules of activated neutrophils might have a role in the regulation of granule-microtubule interactions. When the binding of purified neutrophil granules to reconstituted rat brain microtubules containing approximately 50% tyrosinated tubulin was measured by electron microscopy and compared with granule binding to microtubules that contained no detectable tyrosinated tubulin, granule-microtubule associations were found to be significantly favored by detyrosinated vs. tyrosinated tubulin. These findings indicate that interactions between cytoplasmic granules and microtubules in activated neutrophils may be modulated by rapid changes in the relative content of detyrosinated and tyrosinated tubulin in the microtubule network of the cells. © 1993 Wiley-Liss, Inc.     

Differential Distribution of Posttranslationally Modified Microtubules in Osteoclasts

The differential distribution of microtubules in osteoclasts in culture was examined by using antibodies against acetylated, tyrosinated, or detyrosinated tubulins. Tyrosinated tubulin was found throughout the cytoplasmic microtubules in all cells examined. An expanding protrusion that contained tyrosinated tubulin but none of the detyrosinated or acetylated form was seen in the immature osteoclasts. Detyrosinated or acetylated tubulin was detectable in the peripheral cytoplasm of the mature osteoclasts displaying the loss of the expanding protrusion. Although most of the microtubules were derived from the centrosome, noncentrosomal microtubules were distributed in the expanding protrusion, which was predominantly positive for tyrosinated tubulin. By tracing single microtubules, the authors found that their growing ends were always rich in tyrosinated tubulin subunits. End binding protein 1 bound preferentially to the microtubule ends. Both acetylated and tyrosinated microtubules were shown to be closely associated with podosomes. Microtubules appeared to grow over or into the podosomes; in addition, the growing ends of single microtubules could be observed to target the podosomes. Moreover, a microtubule-associated histone deacetylase 6 was localized in the podosomes of the osteoclast. On the basis of these results, the authors conclude that posttranslational modifications of microtubules may correlate with characteristic changes in podosome dynamics in osteoclasts.     

Post-translational modifications and multiple tubulin isoforms in Nicotiana tabacum L. cells

Distribution of post-translationally modified tubulins in cells of Nicotiana tabacum L. was analysed using a panel of specific antibodies. Polyglutamylated, tyrosinated, nontyrosinated, acetylated and Δ2-tubulin variants were detected on α-tubulin subunits; polyglutamylation was also found on β-tubulin subunits. Modified tubulins were detected by immunofluorescence microscopy in interphase microtubules, preprophase bands, mitotic spindles as well as in phragmoplasts. They were, however, located differently in the various microtubule structures. The antibodies against tyrosinated, acetylated and polyglutamylated tubulins gave uniform staining along all microtubules, while antibodies against nontyrosinated and Δ2-tubulin provided dot-like staining of interphase microtubules. Additionally, immunoreactivity of antibodies against acetylated and Δ2-tubulins was strong in the pole regions of mitotic spindles. High-resolution isoelectric focusing revealed 22 tubulin charge variants in N. tabacum suspension cells. Immunoblotting with antibodies TU-01 and TU-06 against conserved antigenic determinants of α- and β-tubulin molecules, respectively, revealed that 11 isoforms belonged to the α-subunit and 11 isoforms to the β-subunit. Whereas antibodies against polyglutamylated, tyrosinated and acetylated tubulins reacted with several α-tubulin isoforms, antibodies against nontyrosinated and Δ2-tubulin reacted with only one. The combined data demonstrate that plant tubulin is extensively post-translationally modified and that these modifications participate in the generation of plant tubulin polymorphism. Received: 2 May 1996 / Accepted: 16 September 1996     

A small heat shock/alpha-crystallin protein from encysted Artemia embryos suppresses tubulin denaturation

Small heat shock/alpha-crystallin proteins function as molecular chaperones, protecting other proteins from irreversible denaturation by an energy-independent process. The brine shrimp, Artemia franciscana, produces a small heat shock/alpha-crystallin protein termed p26, found in embryos undergoing encystment, diapause, and metabolic arrest. These embryos withstand long-term anoxia and other stresses normally expected to cause death, a property likely dependent on molecular chaperone activity. The association of p26 with tubulin in unfractionated cell-free extracts of Artemia embryos was established by affinity chromatography, suggesting that p26 chaperones tubulin during encystment. To test this possibility, both proteins were purified by modifying published protocols, thereby simplifying the procedures, enhancing p26 yield about 2-fold, and recovering less tubulin than before. The denaturation of purified tubulin as it "aged" and exposed hydrophobic sites during incubation at 35 degrees C was greatly reduced when p26 was present; however, tubulin polymerization into microtubules was reduced. On incubation at 35 degrees C, centrifugation in sucrose density gradients demonstrated the association of purified p26 with tubulin. This is the first study where the relationship between a small heat shock/alpha-crystallin protein and tubulin from the same physiologically stressed organism was examined. The results support the proposal that p26 binds tubulin and prevents its denaturation, thereby increasing the resistance of encysted Artemia embryos to stress. Additional factors are apparently required for release of tubulin from p26 and restoration of efficient assembly, events that would occur as embryos resume development and the need for microtubules is established.     

