Tandem Fluorescent Protein Timers for In Vitro Analysis of Protein Dynamics

Center for Molecular Biology of the University of Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany.
Nature Biotechnology (Impact Factor: 41.51). 06/2012; 30(7):708-14. DOI: 10.1038/nbt.2281
Source: PubMed


The functional state of a cell is largely determined by the spatiotemporal organization of its proteome. Technologies exist for measuring particular aspects of protein turnover and localization, but comprehensive analysis of protein dynamics across different scales is possible only by combining several methods. Here we describe tandem fluorescent protein timers (tFTs), fusions of two single-color fluorescent proteins that mature with different kinetics, which we use to analyze protein turnover and mobility in living cells. We fuse tFTs to proteins in yeast to study the longevity, segregation and inheritance of cellular components and the mobility of proteins between subcellular compartments; to measure protein degradation kinetics without the need for time-course measurements; and to conduct high-throughput screens for regulators of protein turnover. Our experiments reveal the stable nature and asymmetric inheritance of nuclear pore complexes and identify regulators of N-end rule–mediated protein degradation.

Download full-text


Available from: Balca R Mardin, Aug 06, 2014
33 Reads
  • Source
    • "As a first step in testing our hypothesis, we analyzed Pma1 protein localization. There are conflicting reports on Pma1 asymmetry (Smardon et al., 2013; Khmelinskii et al., 2012; Malínská et al., 2003), however we found that Pma1 was asymmetric between mother and daughter cells. Pma1 levels at the plasma membrane were higher in mother cells than daughter cells as indicated by indirect immunofluorescence with antibody to Pma1 (Figure 2A). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Replicative aging in yeast is asymmetric–mother cells age but their daughter cells are rejuvenated. Here we identify an asymmetry in pH between mother and daughter cells that underlies aging and rejuvenation. Cytosolic pH increases in aging mother cells, but is more acidic in daughter cells. This is due to the asymmetric distribution of the major regulator of cytosolic pH, the plasma membrane proton ATPase (Pma1). Pma1 accumulates in aging mother cells, but is largely absent from nascent daughter cells. We previously found that acidity of the vacuole declines in aging mother cells and limits lifespan, but that daughter cell vacuoles re-acidify. We find that Pma1 activity antagonizes mother cell vacuole acidity by reducing cytosolic protons. However, the inherent asymmetry of Pma1 increases cytosolic proton availability in daughter cells and facilitates vacuole re-acidification and rejuvenation. DOI:
    eLife Sciences 09/2014; 3:e03504. DOI:10.7554/eLife.03504 · 9.32 Impact Factor
  • Source
    • "In addition, protein synthesis and degradation are firmly related and therefore represent an important control factor for metabolic regulation (Schwanhäusser et al., 2013). Individual protein turnover rates have been determined in mammalian cells, yeast and bacteria by measuring the incorporation rate of fluorescent tags (Khmelinskii et al., 2012), affinity tags (Belle et al., 2006) or isotopic labels into the proteome (methods reviewed by Hughes & Krijgsveld, 2012; Trötschel et al., 2013). The metabolic incorporation of isotopic labels is currently the most widely used method for cell cultures and conducted using either labelled ammonium (Helbig et al., 2011; Martin et al., 2012), carbon (Cargile et al., 2004) or amino acids (Gerth et al., 2008; Maier et al., 2011; Schwanhäusser et al., 2011). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Protein turnover plays an important role in cell metabolism by regulating metabolic fluxes. Furthermore, the energy costs for protein turnover have been estimated to account for up to a third of the total energy production during cell replication, and hence may represent a major limiting factor in achieving either higher biomass or production yields. This work aims to measure the specific growth rate (μ)-dependent abundance and turnover rate of individual proteins, estimate the ATP cost for protein production and turnover, and compare this with the total energy balance and other maintenance costs. The lactic acid bacteria model organism Lactococcus lactis was used to measure protein turnover rates at μ 0.1 and 0.5 h(-1) in chemostat experiments. Individual turnover rates were measured for approximately 75% of the total proteome. On average, protein turnover increased by sevenfold with the fivefold increase in growth rate while biomass yield increased by 35%. The median turnover rates found were higher than the μ of the bacterium, which suggests relatively high energy consumption for protein turnover. We found that protein turnover costs alone account for 38% and 47% of the total energy produced at μ 0.1 and 0.5 h(-1), respectively, and gene ontology groups Energy metabolism and Translation dominated synthesis costs at both growth rates studied. These results reflect the complexity of metabolic changes that occur in response to changes in environmental conditions and signify the trade-off between biomass yield and the need to produce ATP for maintenance processes.
    Microbiology 04/2014; 160(Pt_7). DOI:10.1099/mic.0.078089-0 · 2.56 Impact Factor
  • Source
    • "Such mechanisms exist for several organelles including mitochondria (Itoh et al., 2002), vacuoles (Hill et al., 1996), peroxisomes (Hoepfner et al., 2001; Fagarasanu et al., 2006), cortical ER (Du et al., 2001; Estrada et al., 2003) and late Golgi (Rossanese et al., 2001). Consistent with the idea that NPCs are also actively trans­ mitted to daughter cells, the use of a tandem fluorescent " timer " protein showed a bias of " old " nups in daughters (Khmelinskii et al., 2012). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Nuclear pore complexes (NPCs) are essential protein assemblies that span the nuclear envelope and establish nuclear-cytoplasmic compartmentalization. We have investigated mechanisms that control NPC number in mother and daughter cells during the asymmetric division of budding yeast. By simultaneously tracking existing NPCs and newly synthesized NPC protomers (nups) through anaphase, we uncovered a pool of the central channel nup Nsp1 that is actively targeted to the bud in association with endoplasmic reticulum. Bud targeting required an intact actin cytoskeleton and the class V myosin, Myo2. Selective inhibition of cytoplasmic Nsp1 or inactivation of Myo2 reduced the inheritance of NPCs in daughter cells, leading to a daughter-specific loss of viability. Our data are consistent with a model in which Nsp1 releases a barrier that otherwise prevents NPC passage through the bud neck. It further supports the finding that NPC inheritance, not de novo NPC assembly, is primarily responsible for controlling NPC number in daughter cells.
    The Journal of Cell Biology 10/2013; 203(2):215-32. DOI:10.1083/jcb.201305115 · 9.83 Impact Factor
Show more