Single Molecule Diffusion Studies Reveal Rapid Translations Across Target Membrane Surfaces for both p40Phox PX domain and Class III PI3K VPS34 Complex 1, the Autophagy Lipid Kinase

Single molecule analyses are providing new insights into the molecular mechanisms by which peripheral membrane proteins approach and translate across a target bilayer surface before stable docking. Here we compare and contrast the translational movements of four diffusers on a representative supported lipid bilayer: (i) the fluorescent lipid LRB-(DO)PE, (ii) the G-protein Rab1A anchored to (DO)PE; (iii) the PI(3)P sensor p40Phox PX domain, and (iv) the autophagy lipid kinase VPS34 Complex 1. The latter enzyme is a Class III PI-3-Kinase and its unusual architecture exhibits two distinct membrane-docking "feet" unique to Class III PI3Ks. Notably, the membrane contact surfaces of the PX domain and autophagy lipid kinase each possess multiple positive charges suitable for electrostatic interactions with their anionic target membrane. The present single molecule tracking studies reveal that both the fluorescent lipid and the lipid-anchored G protein Rab1A exhibit 2-dimensional translation typical of simple lipid diffusion in the bilayer. Single molecule density measurements indicate both PX domain and autophagy lipid kinase exhibit significantly reduced membrane docking when the bilayer negative charge is decreased, confirming the importance of their protein-bilayer electrostatic interactions. Single molecule tracking studies reveal that both PX domain and autophagy lipid kinase exhibit hopping movements across the bilayer surface during their initial bilayer contacts, yielding a significantly higher frequency of long range displacements than allowed by lipid 2-D diffusion. After transitioning from hopping to a stable bilayer-docked state, both PX domain and autophagy lipid kinase exhibit 2-D diffusion slower than lipid diffusion indicating the appearance of increased frictional drag against the bilayer due to new protein-bilayer contacts. PX domain requires its target lipid for the transition from electrostatic hopping to stable bilayer docking, while autophagy lipid kinase can dock spontaneously to anionic lipid bilayers even in the absence of its substrate lipid. In principle, the slowly diffusing, bilayer-docked states of PX domain and autophagy lipid kinase could be interrupted by unbinding-hopping-rebinding events. However, such events are not detected for PX domain in the presence of target lipid (DO)PI(3)P nor for autophagy lipid kinase in the presence of substrate lipid (DO)PI. Instead, unbinding-hopping-rebinding events are only observed for autophagy lipid kinase in the absence of substrate lipid, indicating that the presence of a specific binding lipid reduces the frequency or spatial amplitude of such events. Overall, the findings provide evidence that p40Phox PX domain possesses a search mechanism involving electrostatic hopping that speeds its acquisition of target lipid, as previously observed for PH domains. The evidence indicates that the autophagy lipid kinase also exhibits electrostatic hopping/skating behavior that is proposed to play a functional role by speeding regulatory encounters with autophagy lipid kinase activators and effectors. Moreover, the ability of the lipid kinase to spontaneously dock to anionic membranes without a target lipid could facilitate its distribution across broad regions of the growing autophagosome. Finally, the autophagy lipid kinase exhibits multiple translational modes that we hypothesize are enabled by its two membrane docking feet.