Release of <i>C</i>-terminal tyrosine from tubulin and microtubules at steady state

Arce, Carlos A.; Barra, Héctor S.

doi:10.1042/bj2260311

Cited by 54 publications

(40 citation statements)

References 22 publications

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“…Segmental labelling of microtubules has been described previously (Geuens et al, 1986;Webster et al, 1987). Detyrtubulin stretches can be generated by an individual turnover from tyr-to detyr-tubulin, which is facilitated on pre-existing microtubules (Arce and Barra, 1985). Long stretches of detyrtubulin have been found in the Chlamydomonas flagellum along the outer wall of the B tubules (Johnson, 1998).…”

Section: Discussionmentioning

confidence: 99%

Tubulin detyrosination promotes monolayer formation and apical trafficking in epithelial cells

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Freikamp

et al. 2012

Journal of Cell Science

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SummaryThe role of post-translational tubulin modifications in the development and maintenance of a polarized epithelium is not well understood. We studied the balance between detyrosinated (detyr-) and tyrosinated (tyr-) tubulin in the formation of MDCK cell monolayers. Increased quantities of detyrosinated microtubules were detected during assembly into confluent cell sheets. These tubules were composed of alternating stretches of detyr-and tyr-tubulin. Constant induction of tubulin tyrosination, which decreased the levels of detyr-tubulin by overexpression of tubulin tyrosine ligase (TTL), disrupted monolayer establishment. Detyr-tubulin-depleted cells assembled into isolated islands and developed a prematurely polarized architecture. Thus, tubulin detyrosination is required for the morphological differentiation from non-polarized cells into an epithelial monolayer. Moreover, membrane trafficking, in particular to the apical domain, was slowed down in TTL-overexpressing cells. This effect could be reversed by TTL knockdown, which suggests that detyr-tubulin-enriched microtubules serve as cytoskeletal tracks to guide membrane cargo in polarized MDCK cells.

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Section: Discussionmentioning

confidence: 99%

Tubulin detyrosination promotes monolayer formation and apical trafficking in epithelial cells

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Große

Freikamp

et al. 2012

Journal of Cell Science

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“…Microtubule protein free of tubulin: tyrosine ligase was purified from rat brain by three cycles of assembly-disassembly as previously described [14]. Partially purified tubulin carboxypeptidase was obtained from rat brain as described by Argaraiia et al (231.…”

Section: Other Analytical Proceduresmentioning

confidence: 99%

“…['4C]Tyrosinated tubulin from rat brain was prepared as previously described [14]. The radioactive tubulin was purified by the Method of Murphy and Borisy [22].…”

Section: Other Analytical Proceduresmentioning

confidence: 99%

Relationship between the tyrosination state of tubulin and the activities of tubulin: tyrosine ligase and tubulin carboxypeptidase in rat muscle during development

Ferro

Reinach

1988

European Journal of Biochemistry

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Tubulin can be post-translationally modified by the incorporation or the release of a tyrosine residue at the COOH-terminus of the ct subunit. The present study demonstrates that rat muscle soluble preparations contain tubulin carboxypeptidase besides tubulin : tyrosine ligase.The state of tyrosination of tubulin and the activities of both the ligase and the carboxypeptidase were examined in rat muscle during development. The proportion of tyrosinated tubulin with respect to tyrosinable tubulin (tyrosinated plus detyrosinated tubulin) decreased from 83% (new-born rats) to 28% (adult rats) with the corresponding increase in detyrosinated tubulin. The activities of the enzymes decreased continuously and in a near parallel fashion during development. These results indicate that the changes in the tyrosination state of tubulin can not be explained merely by changes in the enzyme activities.We also compared the ability of rat muscle and brain [14C]tyrosinated tubulin to act as substrate of the carboxypeptidase. Muscle tubulin was found to be a less efficient substrate than brain tubulin.Tubulin is subjected to a post-translational modification by which a tyrosine residue is removed or added back at the COOH-terminus of the ct chain [l -51. Two enzymes are involved in this tyrosination-detyrosination reaction, tubulin : tyrosine ligase and tubulin carboxypeptidase, respectively. The ligase was first found in brain tissue but soon after was shown to be present in other tissues [6] and also in lower eukaryotic organisms [7, 81. On the other hand, the biochemical characterization of the carboxypeptidase was only done in brain. In this paper we show that rat skeletal muscle contains tubulin carboxypeptidase.In vitro, the ligase was shown to act on non-assembled tubulin but not on microtubules [9]. Using brain slices, we were unable. to determine unequivocally the in vivo specificity of this enzyme [lo]. Studies with chicken erythrocytes [ l l ] and cultured cells [12] showed the preference of the ligase for nonassembled tubulin so that this pool in living cells is apparently maintained fully tyrosinated. On the other hand, the carboxypeptidase acts preferentially on microtubules [9, 13, 141 in such a way that the proportions of tyrosinated and detyrosinated tubulin (referred to as the tyrosination state) in these structures vary with time [15]. lmmunofluorescence with specific peptide antibodies has previously shown that tyrosinated and detyrosinated tubulin are present in distinct, but overlapping, subsets of microtubules in cultured cells [16, 171. Total soluble tubulin obtained under depolymerizing conditions is composed by a mixture of the tyrosinated and detyrosinated forms whose relative proportions have been shown to change with development [18 -201. Among the possible mechanisms that could determine the tyrosination state of tubulin we considered the two most likely to be: (a) the

