Abstract
ASAP1 is an Arf GTPase-activating protein (GAP) that functions on membrane surfaces to catalyze the hydrolysis of GTP bound to Arf. ASAP1 contains a tandem of BAR, pleckstrin homology (PH), and Arf GAP domains and contributes to the formation of invadopodia and podosomes. The PH domain interacts with the catalytic domain influencing both the catalytic and Michaelis constants. Tandem BAR-PH domains have been found to fold into a functional unit. The results of sedimentation velocity studies were consistent with predictions from homology models in which the BAR and PH domains of ASAP1 fold together. We set out to test the hypothesis that the BAR domain of ASAP1 affects GAP activity by interacting with the PH and/or Arf GAP domains. Recombinant proteins composed of the BAR, PH, Arf GAP, and Ankyrin repeat domains (called BAR-PZA) and the PH, Arf GAP, and Ankyrin repeat domains (PZA) were compared. Catalytic power for the two proteins was determined using large unilamellar vesicles as a reaction surface. The catalytic power of PZA was greater than that of BAR-PZA. The effect of the BAR domain was dependent on the N-terminal loop of the BAR domain and was not the consequence of differential membrane association or changes in large unilamellar vesicle curvature. The Km for BAR-PZA was greater and the kcat was smaller than for PZA determined by saturation kinetics. Analysis of single turnover kinetics revealed a transition state intermediate that was affected by the BAR domain. We conclude that BAR domains can affect enzymatic activity through intraprotein interactions.
Highlights
ASAP1 is an Arf GTPase-activating protein (GAP) that functions on membrane surfaces to catalyze the hydrolysis of GTP bound to Arf
The nomenclature we use for the recombinant proteins refers to the domain structure: the protein composed of the BAR and pleckstrin homology (PH) domains is called BAR-PH, the protein composed of the BAR, PH, Arf GAP, and Ank repeat domains is called BAR-PZA, the protein composed of the PH, Arf GAP, and Ank repeat domains is called PZA, and the protein composed of the Arf GAP and Ank repeat domains is called ZA
We examined the effect of the BAR domain of ASAP1 on the catalytic activity of the protein
Summary
Proteins—Bacterial expression vectors for His10-ASAP1(325–724) (PZA) [26], ASAP1-(1–724)-His (BAR-PZA) [27] and myristoylated Arf (myrArf1) [28] have been described before. In fixed time point assays and in single turnover kinetics, [␣-32P]GTP bound to Arf was used as a substrate, and the conversion of protein bound [␣-32P]GTP to [␣-32P]GDP was measured. Analytical Ultracentrifugation Sedimentation Velocity— Stock samples of BAR-PZA (6.7 M), BAR-PH (14.1 M), PZA (22.4 M), and ZA (31.8 M), purified by size exclusion with Sephacryl columns (GE Healthcare) in 200 mM K2PO4, pH 7.0, were used to prepare experimental samples at several different loading concentrations by serial dilution using the respective size exclusion chromatography running column buffer. For this analysis, an average of 30 scans were loaded and modeled with a continuous c(s) distribution using a resolution of 200 s-values between 0.2 to 8.0 S and maximum entropy regularization (p ϭ 0.68) [31]. Images were recorded digitally on a 794 Gatan MultiScan charge-coupled device camera with the DigitalMicrograph software package [35]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