Spatial distribution of posttranslationally modified tubulins in polarized cells of developing Artemia

In many differentiated cells, posttranslationally modified tubulins exhibit restricted subcellular distribution, leading to the proposal that they are required for the production and maintenance of polarity. To study this possibility, we used immunological approaches to examine tubulin isoforms in developing Artemia larvae and to determine their location in several types of cells within the organism. The amount of tubulin in relation to total protein remained relatively constant during early larval development while detyrosinated tubulin increased, an event correlated with the differentiation of larval gut muscle cells. Except for epidermal cells of the developing thorax, each type of cell within the Artemia larvae exhibited characteristic staining patterns which were very similar for each antitubulin antibody. Within epidermal cells, microtubules containing acetylated tubulin appeared patchy or punctate in their distribution, an image not seen with the other antibodies. In most polarized cells, staining for tubulin and actin colocalized in discrete areas, demonstrating enrichment of both proteins within the same cellular compartment and suggesting functional interactions. Mitotic figures were stained with qualitatively equal intensity by all of the antitubulin antibodies, but asters were not observed. Midbodies were intensely stained with phalloidin as well as the antibodies to tubulin. It was clear that microtubules exhibited a preferential localization in cells of Artemia but in no case was a tubulin isoform found exclusively in one area of a cell. The results support the contention that microtubules influence the organization of polarized cell structure and function but they do not permit the conclusion that this capability is dependent on the localization of posttranslationally modified tubulins to restricted subcellular positions.     

Tyrosinated, but not detyrosinated, α-tubulin is present in root tip cells

Summary The distribution of tyrosinated and detyrosinated tubulin in microtubule arrays of pine and onion cells was investigated by immunofluorescence techniques. Staining of isolated cells and methacrylate sections ofPinus radiata andAllium cepa root tips indicated that all microtubule structures contained tyrosinated tubulin but not the posttranslationally modified detyrosinated tubulin. The detyrosinated tubulin epitope was, however, created in vitro by treating both sections and fixed whole cells with carboxypeptidase A.     

Developmental and comparative aspects of brine shrimp tubulin.

Tubulin from embryos of the brine shrimp Artemia has been purified to apparent homogeneity by chromatography on phosphocellulose P11 and DEAE-cellulose, (NH4)2SO4 fractionation and assembly-disassembly of microtubules. Peptide mapping indicated that Artemia and bovine brain tubulin were very similar in spite of differences in the electrophoretic behaviour of tubulin from these two organisms. Isoelectric focusing and two-dimensional gel electrophoresis were used to resolve and identify several Artemia isotubulins . The isotubulin composition and the quantity of tubulin did not change during pre-emergence development of Artemia embryos. Formation of microtubules with tubulin purified from embryos at different stages of development did not require glycerol or microtubule-associated proteins and formation of structurally normal microtubules was actually hindered by glycerol and Mg2+. The characteristics of Artemia tubulin, in concert with the unusual life history of Artemia, suggest that this organism will be very useful for the study of tubulin gene expression and tubulin utilization during embryo development.     

Tyrosination State of Tubulin and the Activity of Tubulin:Tyrosine Ligase and Tubulin Carboxypeptidase in the Developing Retina of the Chick

The tyrosination state of tubulin and the enzymes involved in the tubulin tyrosination/detyrosination cycle--tubulin:tyrosine ligase and tubulin carboxypeptidase--were determined in chick retina during development. The amount of tyrosinable (tyrosinated plus detyrosinated) tubulin increased approximately 110% from embryonic day 7 to 14. Then it decreased, and by day 19 it was similar to the value on day 7. This result did not change after hatching, at least up to day 20. The proportion of tyrosinated and detyrosinated tubulin significantly changed with the development of the animal. At embryonic day 7, these tubulin species were at a proportion of 70 and 30%, respectively, and after hatching, the values inverted, to 30 and 70%, respectively. This change did not correlate with the activity of the ligase relative to that of the carboxypeptidase, as measured in vitro. This observation suggested that a change in the turnover rate of microtubules, in the proportion of assembled and nonassembled tubulin pools, or in both had occurred. Coincident with the last possibility, the proportion of assembled tubulin was found to increase during the development of the animal. This finding suggests that the tyrosination state of tubulin may be determined, at least in part, by the assembly state.     

