Abstract
Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a key enzyme of the Golgi system because it produces its lipid hallmark - the phosphatidylinositol 4-phosphate (PI4P). It is recruited to Golgi by the Golgi resident ACBD3 protein, regulated by 14-3-3 proteins and it also serves as an adaptor because it recruits the small GTPase Rab11. Here, we analyzed the protein complexes formed by PI4KB in vitro using small angle x-ray scattering (SAXS) and we discovered that these protein complexes are highly flexible. The 14-3-3:PI4KB:Rab11 protein complex has 2:1:1 stoichiometry and its different conformations are rather compact, however, the ACBD3:PI4KB protein complex has both, very compact and very extended conformations. Furthermore, in vitro reconstitution revealed that the membrane is necessary for the formation of ACBD3:PI4KB:Rab11 protein complex at physiological (nanomolar) concentrations.
Highlights
14-3-3 proteins are known to regulate hundreds of proteins in phosphorylation dependent manner[24] including regulators of G-protein signaling[25], transcription factors such as FOXO26,27 and multiple other enzymes
Even the interpretation of simple molecular envelopes is difficult without the knowledge of the exact stoichiometry
Previous crystallographic studies suggest that PI4KB:Rab[11] form a 1:1 complex[17] and the stoichiometry of 14-3-3:PI4KB:Rab[11] protein complex was never experimentally tackled based on the known stoichiometries of PI4KB:Rab[11] (1:1) and 14-3-3:PI4KB (2:2) protein complexes we expected a 2:2:2 14-3-3:PI4KB:Rab[11] complex to be formed
Summary
14-3-3 proteins are known to regulate hundreds of proteins in phosphorylation dependent manner[24] including regulators of G-protein signaling[25], transcription factors such as FOXO26,27 and multiple other enzymes. ACBD3 protein is Golgi localized and serves as a signaling hub[34] The GOLD domain anchors ACBD3 to the Golgi via its interaction with giantin[35] while the Q domain serves to bind the very N-terminal helix of PI4KB bringing it to close vicinity of the membrane and activating it[7]. Viral 3A proteins bind the GOLD domain and literally pin it down to target membrane[36] in order to recruit PI4KB to viral replication sites[37,38,39]. Its interaction with PI4KB was structurally characterized and revealed that PI4KB does not affect neither switch I nor switch II region of Rab1117 and most like just serves to recruit Rab[11] and its effectors to proper (Golgi) membrane[11]
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