title: Incomplete proteasomal degradation of green fluorescent proteins in the context of tandem fluorescent protein timers.
abstract: Tandem fluorescent protein timers (tFTs) report on protein age through time-dependent change in color, which can be exploited to study protein turnover and trafficking. Each tFT, composed of two fluorescent proteins (FPs) that differ in maturation kinetics, is suited to follow protein dynamics within a specific time range determined by the maturation rates of both FPs. So far, tFTs have been constructed by combining slower-maturing red fluorescent proteins (redFPs) with the faster-maturing superfolder green fluorescent protein (sfGFP). Toward a comprehensive characterization of tFTs, we compare here tFTs composed of different faster-maturing green fluorescent proteins (greenFPs) while keeping the slower-maturing redFP constant (mCherry). Our results indicate that the greenFP maturation kinetics influences the time range of a tFT. Moreover, we observe that commonly used greenFPs can partially withstand proteasomal degradation due to the stability of the FP fold, which results in accumulation of tFT fragments in the cell. Depending on the order of FPs in the timer, incomplete proteasomal degradation either shifts the time range of the tFT toward slower time scales or precludes its use for measurements of protein turnover. We identify greenFPs that are efficiently degraded by the proteasome and provide simple guidelines for the design of new tFTs.
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg and Deutsches Krebsforschungszentrum, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany.
authors.forename: Anton
authors.initials: A
authors.lastname: Khmelinskii
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg and Deutsches Krebsforschungszentrum, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Meurer
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg and Deutsches Krebsforschungszentrum, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany.
authors.forename: Chi-Ting
authors.initials: CT
authors.lastname: Ho
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg and Deutsches Krebsforschungszentrum, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany.
authors.forename: Birgit
authors.initials: B
authors.lastname: Besenbeck
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg and Deutsches Krebsforschungszentrum, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany.
authors.forename: Julia
authors.initials: J
authors.lastname: Füller
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg and Deutsches Krebsforschungszentrum, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany.
authors.forename: Marius K
authors.initials: MK
authors.lastname: Lemberg
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg and Deutsches Krebsforschungszentrum, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany.
authors.forename: Bernd
authors.initials: B
authors.lastname: Bukau
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg and Deutsches Krebsforschungszentrum, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany.
authors.forename: Axel
authors.initials: A
authors.lastname: Mogk
authors.affiliation: Zentrum für Molekulare Biologie der Universität Heidelberg and Deutsches Krebsforschungszentrum, DKFZ-ZMBH Alliance, 69120 Heidelberg, Germany m.knop@zmbh.uni-heidelberg.de.
authors.forename: Michael
authors.initials: M
authors.lastname: Knop
mesh_terms: D049452:Green Fluorescent Proteins / Q000378:metabolism*; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D021381:Protein Transport; D059748:Proteolysis; D011993:Recombinant Fusion Proteins; D015003:Yeasts / Q000235:genetics / Q000378:metabolism; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D046988:Proteasome Endopeptidase Complex; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein