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Tyrosination of microtubules and non-assembled tubulin in brain slices
Abstract:
Brain slices were used to examine comparatively the incorporation of [14C]tyrosine into the C terminus of alpha-tubulin of the microtubule and non-assembled tubulin pools. We found that the incorporation of [14C]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 tubulin: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.
Insights
This study reveals that tubulin tyrosine ligase preferentially binds to microtubules in vivo, unlike previous in vitro findings. This suggests microtubules, not tubulin dimers, are the primary substrate for this enzyme.
Area of Science:
- Cell Biology
- Neuroscience
- Protein Biochemistry
Background:
- Tubulin is a key component of microtubules, essential for cellular structure and function.
- The C-terminal tyrosine of alpha-tubulin is dynamically regulated by tubulin carboxypeptidase and tubulin:tyrosine ligase.
- Understanding the localization and substrate preference of tubulin:tyrosine ligase is crucial for comprehending microtubule dynamics.
Purpose of the Study:
- To investigate the substrate preference of tubulin:tyrosine ligase in brain slices.
- To determine whether microtubules or non-assembled tubulin are the primary in vivo substrate for tubulin:tyrosine ligase.
- To examine the association of tubulin:tyrosine ligase with microtubules under different homogenization conditions.
Main Methods:
- Utilized [14C]tyrosine incorporation assays in brain slices to quantify tubulin modification.
- Performed differential homogenization techniques (microtubule-preserving vs. non-preserving) to assess enzyme localization.
- Conducted in vitro assays to evaluate the effect of microtubule assembly on tubulin:tyrosine ligase activity and sedimentation.
Main Results:
- [14C]tyrosine incorporation into alpha-tubulin was significantly higher in polymerized microtubules compared to non-assembled tubulin pools.
- Tubulin:tyrosine ligase was found to be primarily associated with microtubules under conditions preserving microtubule integrity.
- Soluble tubulin:tyrosine ligase did not associate with newly formed microtubules in vitro, indicating a specific in vivo association.
Conclusions:
- Microtubules, rather than soluble tubulin dimers, are likely the major in vivo substrate for tubulin:tyrosine ligase.
- The findings challenge previous in vitro studies suggesting a preference for non-assembled tubulin.
- This work provides critical insights into the in vivo regulation of tubulin carboxytagmentation and microtubule dynamics.