WASP (Wiskott-Aldrich syndrome protein) has been proposed to adopt a closed conformation (autoinhibited conformation) due to interaction between the carboxy terminal and the GTPase binding domain. Various WASP-interacting proteins have been suggested to relieve this autoinhibition. We have used the split YFP (Yellow Fluorescent Protein) to analyze the conformation of WASP. Saccharomyces cerevisiae cells expressing YFP1-154-WASP-YFP155-238 were found to exhibit YFP fluorescence while cells expressing of YFP1-154-WASP and WASP-YFP155-238 did not suggesting an intramolecular complementation of the YFP molecule. The fluorescence signal of YFP1-154-WASP-YFP155-238 was enhanced in the presence of WIP (WASP-interacting protein) however this is not due to the increased stability of YFP1-154-WASP-YFP155-238. Expression of Toca-1 and Nck1 reduced the YFP fluorescence from YFP1-154-WASP-YFP155-238 even in the presence of WIP suggesting that binding of Toca-1 or Nck1 altered the conformation of YFP1-154-WASP-YFP155-238. Thus both Nek1 and Toca-1 can relieve the autoinhibition of the WASP molecule. (C) 2007 Elsevier Inc. All rights reserved.