Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Summary

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

Abstract

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Introduction

Membrane fusion is a critical process in many biological reactions. Under biological conditions, membrane fusion is not spontaneous and requires specialized fusion proteins to catalyze such reactions¹. ER homotypic membrane fusion is mediated in animals by the dynamin related GTPase atlastin². Atlastin’s role in homotypic fusion is fundamental for three-way junctions in peripheral ER, which constitutes a large interconnected network of tubules that extend throughout the cell. Atlastins have a conserved domain morphology consisting of a large GTPase, a three helix bundle middle domain, a hydrophobic membrane anchor,....

Protocol

1. Purification of GST-DAtl-His8

1. Protein expression and lysate preparation
   1. Transform BL21 (DE3) E. coli with the GST-DAtl-His8 construct in pGEX4-T3² and select on an ampicillin plate.
   2. Select a single transformant and inoculate 5 mL of LB + ampicillin (5 µL of 100 mg/mL ampicillin) in a 14 mL culture tube and incubate at 25 °C with shaking at 200 rpm for 6-8 h.
      NOTE: Due to leaky expression, it is not recommended to incubate at higher temperatures. Growth at 25 °C reduces aggregation of protein during this growth period.
   3. Inoculate 200 mL of LB + a....

Results

The efficiency of atlastin reconstitution is presented in Figure 2. Reconstituted proteoliposomes were floated in an iohexol discontinuous gradient. Unincorporated protein was sedimented in the bottom layer (B) or in the middle layer (M). Reconstituted protein would float to the top layer (T). Samples of the gradient were harvested and analyzed by SDS-PAGE and Coomassie staining. The quantification of the gel by densitometry shows a very high efficiency of reconstitution with negligible lose.......

Discussion

The methods here delineate an efficient method for purifying, reconstituting, and measuring fusion activity of recombinant atlastin. To ensure high yields of functional atlastin some critical steps must be considered. Expression of atlastin must be done at low temperatures (16 °C) to avoid aggregation and one should aim for a final concentration between 0.4–1.5 mg/mL. Very dilute protein will not be reconstituted optimally at a 1:400 protein to lipid ratio. Reconstitution efficiency can be optionally analyzed .......

Materials

Name	Company	Catalog Number	Comments
10 mL poly-prep chromatography columns	Biored	731-1550
10 x 75 mm Flint glass tubes	VWR	608225-402
47 mm diameter, 0.45um pore whatman sterile membrane filters	Whatman	7141 104
96 well white plate	NUNC	437796
Anapoe X-100	Anatrace	9002-931-1
Cell disrupter	Avestin	Avestin Emulsiflex C3
DOPS (1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt))	Avanti	840035P-10mg	DOPS
EDTA	Research organics inc.	6381-92-6	Ethylenediaminetetraacetic acid
EDTA-free protease inhibitor cocktail	Roche	11873580001	Complete protease inhibitor
Extruder	Sigma Aldrich	Z373400	Liposofast Basic Extruder
GE Akta Prime liquid chromatography system	GE Pharmacia	8149-30-0006
Glutathione agarose beads	Sigma aldrich	G4510-50ml
Glycerol	EMD	GX0185-5
GTP	Sigma Aldrich	36051-31-7	Guanosine 5' triphosphate sodium salt hydrate
HEPES, acid free	Omnipur	5330
Imidazole	fluka	5670
Immobilized metal affinity chromatography (IMAC) resin column	GE Healthcare	17040801	1 mL HiTrap Chelating HP immobilized metal affinity chromatography columns
Iohexol	Accurate chemical and scientific corporation	AN 7050 BLK	Accudenz/Nycodenz
IPTG	Research products international corp.	I56000-100.0	IPTG, dioxane free
L-Glutathione reduced	Sigma-Aldrich	G4251-5g
Magnesium chloride	Fisher	7791-18-6
Methanol	Omnisolv	MX0488-1
NBD-DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt))	Avanti	236495	NBD-DPPE
n-Dodecyl β-D-maltoside	Chem-Impex International	21950
Nonpolar polystyrene adsorbent beads	BioRad	152-3920	SM2 Biobeads
Nuclepore track-etch polycarbonate 19 mm 0.1 um pore membrane	Whatman	800309
Optima LE80K Ultra centrifuge	Beckman Coulter
Phosphatidylcholine, L-α-dipalmitoyl [choline methyl-3H]	ARC	ART0284	Titriated lipids
Plate reader	TECAN	TECAN infinite M200 plate reader
POPC (1-palmitoyl-2-oleoyl-glycero-3-phosphocholine)	Avanti	850457C-25mg	POPC
Potassium chloride	MP	151944
Rh-DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt))	Avanti	236495	Rh-DPPE
Scintillation Cocktail	National Diagnostics	LS-272	Ecoscint XR Scintillation solution for aqueous or non-aqueous samples
Scintillation vials	Beckman	592928	Fast turn cap Mini Poly-Q Vial
Thrombin	Sigma	T1063-1kU	Thrombin from human plasma
Triton X-100	Fisher	BP151-500
Ultra-clear centrifuge tubes 5 x 41 mm	Beckman	344090
Vortex 9 to 13mm Tube Holder	VWR	58816-138	Insert for vortexing flint glass tubes
Vortex Insert Retainer	VWR	58816-132	Retainer needed for vortex tube holder
Vortexer	VWR	2.235074	Vortex Genie 2 model G560
β-mercaptoethanol molecular biology grade	Calbiochem	444203