title: Conversion of green fluorescent protein into a toxic, aggregation-prone protein by C-terminal addition of a short peptide.
abstract: A non-natural 16-residue "degron" peptide has been reported to convey proteasome-dependent degradation when fused to proteins expressed in yeast (Gilon, T., Chomsky, O., and Kulka, R. (2000) Mol. Cell. Biol. 20, 7214-7219) or when fused to green fluorescent protein (GFP) and expressed in mammalian cells (Bence, N. F., Sampat, R. M., and Kopito, R. R. (2001) Science 292, 1552-1555). We find that expression of the GFP::degron in Caenorhabditis elegans muscle or neurons results in the formation of stable perinuclear deposits. Similar perinuclear deposition of GFP::degron was also observed upon transfection of primary rat hippocampal neurons or mouse Neuro2A cells. The generality of this observation was supported by transfection of HEK 293 cells with both GFP::degron and DsRed(monomer)::degron constructs. GFP::degron expressed in C. elegans is less soluble than unmodified GFP and induces the small chaperone protein HSP-16, which co-localizes and co-immunoprecipitates with GFP::degron deposits. Induction of GFP::degron in C. elegans muscle leads to rapid paralysis, demonstrating the in vivo toxicity of this aggregating variant. This paralysis is suppressed by co-expression of HSP-16, which dramatically alters the subcellular distribution of GFP::degron. Our results suggest that in C. elegans, and perhaps in mammalian cells, the degron peptide is not a specific proteasome-targeting signal but acts instead by altering GFP secondary or tertiary structure, resulting in an aggregation-prone form recognized by the chaperone system. This altered form of GFP can form toxic aggregates if its expression level exceeds the capacity of chaperone-based degradation pathways. GFP::degron may serve as an instructive "generic" aggregating control protein for studies of disease-associated aggregating proteins, such as huntingtin, alpha-synuclein, and the beta-amyloid peptide.
authors.affiliation: Institute for Behavioral Genetics, University of Colorado, Boulder, Colorado 80309, USA. linkc@colorado.edu
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Link
authors.forename: Virginia
authors.initials: V
authors.lastname: Fonte
authors.forename: Brian
authors.initials: B
authors.lastname: Hiester
authors.forename: John
authors.initials: J
authors.lastname: Yerg
authors.forename: Jmil
authors.initials: J
authors.lastname: Ferguson
authors.forename: Susan
authors.initials: S
authors.lastname: Csontos
authors.forename: Michael A
authors.initials: MA
authors.lastname: Silverman
authors.forename: Gretchen H
authors.initials: GH
authors.lastname: Stein
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017173:Caenorhabditis elegans; D003001:Cloning, Molecular; D017931:DNA Primers; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000633:toxicity; D012995:Solubility
chemical_list: D017931:DNA Primers; D010455:Peptides; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins