title: Out with the old, in with the new? Comparing methods for measuring protein degradation.
abstract: Protein degradation is a critical factor in controlling cellular protein abundance. Here, we compare classical methods for determining protein degradation rates to a novel GFP (green fluorescent protein) fusion protein based method that assesses the intrinsic stability of cloned cDNA library products by flow cytometry [Yen et al. (2008) Science 322, 918]. While no method is perfect, we conclude that chimeric gene reporter approaches, though powerful, should be applied cautiously, due principally to GFP (or other reporter tag) interference with protein organelle targeting or incorporation into macromolecular assemblies, both of which cause spuriously high degradation rates.
authors.affiliation: Cell Biology Section, Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases/NIH, Bethesda, MD 20892, U.S.A. jyewdell@nih.gov
authors.forename: Jonathan W
authors.initials: JW
authors.lastname: Yewdell
authors.forename: Joshua R
authors.initials: JR
authors.lastname: Lacsina
authors.forename: Martin C
authors.initials: MC
authors.lastname: Rechsteiner
authors.forename: Christopher V
authors.initials: CV
authors.lastname: Nicchitta
mesh_terms: D000818:Animals; D001671:Biochemistry / Q000379:methods*; D003001:Cloning, Molecular / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D011506:Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins