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Functionally distinct isoforms of dynactin are expressed in human neurons
M K Tokito1, D S Howland, V M Lee
1Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia 19104-6046, USA.
Molecular Biology of the Cell
|August 1, 1996
Summary
Researchers identified two human isoforms of p150Glued, a key dynactin protein. One isoform binds microtubules, while the other does not, suggesting a novel neuronal function for dynactin complexes lacking direct microtubule interaction.
Area of Science:
- Cell Biology
- Neuroscience
- Molecular Biology
Background:
- P150Glued is the largest subunit of dynactin, essential for cytoplasmic dynein binding and microtubule-based vesicle transport.
- Dynactin is a multi-subunit protein complex crucial for intracellular transport in eukaryotic cells.
Purpose of the Study:
- To investigate the functional differences between p150Glued and its p135 isoform, particularly their interaction with microtubules and cytoplasmic dynein.
- To characterize the assembly and cellular localization of these dynactin isoforms in human brain.
Main Methods:
- Isolation of human cDNAs encoding p150Glued and p135 isoforms.
- Transient transfection assays to assess protein expression and localization.
- In vitro microtubule-binding assays to determine microtubule interaction.
- Sequential immunoprecipitation assays to analyze protein complex formation.
Main Results:
- Alternative mRNA splicing of the DCTN1 gene generates p150Glued and p135 isoforms in human brain.
- The p150Glued isoform binds to microtubules, whereas the p135 isoform lacks this ability due to the absence of a microtubule-binding motif.
- Both isoforms bind to cytoplasmic dynein and partition similarly into cytosolic and membrane fractions.
- Distinct dynactin complexes, sedimenting at approximately 20 S, are formed by these isoforms in the brain.
Conclusions:
- The p135 isoform represents a distinct form of dynactin that does not directly bind microtubules.
- These findings suggest a conserved neuronal function for dynactin complexes that interact with cytoplasmic dynein but not microtubules.
- The differential binding properties of dynactin isoforms may play a role in regulating intracellular transport in neurons.