Dataset Information

An improved protein lipid overlay assay for studying lipid-protein interactions.

ABSTRACT: Background:Lipids perform multiple functions in the cell, and lipid-protein interactions play a key role in metabolism. Although various techniques have been developed to study lipid-protein interactions, the interacting protein partners that bind to most lipids remain unknown. The protein lipid overlay (PLO) assay has revealed numerous lipid-protein interactions, but its application suffers from unresolved technical issues. Results:Herein, we found that blocking proteins may interfere with interactions between lipids and their binding proteins if a separate blocking step is carried out before the incubation step in the PLO assay. To overcome this, we modified the PLO assay by combining an incubation step alongside the blocking step. Verification experiments included phosphatidylinositol-3-phosphate (PI3P) and its commercially available interacting protein G302, C18:1, C18:2, C18:3 and the Arabidopsis plasma membrane H+-ATPase (PM H+-ATPase) AHA2 C-terminus, phosphatidylglycerol (PG) and AtROP6, and phosphatidylserine (PS) and the AHA2 C-terminus. The lipid-protein binding signal in the classical PLO (CPLO) assay was weak and not reproducible, but the modified PLO (MPLO) assay displayed significantly improved sensitivity and reproducibility. Conclusions:This work identified a limitation of the CPLO assay, and both sensitivity and reproducibility were improved in the modified assay, which could prove to be more effective for investigating lipid-protein interactions.

SUBMITTER: Han X

PROVIDER: S-EPMC7060618 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

An improved protein lipid overlay assay for studying lipid-protein interactions.

Han Xiuli X Yang Yongqing Y Zhao Fengyun F Zhang Tianren T Yu Xiang X

Plant methods 20200306

<h4>Background</h4>Lipids perform multiple functions in the cell, and lipid-protein interactions play a key role in metabolism. Although various techniques have been developed to study lipid-protein interactions, the interacting protein partners that bind to most lipids remain unknown. The protein lipid overlay (PLO) assay has revealed numerous lipid-protein interactions, but its application suffers from unresolved technical issues.<h4>Results</h4>Herein, we found that blocking proteins may inte ...[more]

PMID: 32165912

Similar Datasets

Project description:A critical challenge for all organisms is to carefully control the amount of lipids they store. An important node for this regulation is the protein coat present at the surface of lipid droplets (LDs), the intracellular organelles dedicated to lipid storage. Only limited aspects of this regulation are understood so far. For the probably best characterized case, the regulation of lipolysis in mammals, some of the major protein players have been identified, and it has been established that this process crucially depends on an orchestrated set of protein-protein interactions. Proteomic analysis has revealed that LDs are associated with dozens, if not hundreds, of different proteins, most of them poorly characterized, with even fewer data regarding which of them might physically interact. To comprehensively understand the mechanism of lipid storage regulation, it will likely be essential to define the interactome of LD-associated proteins.Previous studies of such interactions were hampered by technical limitations. Therefore, we have developed a split-luciferase based protein-protein interaction assay and test for interactions among 47 proteins from Drosophila and from mouse. We confirmed previously described interactions and identified many new ones. In 1561 complementation tests, we assayed for interactions among 487 protein pairs of which 92 (19%) resulted in a successful luciferase complementation. These results suggest that a prominent fraction of the LD-associated proteome participates in protein-protein interactions.In targeted experiments, we analyzed the two proteins Jabba and CG9186 in greater detail. Jabba mediates the sequestration of histones to LDs. We successfully applied our split luciferase complementation assay to learn more about this function as we were e.g. able to map the interaction between Jabba and histones. For CG9186, expression levels affect the positioning of LDs. Here, we reveal the ubiquitination of CG9186, and link this posttranslational modification to LD cluster induction.