Relationship between the Golgi complex and microtubules enriched in detyrosinated or acetylated α-tubulin: studies on cells recovering from nocodazole and cells in the terminal phase of cytokinesis

Double immunofluorescence microscopy was used to study the relationship between the Golgi complex and microtubules enriched in posttranslationally modified tubulins in cultured mouse L929 fibroblasts. In interphase cells, the elements of the Golgi complex were grouped around the microtubule-organizing center. From here, tyrosinated microtubules extended to the periphery of the cells, whereas the distribution of detyrosinated and acetylated microtubules largely overlapped with that of the Golgi complex. Treatment of cells with 10 M nocodazole led to the disruption of all microtubules and dispersion of the Golgi elements. Following withdrawal of the drug, tyrosinated microtubules reformed first, followed by acetylated and then detyrosinated microtubules. In parallel, the Golgi elements moved back toward the juxtanuclear region and reestablished a close spatial relationship first with the acetylated and later also with the detyrosinated microtubules. Long-term recovery in the presence of 0.15 or 0.3 M nocodazole allowed partial reformation of tyrosinated and acetylated microtubules, whereas no or only a few detyrosinated microtubules were detected. At the same time, the Golgi elements were grouped closer together around or on one side of the nucleus in close relation to acetylated microtubules. In synchronized cells released from a mitotic block, a radiating array of tyrosinated microtubules was first formed, followed by acetylated and detyrosinated microtubules. The Golgi elements initially came together in a few groups and thereafter took an overall morphology similar to that in interphase cells. During this reunification, they showed a close spatial relationship to acetylated microtubules, whereas detyrosinated microtubules appeared only later. Microtubules enriched in acetylated and/or detyrosinated tubulin thus appear to take part in establishing and maintaining the organization of the Golgi elements within an interconnected supraorganellar system. Whether the acetylation and detyrosination of tubulin are directly involved in this process or merely represent two modifications within this subpopulation of microtubules remains unknown.On leave of absence from the Department of Histology and Embryology, Institute of Biostructure, Medical School, Warsaw, Poland     

Tyrosination of α‐tubulin controls the initiation of processive dynein–dynactin motility

Post‐translational modifications (PTMs) of α/β‐tubulin are believed to regulate interactions with microtubule‐binding proteins. A well‐characterized PTM involves in the removal and re‐ligation of the C‐terminal tyrosine on α‐tubulin, but the purpose of this tyrosination–detyrosination cycle remains elusive. Here, we examined the processive motility of mammalian dynein complexed with dynactin and BicD2 (DDB) on tyrosinated versus detyrosinated microtubules. Motility was decreased ~fourfold on detyrosinated microtubules, constituting the largest effect of a tubulin PTM on motor function observed to date. This preference is mediated by dynactin's microtubule‐binding p150 subunit rather than dynein itself. Interestingly, on a bipartite microtubule consisting of tyrosinated and detyrosinated segments, DDB molecules that initiated movement on tyrosinated tubulin continued moving into the segment composed of detyrosinated tubulin. This result indicates that the α‐tubulin tyrosine facilitates initial motor–tubulin encounters, but is not needed for subsequent motility. Our results reveal a strong effect of the C‐terminal α‐tubulin tyrosine on dynein–dynactin motility and suggest that the tubulin tyrosination cycle could modulate the initiation of dynein‐driven motility in cells.     

Partial purification and characterization of microtubular protein from Trypanosoma brucei

The tubulin proteins of the parasitic hemoflagellate Trypanosoma brucei brucei were purified and characterized. Cytoskeletal microtubules of trypanosomes do not disrupt under conditions used to solubilize brain tubulins. Trypanosomal tubulins, solubilized by extensive sonication, were partially purified from the crude cell extracts by taxol-mediated polymerization. Taxolinduced microtubules were identified by electron microscopy and analyzed biochemically. They consist predominantly of two proteins of about 52,000 and 56,000 Da. Their mobilities on sodium dodecyl sulfate gels differ slightly from those of bovine brain tubulins. Immunological cross-reactivity with antibodies raised against bovine brain tubulins confirmed the nature of the trypanosomal proteins. Peptide mapping of bovine and trypanosomal alpha- and beta-tubulins was performed by enzymatic digestion with staphylococcal protease V8 and chemical cleavage with N-chlorosuccinimide. In both cases, the peptide patterns generated from the trypanosomal alpha- and beta-tubulins were closely related to each other. This suggests that the trypanosomal alpha- and beta-tubulins may have remained more conserved during evolution than the tubulins from higher eukaryotes. The trypanosomal alpha-tubulin is post-translationally modified in vivo by the reversible addition of a tyrosine residue at its COOH terminus. As in higher eukaryotes, this reaction is completely specific for the alpha-polypeptide chain. Our observation represents the first documentation of the occurrence of COOH-terminal tyrosinolation of alpha-tubulin in an eukaryotic microorganism.     

