Forms bearing four to six units were recovered specifically in the polymeric fraction, whereas those bearing one to three units were distributed evenly between polymeric and soluble fractions. Partition of glutamylated alpha-tubulin isoforms was also correlated with the length of the polyglutamyl chain.
Evidence was obtained that deglutamylation of alpha-tubulin operates following two distinct rates depending on the length of the polyglutamyl chain, the distal units (4th-6th) being removed rapidly whereas the proximal ones (1st-3rd) appearing much more resistant to deglutamylation. Chase experiments revealed the existence of a reversal reaction that, in the case of alpha-tubulin, was not affected by microtubule drugs, suggesting that deglutamylation of this subunit can occur on both polymers and soluble tubulin. These results strongly suggest that microtubule polymers are the preferred substrate for polyglutamylation. Nocodazole markedly inhibited incorporation of glutamate into alpha- and beta-tubulin, whereas taxol had no effect for alpha-tubulin and a stimulating effect for beta-tubulin. Selective posttranslational labeling with glutamate and immunoblotting with a specific monoclonal antibody (GT335) enabled us to analyze polyglutamylation of both alpha and beta subunits. The relationship between microtubule dynamics and polyglutamylation of tubulin was investigated in young differentiating mouse brain neurons.
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