The conversion of tubulin carboxyl groups to amides has a stabilizing effect on microtubules.
In a recent study, we demonstrated that the conversion of carboxyl residues in the C-termini of tubulin to neutral amides with glycine ethyl ester enhanced the ability of the protein to assemble into microtubules and decreased its interaction with microtubule-associated proteins (MAPs). In this work, we investigated the effects of carboxyl modification on the dynamic behavior of microtubules at polymer mass steady state. After steady state, microtubules assembled from unmodified tubulin were sheared, and the mean polymer lengths decreased to 5 microns and then increased to 29 microns within 130 min. In contrast, lengths of sheared microtubules polymerized from tubulin containing 23 modified carboxyl groups increased by only 2-fold. Stabilization of polymer lengths was also observed directly by video-enhanced light microscopy of microtubules grown off of axonemes. Rapid shortening was seen in microtubules composed of unmodified but not modified tubulin. Further evidence for the less dynamic behavior of microtubules as a result of carboxyl modification was obtained from kinetic studies of the elongation phase during assembly which showed a 3-fold lower off-rate constant, k-, for modified microtubules. Another effect of the modification was a 12-fold reduction in the steady-state rate constant for GTP hydrolysis (165 s-1 for unmodified and 14 s-1 for modified). These results suggest that reduction of the negative charges in the C-termini by modification of the acidic residues stabilizes microtubules against depolymerization. MAPs may stabilize microtubules in an analogous manner.
Mejillano
M R
MR
Department of Biochemistry, University of Kansas, Lawrence 66045-2106.
Tolo
E T
ET
Williams
R C
RC
Himes
R H
RH
D000577:Amides / Q000737:chemistry*; D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D005998:Glycine / Q000031:analogs & derivatives; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008853:Microscopy; D008854:Microscopy, Electron; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism; D011108:Polymers / Q000737:chemistry*; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism
D000577:Amides; D011108:Polymers; D014404:Tubulin; C022010:glycine ethyl ester; D006160:Guanosine Triphosphate; D005998:Glycine