A facile, sensitive and quantitative membrane‐binding assay for proteins

Jose, Gregor P.; Gopan, Shilpa; Bhattacharyya, Soumya; Pucadyil, Thomas J.

doi:10.1111/tra.12719

Cited by 14 publications

(18 citation statements)

References 37 publications

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“…The previously reported interaction between Drp1 and DGS-NTA(Ni 2+ ) was assayed using liposome sedimentation (Koirala et al 2013). To confirm this interaction using sensitive methods, we turned to a recently developed crosslinking approach called Proximity-based Labeling of Membrane-Associated Proteins (PLiMAP) (Jose and Pucadyil 2020; Jose et al 2020). PLiMAP utilizes liposomes containing a small fraction (1 mol%) of a UV-activable, diazirine-containing fluorescent lipid probe (BODIPY-diazirine PE).…”

Section: Resultsmentioning

confidence: 99%

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Metal-binding propensity in the mitochondrial dynamin-related protein 1

Roy

Pucadyil

2022

Preprint

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Dynamin-related protein1 (Drp1) functions to divide mitochondria and peroxisomes by binding specific adaptor proteins and lipids, both of which are integral to the limiting organellar membrane. In efforts to understand how such multivalent interactions regulate Drp1 functions, in vitro reconstitution schemes rely on recruiting soluble portions of the adaptors appended with genetically encoded polyhistidine tags onto membranes containing Ni2+-bound chelator lipids. These strategies are facile and circumvent the challenge in working with membrane proteins but assume that binding is specific to proteins carrying the polyhistidine tag. Here, we find using chelator lipids and chelator beads that both native and recombinant Drp1 directly bind Ni2+ ions. Unlike that seen with the native mitochondrial lipid cardiolipin, metal-bound chelator lipids recruit Drp1 to the membrane but is rendered functionally inactive in membrane fission. Metal-bound chelator beads also recruit Drp1 and represents a potential strategy to deplete or purify the protein from native tissue lysates.

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Section: Resultsmentioning

confidence: 99%

“…DOPC was the bulk lipid in all experiments. The UV-activable, diazirine-containing fluorescent lipid probe (BODIPY-diazirine PE) was prepared according to published protocols (Jose and Pucadyil 2020; Jose et al 2020). Desired lipids were aliquoted in the required amount into a glass tube and dried under high vacuum for 30 mins to a thin film.…”

Section: Methodsmentioning

confidence: 99%

Metal-binding propensity in the mitochondrial dynamin-related protein 1

Roy

Pucadyil

2022

Preprint

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“… 2007 ), or Proximity-based Labeling of Membrane-Associated Proteins (PLiMAP) assay (Jose et al. 2020 ) may be employed for measuring the protein affinity to the membranes of different mean curvatures. Liposomes of different curvatures are synthesized in these assays by extruding them through filters with predefined pore sizes, followed by incubation with proteins.…”

Section: Assays To Measure Idps/idprs–membrane Interactionsmentioning

confidence: 99%

Insights into Membrane Curvature Sensing and Membrane Remodeling by Intrinsically Disordered Proteins and Protein Regions

2022

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Cellular membranes are highly dynamic in shape. They can rapidly and precisely regulate their shape to perform various cellular functions. The protein’s ability to sense membrane curvature is essential in various biological events such as cell signaling and membrane trafficking. As they are bound, these curvature-sensing proteins may also change the local membrane shape by one or more curvature driving mechanisms. Established curvature-sensing/driving mechanisms rely on proteins with specific structural features such as amphipathic helices and intrinsically curved shapes. However, the recent discovery and characterization of many proteins have shattered the protein structure–function paradigm, believing that the protein functions require a unique structural feature. Typically, such structure-independent functions are carried either entirely by intrinsically disordered proteins or hybrid proteins containing disordered regions and structured domains. It is becoming more apparent that disordered proteins and regions can be potent sensors/inducers of membrane curvatures. In this article, we outline the basic features of disordered proteins and regions, the motifs in such proteins that encode the function, membrane remodeling by disordered proteins and regions, and assays that may be employed to investigate curvature sensing and generation by ordered/disordered proteins. Graphical Abstract

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“…This domain displays low binding affinity for ATP ( K M ∼10–80 μM) 9,73 and hydrolyzes ATP at a basal rate of ∼1 μM min −1 9,23,73 . EHDs exist as dimers in solution and readily bind membranes that contain anionic phospholipids with little specificity for the lipid head group chemistry 20,23,73–78 . Negative‐stained and cryoEM analyses on anionic liposomes reveal EHDs to be present as loosely ordered electron‐dense coats on tubular membranes implying that they can organize into scaffolds that distend spherical liposomes into tubules 69,73,79,80 .…”

Section: Intrinsic Functions Of Ehdsmentioning

confidence: 99%

Cellular functions and intrinsic attributes of the ATP‐binding Eps15 homology domain‐containing proteins

Bhattacharyya

Pucadyil

2020

Protein Science

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Several cellular processes rely on a cohort of dedicated proteins that manage tubulation, fission, and fusion of membranes. A notably large number of them belong to the dynamin superfamily of proteins. Among them is the evolutionarily conserved group of ATP-binding Eps15-homology domain-containing proteins (EHDs). In the two decades since their discovery, EHDs have been linked to a range of cellular processes that require remodeling or maintenance of specific membrane shapes such as during endocytic recycling, caveolar biogenesis, ciliogenesis, formation of T-tubules in skeletal muscles, and membrane resealing after rupture. Recent work has shed light on their structure and the unique attributes they possess in linking ATP hydrolysis to membrane remodeling. This review summarizes some of these recent developments and reconciles intrinsic protein functions to their cellular roles.

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A facile, sensitive and quantitative membrane‐binding assay for proteins

Cited by 14 publications

References 37 publications

Metal-binding propensity in the mitochondrial dynamin-related protein 1

Metal-binding propensity in the mitochondrial dynamin-related protein 1

Insights into Membrane Curvature Sensing and Membrane Remodeling by Intrinsically Disordered Proteins and Protein Regions

Cellular functions and intrinsic attributes of the ATP‐binding Eps15 homology domain‐containing proteins

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