Project description:The regulation of organismic lipid storage amounts is a central goal of all organisms. Proteins associated with the dedicated lipid storage organelles, the so-called lipid droplets (LDs), are instrumental to this regulation. LD proteomes of diverse organisms have been characterized by mass spectrometry. Yet, the functional characterization of most of these proteins is still lagging behind. Moreover, knowledge of interactions among LD-associated proteins is sparse and mostly limited to proteins involved in mammalian lipolysis regulation. This process crucially depends on an orchestrated set of protein-protein interactions which limit the accessibility, and thus remobilization, of the lipid stores. Thus, a better knowledge of the LD-associated protein interactome likely reveals entry points to deeper understand lipid storage regulation on a mechanistic level. Here, we utilize a split-luciferase based protein-protein interaction assay to identify interactions among 46 LD-associated proteins and protein variants of which 40 are Drosophila and 6 mouse proteins. In 1531 complementation tests, we assayed for interactions among 487 luciferase fragment fusion protein pairs (covered by 830 different construct combinations) of which 85 (17.45 %) resulted in a significant luciferase complementation. These results suggest, that a prominent fraction of the LD-associated proteome participates in protein-protein interactions, and provide a basis for further functional studies. Besides confirming previously described interactions, we identified several new protein interaction pairs. Of these, we characterize the following three in greater detail: (i) Jabba and CG9186, (ii) Jabba and Histones and (iii) Ubiquitin and CG9186. The interaction among Jabba and Histones is of physiological significance and we provide data suggesting that Jabba alone is sufficient to recruit Histones to LDs. Further, we identified a positively charged amino acid stretch of 16 amino acids within the Jabba protein which is necessary for the Jabba-Histone interaction. CG9186 is an annotated esterase which is involved in lipid storage amount regulation and positioning of LDs. We present data supporting the ubiquitination of at least two lysine residues of CG9186 and demonstrate a role of CG9186 ubiquitination in the induction of LD clusters linked to the overexpression of CG9186.

Project description:BACKGROUND:The Arabidopsis thaliana-Pseudomonas syringae model pathosystem is one of the most widely used systems to understand the mechanisms of microbial pathogenesis and plant innate immunity. Several inoculation methods have been used to study plant-pathogen interactions in this model system. However, none of the methods reported to date are similar to those occurring in nature and amicable to large-scale mutant screens. RESULTS:In this study, we developed a rapid and reliable seedling flood-inoculation method based on young Arabidopsis seedlings grown on MS medium. This method has several advantages over conventional soil-grown plant inoculation assays, including a shorter growth and incubation period, ease of inoculation and handling, uniform infection and disease development, requires less growth chamber space and is suitable for high-throughput screens. In this study we demonstrated the efficacy of the Arabidopsis seedling assay to study 1) the virulence factors of P. syringae pv. tomato DC3000, including type III protein secretion system (TTSS) and phytotoxin coronatine (COR); 2) the effector-triggered immunity; and 3) Arabidopsis mutants affected in salicylic acid (SA)- and pathogen-associated molecular pattern (PAMPs)-mediated pathways. Furthermore, we applied this technique to study nonhost resistance (NHR) responses in Arabidopsis using nonhost pathogens, such as P. syringae pv. tabaci, pv. glycinea and pv. tomato T1, and confirmed the functional role of FLAGELLIN-SENSING 2 (FLS2) in NHR. CONCLUSIONS:The Arabidopsis seedling flood-inoculation assay provides a rapid, efficient and economical method for studying Arabidopsis-Pseudomonas interactions with minimal growth chamber space and time. This assay could also provide an excellent system for investigating the virulence mechanisms of P. syringae. Using this method, we demonstrated that FLS2 plays a critical role in conferring NHR against nonhost pathovars of P. syringae, but not to Xanthomonas campestris pv. vesicatoria. This method is potentially ideal for high-throughput screening of both Arabidopsis and pathogen mutants.

Dataset Information

An improved protein lipid overlay assay for studying lipid-protein interactions.

Publications

An improved protein lipid overlay assay for studying lipid-protein interactions.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets