Tubulin folding is altered by mutations in a putative GTP binding motif.
Tubulins contain a glycine-rich loop, that has been implicated in microtubule dynamics by means of an intramolecular interaction with the carboxy-terminal region. As a further extension of the analysis of the role of the carboxy-terminal region in tubulin folding we have mutated the glycine-rich loop of tubulin subunits. An alpha-tubulin point mutant with a T150-->G substitution (the corresponding residue present in beta-tubulin) was able to incorporate into dimers and microtubules. On the other hand, four beta-tubulin point mutants, including the G148-->T substitution, did not incorporate into dimers, did not release monomers, but were able to form C900 and C300 complexes (intermediates in the process of tubulin folding). Three other mutants within this region (which approximately encompasses residues 137-152) were incapable of forming dimers and C300 complexes but gave rise to the formation of C900 complexes. These results suggest that tubulin goes through two sequential folding states during the folding process, first in association with TCP1-complexes (C900) prior to the transfer to C300 complexes. It is this second step that implies binding/hydrolysis of GTP, reinforcing our previous proposed model for tubulin folding and assembly.
Zabala
J C
JC
Departamento de Biologia Molecular, Facultad de Medicina, Universidad de Cantabria, Spain.
Fontalba
A
A
Avila
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D017931:DNA Primers / Q000235:genetics; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014404:Tubulin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D014404:Tubulin; D006160:Guanosine Triphosphate