Atypical Binding Behavior of Epsin‐like Clathrin Adaptor 1 to Diacylglycerol Pyrophosphate

Abstract

Epsin N‐terminal homology (ENTH) and AP‐180 N‐terminal homology (ANTH) domain proteins are membrane binding proteins known to interact with polyphosphoinositides (PIPs). They share function, structure and sequence similarity, and are grouped together as A/ENTH domain proteins. The ENTH domain contains a distinctive N‐terminal helix (Ho) region that plays a crucial role in membrane bending. This Ho helix is not usually found in ANTH domains. Epsin‐like Clathrin Adaptor 1 (ECA1) is one such A/ENTH protein that acts as an adaptor protein in clathrin mediated endocytosis. It interacts with clathrin via its C‐terminus, and PIPs via its N‐terminus. In addition to having ANTH domain sequence similarity, ECA1 also contains the distinctive Ho helix region that is generally found in the ENTH domain. ECA1 is therefore best characterized as an N‐ANTH domain protein with a unique combination of both ANTH and ENTH regions. ECA1 is known to play a role in stress signaling in plants and also binds to phosphatidic acid (PA) upon salt stress. Like proteins, membrane lipids undergo drastic changes during stress stimuli. PA is a lipid second messenger that rapidly and transiently increases under stress stimuli. Upon rise of PA levels another lipid, diacylglycerol pyrophosphate (DGPP) starts to accumulate. DGPP is phosphorylated PA. It is suggested to be synthesized at higher levels as a signal for PA attenuation during stress. We recently showed in vitro that ECA1‐PA ‐affinity is modulated as a function of membrane curvature stress. In this research, we investigate ECA1 affinity to DGPP. We hypothesize that the PA target ECA1 has affinity for its metabolite DGPP which is highly negatively charged and this affinity is variable at varying pH. We tested ECA1 affinity to DGPP using liposome binding assay along with other anionic lipids; i.e., phosphatidylserine, phosphatidylinositol (4)‐phosphate and phosphatidylinositol (4,5)‐bisphosphate in the presence and absence of phosphatidylethanolamine. Our results show striking evidence that protein‐lipid interactions are not simply based on electrostatics. We also show that higher charge density on membranes does not always correspond to higher protein affinity. In fact, DGPP showed lesser protein binding to ECA1 compared to PA although DGPP has a higher negative charge. This study brings us a step closer towards understanding the complexity of protein interactions with anionic lipids and opens the discussion to lipid homeostasis upon salt stress.Support or Funding InformationDepartment of Biological Science, Kent State University and NSF CHE 1412920This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.