Immunocytochemical localization of tubulins in spermatids and spermatozoa of Euptoieta hegesia (Lepidoptera: Nymphalidae)

A comparative analysis of the distribution of tubulin types in apyrene and eupyrene sperm of Euptoieta hegesia butterflies was carried out, also verifying the presence of tubulin in lacinate appendages of the eupyrene sperm. Ultrathin sections of LR White embedded spermatids and spermatozoa were labeled for alpha, beta, gamma, alpha-acetylated and alpha-tyrosinated tubulins. Apyrene and eupyrene spermatids show the same antibody recognition pattern for tubulins. All tubulin types were detected in axonemal microtubules. Alpha and gamma tubulins were also detected on the cytoplasmic microtubules. However, for beta and tyrosinated tubulins only scattered labeling was detected on cytoplasmic microtubules and acetylated tubulin was not detected. In apyrene and eupyrene spermatozoa only the axoneme labeling was analyzed since cytoplasmic microtubules no longer exist in these cells. Alpha, beta and tyrosinated tubulins were easily detected on the apyrene and eupyrene axoneme; gamma tubulin was strongly marked on eupyrene axonemes but was scattered on the apyrene ones. Acetylated tubulin appeared with scattered labeling on the axoneme of both sperm types. Our results demonstrate significant differences in tubulin distribution in apyrene and eupyrene axonemal and cytoplasmic microtubules. Extracellular structures, especially the lacinate appendages, were not labeled by antibodies for any tubulin.     

DNA-dependent RNA-polymerases from Artemia embryos. characterization of polymerases I and II from nauplius larvae.

Partial purification and characterization of DNA-dependent RNA-polymerases from nauplius larvae of the brine shrimp, Artemia salina, are described. Fractionation of solubilized RNA-polymerases on columns of DEAE-cellulose yielded partially purified preparations of RNA polymerases I and II. The properties of these enzymes were found to be similar to properties of corresponding enzymes from other animal sources. A significant change in the relative amounts of polymerases I and II occurs between 36 and 72 hr of development. Polymerase activity obtained from 36-hr nauplii consisted of approximately equal amounts of polymerases I and II, whereas polymerase II accounted for more than 80% of the activity recovered from 72-hr nauplii. Total polymerase activity was lower at 72 than at 36 hr. The significance of these changes in relation to the decrease in RNA synthesis in vivo that occurs after 36 hr is discussed.     

Tyrosinated,detyrosinated and acetylated tubulin isotypes in rat brain membranes. Their proportions in comparison with those in cytosol

The heterogeneity of -tubulin and the relative proportions of the tubulin isotypes were investigated in brain membranes of rats of 1, 25 and 180 days of age by using four anti--tubulin antibodies: a) the monoclonal DM1A antibody, specific for -tubulin; b) the monoclonal 6-11B-1 antibody, specific for acetylated tubulin; c) a polyclonal antibody (Glu antibody), specific for detyrosinated tubulin; and d) a polyclonal antibody (Tyr antibody), specific for tyrosinated tubulin. We found that rat brain membranes contain the three tubulin isotypes mentioned above. The proportions of tyrosinated and detyrosinated tubulin relative to total -tubulin were somewhat lower in membrane than in cytosol in animals of 25 and 180 days of age. At day one of development, the proportions in membrane were similar to those found in cytosol. With respect to the acetylated form, it was about 20 times higher in membrane than in cytosol at the three ages studied. The proportion of acetylated tubulin was determined in different subcellular fractions: myelin, synaptic vesicles, mitochondria, microsomes, and plasma membrane. While the amount of total tubulin differed between the different subcellular fractions, the proportion of acetylated tubulin relative to total -tubulin was constant and similar to that found in total membranes. The proportion of acetylated tubulin was also investigated in non-neural tissues (kidney, liver and lung). Although values for cytosol were about 10-fold higher than that found in brain cytosol, no detectable values for membranes could be obtained in these organs.