Tubulin Binding Sites on -Tubulin: Identification and Molecular Characterization

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• 作者：Roxana Llanos ; V&eacute ; ronique Chevrier ; Michel Ronjat ; Patricia Meurer-Grob ; Pascal Martinez ; Ronald Frank ; Michel Bornens ; Richard H. Wade ; Juergen Wehland ; and Didier Job
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：November 30, 1999
• 年：1999
• 卷：38
• 期：48
• 页码：15712 - 15720
• 全文大小：767K
• 年卷期：v.38,no.48(November 30, 1999)
• ISSN：1520-4995

文摘

C="/images/gifchars/gamma.gif" BORDER=0 >-Tubulin is essential to microtubule organization in eukaryotic cells. It is believed that chars/gamma.gif" BORDER=0 >-tubulininteracts with tubulin to accomplish its cellular functions. However, such an interaction has been difficultto demonstrate and to characterize at the molecular level. chars/gamma.gif" BORDER=0 >-Tubulin is a poorly soluble protein, not amenableto biochemical studies in a purified form as yet. Therefore basic questions concerning the existence andproperties of tubulin binding sites on chars/gamma.gif" BORDER=0 >-tubulin have been difficult to address. Here we have performeda systematic search for tubulin binding sites on chars/gamma.gif" BORDER=0 >-tubulin using the SPOT peptide technique. We find aspecific interaction of tubulin with six distinct domains on chars/gamma.gif" BORDER=0 >-tubulin. These domains are clustered in thecentral part of the chars/gamma.gif" BORDER=0 >-tubulin primary amino acid sequence. Synthetic peptides corresponding to the tubulinbinding domains of chars/gamma.gif" BORDER=0 >-tubulin bind with nanomolar K_ds to tubulin dimers. These peptides do not interferemeasurably with microtubule assembly in vitro and associate with microtubules along the polymer length.On the tertiary structure, the chars/gamma.gif" BORDER=0 >-tubulin peptides cluster to surface regions on both sides of the molecule.Using SPOT analysis, we also find peptides interacting with chars/gamma.gif" BORDER=0 >-tubulin in both the chars/alpha.gif" BORDER=0>- and chars/beta2.gif" BORDER=0 ALIGN="middle">-tubulin subunits.The tubulin peptides cluster to surface regions on both sides of the chars/alpha.gif" BORDER=0>- and chars/beta2.gif" BORDER=0 ALIGN="middle">- subunits. These data establishchars/gamma.gif" BORDER=0 >-tubulin as a tubulin ligand with unique tubulin-binding properties and suggests that chars/gamma.gif" BORDER=0 >-tubulin and tubulindimers associate through lateral interactions.