title: Defining the oligomerization state of γ-synuclein in solution and in cells.
abstract: γ-Synuclein is expressed at high levels in neuronal cells and in multiple invasive cancers. Like its family member α-synuclein, γ-synuclein is thought to be natively unfolded but does not readily form fibrils. The function of γ-synuclein is unknown, but we have found that it interacts strongly with the enzyme phospholipase Cβ (PLCβ), altering its interaction with G proteins. As a first step in determining its role, we have characterized its oligomerization using fluorescence homotransfer, photon-counting histogram analysis, and native gel electrophoresis. We found that when its expressed in Escherichia coli and purified, γ-synuclein appears monomeric on chromatographs under denaturing conditions, but under native conditions, it appears as oligomers of varying sizes. We followed the monomer-to-tetramer association by labeling the protein with fluorescein and following the concentration-dependent loss in fluorescence anisotropy resulting from fluorescence homotransfer. We also performed photon-counting histogram analysis at increasing concentrations of fluorescein-labeled γ-synuclein and found concentration-dependent oligomerization. Addition of PLCβ2, a strong γ-synuclein binding partner whose cellular expression is correlated with γ-synuclein, results in disruption of γ-synuclein oligomers. Similarly, its binding to lipid membranes promotes the monomer form. When we exogenously express γ-synuclein or microinject purified protein into cells, the protein appears monomeric. Our studies show that even though purified γ-synuclein form oligomers, when binding partners are present, as in cells, it dissociates to a monomer to bind these partners, which in turn may modify protein function and integrity.
authors.affiliation: Department of Physiology & Biophysics, Stony Brook University , Stony Brook, New York 11794, United States.
authors.forename: Urszula
authors.initials: U
authors.lastname: Golebiewska
authors.forename: Cassandra
authors.initials: C
authors.lastname: Zurawsky
authors.forename: Suzanne
authors.initials: S
authors.lastname: Scarlata
mesh_terms: D045744:Cell Line, Tumor; D002477:Cells / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D054799:Phospholipase C beta / Q000737:chemistry / Q000378:metabolism; D012996:Solutions; D051845:gamma-Synuclein / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D012996:Solutions; D051845:gamma-Synuclein; D054799:Phospholipase C beta