TUBULIN POLYGLYCYLATION - DIFFERENTIAL POSTTRANSLATIONAL MODIFICATIONOF DYNAMIC CYTOPLASMIC AND STABLE AXONEMAL MICROTUBULES IN PARAMECIUM

Polyglycylation, a posttranslational modification of tubulin, was disc overed in the highly stable axonemal microtubules of Paramecium cilia where it involves the lateral linkage of up to 34 glycine units per tu bulin subunit. The observation of this type of posttranslational modif ication mainly in axonemes raises the question as to its relationship with axonemal organization and with microtubule stability. This led us to investigate the glycylation status of cytoplasmic microtubules tha t correspond to the dynamic microtubules in Paramecium. Two anti-glycy lated tubulin monoclonal antibodies (mAbs), TAP 952 and AXO 49, are sh own here to exhibit different affinities toward mono- and polyglycylat ed synthetic tubulin peptides. Using immunoblotting and mass spectrome try, we show that cytoplasmic tubulin is glycylated. In contrast to th e highly glycylated axonemal tubulin, which is recognized by the two m Abs, cytoplasmic tubulin reacts exclusively with TAP 952, and the alph a- and beta-tubulin subunits are modified by only 1-5 and 2-9 glycine units, respectively. Our analyses suggest that most of the cytoplasmic tubulin contains side chain lengths of 1 or 2 glycine units distribut ed on several glycylation sites. The subcellular partition of distinct polyglycylated tubulin isoforms between cytoplasmic and axonemal comp artments implies the existence of regulatory mechanisms for glycylatio n. By following axonemal tubulin immunoreactivity with anti-glycylated tubulin mAbs upon incubation with a Paramecium cellular extract, the presence of a deglycylation enzyme is revealed in the cytoplasm of thi s organism. These observations establish that polyglycylation is rever sible and indicate that, in vivo, an equilibrium between glycylating a nd deglycylating enzymes might be responsible for the length of the ol igoglycine side chains of tubulin.