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“…Unlabelled three-cycle microtubule protein was obtained from rat brain as previously described [12].…”

Section: Other Analytical Proceduresmentioning

confidence: 99%

“…Assembly-competent [14C]tyrosinated tubulin of high specific radioactivity was prepared by incorporation of [14C]tyrosine into tubulin of a soluble brain extract [12] and purified by one cycle of assembly/disassembly and chromatography of phosphocellulose as previously described [18].…”

Section: Other Analytical Proceduresmentioning

confidence: 99%

Tyrosination of microtubules and non‐assembled tubulin in brain slices

Beltramo

Arce

Barra

1987

European Journal of Biochemistry

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Brain slices were used to examine comparatively the incorporation of ['4C]tyrosine into the C terminus of a-tubulin of the microtubule and non-assembled tubulin pools. We found that the incorporation of ['4C]tyrosine from 5 min up to 60 min of incubation was higher in microtubules than in non-assembled tubulin. The possibility that this result was due to the activity of tubulin carboxypeptidase or tubu1in:tyrosine ligase during the in vitro isolation of tubulin was discarded.We also found that tubulin: tyrosine ligase was mainly associated with microtubules when brain slices were homogenized under microtubule-preserving conditions. Conversely the enzyme behaved as a soluble entity when homogenization was performed under conditions that do not preserve microtubules. In addition, soluble tubulin: tyrosine ligase did not become sedimentable when in vitro conditions were changed to induce the formation of microtubules.The results presented in this work indicate the possibility that, in vivo, microtubules and not tubulin dimers are the major substrate for tubulin: tyrosine ligase. This is in contrast with previous findings from in vitro experiments, which showed a preference of the ligase for non-assembled tubulin.The C terminus of the 01 chain of tubulin can be modified in such a way that the terminal tyrosine residue can be released by tubulin carboxypeptidase [2, 31 and incorporated again by tubulin: tyrosine ligase (TTLase) [4-61. According to studies in vitro [7, 81 this cyclic tyrosination/detyrosination reaction does not regulate the microtubule assembly. Despite the work from different laboratories, the significance of this reaction remains unknown. We have focused our attention on the regulation of the state of tyrosination of tubulin and microtubules, that is, the proportion of tyrosinated and nontyrosinated tubulin molecules in both fractions. Rodriguez and Borisy [9] showed that in chick brain the proportion of tyrosinated tubulin in the microtubule and non-assembled tubulin fractions changes with the development of the animal. It is possible that the tyrosination state of the microtubule and non-assembled tubulin pools depends, at least in part, on the relative capabilities of both states of aggregation to act as substrates of TTLase and tubulin carboxypeptidase. It was previously found that in vitro TTLase acts mainly on nonassembled tubulin 17, 101 and tubulin carboxypeptidase on microtubules [7,11, 121. In this work we used brain slices to investigate comparatively the incorporation of [14C]tyrosine into the microtubule and non-assembled tubulin fractions and the distribution of TTLase between both pools.

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Release of C-terminal tyrosine from tubulin and microtubules at steady state

Cited by 54 publications

References 22 publications

Tubulin detyrosination promotes monolayer formation and apical trafficking in epithelial cells

Tubulin detyrosination promotes monolayer formation and apical trafficking in epithelial cells

Relationship between the tyrosination state of tubulin and the activities of tubulin: tyrosine ligase and tubulin carboxypeptidase in rat muscle during development

Tyrosination of microtubules and non‐assembled tubulin in brain slices

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