Tubulin binding sites on gamma-tubulin: identification and molecular characterization.
gamma-Tubulin is essential to microtubule organization in eukaryotic cells. It is believed that gamma-tubulin interacts with tubulin to accomplish its cellular functions. However, such an interaction has been difficult to demonstrate and to characterize at the molecular level. gamma-Tubulin is a poorly soluble protein, not amenable to biochemical studies in a purified form as yet. Therefore basic questions concerning the existence and properties of tubulin binding sites on gamma-tubulin have been difficult to address. Here we have performed a systematic search for tubulin binding sites on gamma-tubulin using the SPOT peptide technique. We find a specific interaction of tubulin with six distinct domains on gamma-tubulin. These domains are clustered in the central part of the gamma-tubulin primary amino acid sequence. Synthetic peptides corresponding to the tubulin binding domains of gamma-tubulin bind with nanomolar K(d)s to tubulin dimers. These peptides do not interfere measurably with microtubule assembly in vitro and associate with microtubules along the polymer length. On the tertiary structure, the gamma-tubulin peptides cluster to surface regions on both sides of the molecule. Using SPOT analysis, we also find peptides interacting with gamma-tubulin in both the alpha- and beta-tubulin subunits. The tubulin peptides cluster to surface regions on both sides of the alpha- and beta- subunits. These data establish gamma-tubulin as a tubulin ligand with unique tubulin-binding properties and suggests that gamma-tubulin and tubulin dimers associate through lateral interactions.
Llanos
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R
Département de Biologie Moléculaire et Structurale, CEA de Grenoble, 17 Rue des Martyrs, 38054 Grenoble Cedex 9, France.
Chevrier
V
V
Ronjat
M
M
Meurer-Grob
P
P
Martinez
P
P
Frank
R
R
Bornens
M
M
Wade
R H
RH
Wehland
J
J
Job
D
D
D000595:Amino Acid Sequence; D001665:Binding Sites; D019281:Dimerization; D006801:Humans; D008870:Microtubules / Q000737:chemistry / Q000648:ultrastructure; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D020033:Protein Isoforms / Q000737:chemistry; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance; D014404:Tubulin / Q000032:analysis / Q000737:chemistry*
D010446:Peptide Fragments; D020033:Protein Isoforms; D014404:Tubulin