Expression, Purification, and Liposome Binding of Budding Yeast SNX-BAR Heterodimers

Summary

Here, we present a workflow for the expression, purification and liposome binding of SNX-BAR heterodimers in yeast.

Abstract

SNX-BAR proteins are an evolutionarily conserved class of membrane remodeling proteins that play key roles in sorting and trafficking of protein and lipids during endocytosis, sorting within the endosomal system, and autophagy. Central to SNX-BAR protein function is the ability to form homodimers or heterodimers that bind membranes using highly conserved phox-homology (PX) and BAR (Bin/Amphiphysin/Rvs) domains. In addition, oligomerization of SNX-BAR dimers on membranes can elicit the formation of membrane tubules and vesicles and this activity is thought to reflect their functions as coat proteins for endosome-derived transport carriers. Researchers have long utilized in vitro binding studies using recombinant SNX-BAR proteins on synthetic liposomes or giant unilamellar vesicles (GUVs) to reveal the precise makeup of lipids needed to drive membrane remodeling, thus revealing their mechanism of action. However, due to technical challenges with dual expression systems, toxicity of SNX-BAR protein expression in bacteria, and poor solubility of individual SNX-BAR proteins, most studies to date have examined SNX-BAR homodimers, including non-physiological dimers that form during expression in bacteria. Recently, we have optimized a protocol to overcome the major shortcomings of a typical bacterial expression system. Using this workflow, we demonstrate how to successfully express and purify large amounts of SNX-BAR heterodimers and how to reconstitute them on synthetic liposomes for binding and tubulation assays.

Introduction

Membrane-bound organelles such as the plasma membrane, the endoplasmic reticulum, the Golgi apparatus, lysosome (yeast vacuole), and endosome comprise the endomembrane system of the eukaryotic cell. Most organelles have the ability to communicate and exchange material with other organelles through vesicle transport carriers. How the cell coordinates the packaging and formation of vesicle transport carriers within the endomembrane system is not well understood. However, the proteins and lipids that constitute much of the endomembrane system are known to originate from internalizing endocytic vesicles from the plasma membrane (PM). The endosome is the primary acceptor o....

Protocol

1. Yeast Strain Construction

1. Begin with TVY614 (pep4Δ::LEU2 prb1Δ::hisG prc1Δ::HIS3)¹¹ as the parent strain. This strain is deficient for vacuolar proteases, which contribute to the majority of protein degradation after cell lysis, and therefore allows for a cleaner and more efficient purification.
2. Design primers¹² and integrate tandem affinity purification (TAP) tag at the C-terminus of Atg20 (SNX-BAR ORF 1) using homologous recombination. Use polymerase chain reaction (PCR) to confirm integrations (Figure 2).
3. Perform....

Results

This protocol describes a method for reproducible and robust production of endogenous yeast SNX-BAR complexes that can be used for downstream membrane remodeling assays (Figure 1). The construction of the yeast strain used for purification takes advantage of the efficiency of homologous recombination in budding yeast, allowing for modifications at the genomic loci of the targeted SNX-BARs (Figure 2). This design has two advantage.......

Discussion

Here, we demonstrate an optimized workflow to purify SNX-BAR dimers in yeast and two assays to evaluate their biophysical properties on synthetic liposomes. The main advantage over typical recombinant protein expression in Escherichia coli or other systems is the ability to evenly express SNX-BAR proteins in a native host, thus avoiding the toxicity and insolubility issues found in purifying SNX-BARs in other systems. It is also notable that our system does not require molecular cloning or the harboring of multi.......

Materials

Name	Company	Catalog Number	Comments
0.2 micrometer PC Membranes	Avanti	610006
10 mL Poly-Prep Chromatography column (Bio-Rad)	Bio-Rad	731-1550
27 Gauge needle	BD Biosciences	301629
Amicon Ultra Centrifugal Filter with 10K cutoff	Amicon	UFC501024
Avestin EmulsiFlex-C3 Homogenizer	Avestin	EF-C3
BCA assay	Pierce	23225
Beckman Optima MAX-XP Ultracentrifuge	Beckman Coulter	393315
cOmplete Protease Inhibitor Cocktail	Roche	4693116001
DOPC	Avanti	850375
DOPS	Avanti	840035
ergosterol (Sigma)	Sigma	47130-U
Extruder Set with Block 0.2 microlter/1mL	Avanti	610000
FEI Tecnai F20 transmission electron microscope (200 kV)
Glass culture tubes	VWR	47729-570
IgG sepharose beads (GE Healthcare)	GE Healthcare	17-0969-01
Microlter glass syringes	Hamilton	7637-01
New Brunswick Excella E25	Eppendorf	M1353-0000	or equivalent shaking 30 C
Ni-NTA Magnetic Agarose Beads	Pierce	78605
Optima XE-90 Ultracentrifuge	Beckman Coulter	A94516
Parafilm M	VWR	52858-076
PI3P	Echelon	P-3016	or Echelon equivalent
Polycarbonate bottle assembly	Beckman Coulter	355622
TLA-100 Fixed-Angle Rotor	Beckman Coulter	343840
Type 45 Ti Rotor	Beckman Coulter
Vacuum Desiccator, Bottom and Lid with Socket Valve	VWR	75871-436
Vacuum Pump Alcatel (Pascal 2005 C1)	A&J Vacuum	PN07050
Vortex with foam holder	VWR	10153-838
VWR KIT MICROTUBE	VWR	12620-880