© 2012 Nature America, Inc. All rights reserved. equivalent equivalent support for this work. should Correspondence be addressed to or M.K. E.S. ([email protected]) ([email protected]). 6 Molecular Biology Laboratory, Genome Biology Unit, Heidelberg, Germany. Cell Biology and Biophysics Unit, Heidelberg, Germany. 1 parallel in proteins multiple of kinetics degradation the determine to spectrometry mass with combined be can labeling Metabolic of labeling. a period after short remaining protein specific labeling immun by followed metabolic pulse-chase with analyzed typically is turnover Protein resolution. to down subcellular and trafficking pro turnover tein measuring systematically for exist techniques few cells, living for interference targeted to clinical treat such diseases strategies new provide could mobility and turnover protein standing Alzheimer’s, Parkinson’s and Huntington’s as such disorders neurodegenerative diseases to linked is proteins specific of fibrosis cystic causes channel ion chloride cancers the p53 tumor suppressor plays a critical role in many forms of human diseases. Deregulated degradation of cell cycle control proteins various such asunderlie and organisms aging in observed are turnover tein tuned in response to intrinsic and inputs.extrinsic Alterations in pro degradation, lysosomal and proteasomal as such processes through degradation, protein and translation, and transcription through sis, protein synthe between balance a as now is understood homeostasis or of complex constitution, are in a steady state of rapid flux” constituents of living matter, whether functional or structural, of simple turnover was introduced in 1942 protein by Schönheimer, of who noted that “all importance The scales. population and single-cell the at trafficking subcellular proteinofand turnover measurement taneous simul require would dynamics proteome of monitoring Systematic and asymmetric inheritance of nuclear pore complexes and identify regulators of N-end rule–mediated protein measurements;degradation. and to conduct high-throughput screens for regulators of protein turnover. Our experiments mobilityreveal ofthe proteinsstable naturebetween subcellular compartments; to measure protein degradation kinetics without the needliving for cells.time-course We fuse tFTs to proteins in yeast to study the longevity, segregation single-colorand inheritance fluorescent of cellularproteins componentsthat mature and withthe different kinetics, which we use to analyze protein turnoverscales andis possiblemobility onlyin by combining several methods. Here we describe tandem fluorescent protein timers measuring(tFTs), particular fusionsaspects of of two protein turnover and localization, but comprehensive analysis of protein dynamicsThe functionalacross differentstate of a cell is largely determined by the spatiotemporal organization of its proteome. Technologies exist for Wolfgang Huber R Balca Mardin Anton Khmelinskii of protein dynamics T nature biotechnology nature Received 28 March; accepted 19 May; published online 24 June 2012; Present Present address: ETH Zurich, Light Microscopy and Screening Center, Zurich, Switzerland. Center for Molecular Biology of the University of Heidelberg (ZMBH), DKFZ-ZMBH Alliance, Heidelberg, Germany. Although there are many ways to measure protein abundance in in abundance protein measure to ways many are there Although andem andem fluorescent protein timers for 2 . . Abnormal and trafficking degradation of a mutant form of a 1 o , , Andreas Kaufmann 4 precipitation, which measures the amount of a of amount the measures which precipitation, , , Elmar Schiebel 1 , 2

, 7 , , Philipp J Keller advance online publication online advance 1 3 . Moreover, . accumulation & Michael Knop 2 3 , 6 Janelia Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA. 2 , , Susanne Trautmann 4 , . Therefore, under 3 , 7 6 5 . However, this this However, . , , Anna Bartosik . 1 . . Protein

doi:10.1038/nbt.228 5 German German Cancer Research Center (DKFZ), DKFZ-ZMBH Alliance, Heidelberg, Germany. 1 , 2 , 5 - - - - - tagged proteins, or by their low brightness. low by their or proteins, tagged oligomers form to tendency their by either hampered been far so has timers fluorescent of tion applica However, mobility. widespread and turnover protein follow ( fluorescent states, or intensity ratio, provides a measure of protein age tions color over time as its fluorophore undergoes successive chemical reac A conventional fluorescent cells. timer is a living fluorescent protein in that switches trafficking subcellular and turnover protein both on cells. living to applicable not are they but used, by been have Westernfollowed also blot analysis, fractionation time over them follow to phot or photoactivation with proteins fusion protein fluorescent- label selectively to is trafficking subcellular studying of method common most The dynamics. protein subcellular of studies substrates ligase protein similar method profiling stability protein global using mRNA, same the from of fluorescent-protein abundance fusions tothe a reference normalizing fluorescent by protein expressedmeasurements time-point single rescent signal over time the change of and fluo measuring then or photobleaching activation measured be can proteins photo fusion through fluorescent-protein by cells pulse-labeling living in turnover protein of rate The living cells. in turnover protein of monitoring allow not does technique Fig. Fig. 1 1 , So-called ‘fluorescent timer’ proteins have the potential to report report to potential the have proteins timer’ ‘fluorescent So-called 2 2 , 8 1 , 5 , , Matthias Meurer 7 4 a These These authors contributed equally to this work. , , Malte Wachsmuth . The ratio of fluorescence intensities of the second and first first and second the of intensities fluorescence of ratio The . 1 ), ), which should enable the use of fluorescent protein timers to 10 . . This approach was applied to identify E3 7 , 11 8 . Turnover rates can also be determined with 2 12 in in vivo , but the use of a soluble reference hinders hinders reference soluble a of use the but , European European Molecular Biology Laboratory (EMBL), , 13 1 14– , . Biochemical approaches, such as cell cell as such approaches, Biochemical . 2 2 , 16 8 , , Gislene Pereira , , Joseph D Barry , which can perturb the behavior of behavior the perturb can which , analysis 8 These These authors provided ticles e l c i rt A 5 4 o , 4 , bleaching and and bleaching European European 8

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ubiquitin- 9 or a or  - - - ­

© 2012 Nature America, Inc. All rights reserved. conservative manner.a conservative During mitosis in the preexisting (old) SPB is seg duplicate and centrosome the of (SPBs). equivalent yeast bodies the are pole SPBs spindle of inheritance the examined we First, yeast budding in the localization subcellular age-dependent with proteins to mCherry-sfGFP fused tFTs, we of usefulness the Todemonstrate components cellular of Dynamics ( pool protein the of age the of function a is fluorescence over red time, such that the acquire ratio of red to gradually green fluorescence and synthesis after shortly green-fluorescent synthesis of minutes within fluorescent sfGFP, protein becomes rescent which of half-time a with min ~40 matures which mCherry, protein fluorescent ( tFT a of range time useful the defines conventional largely protein fluorescent maturing slowly more of from the kinetics tion distinct properties fluorescent timers ( with timers fluorescent new generate quickly to exploited be may proteins fluorescent pool color a of age the ( of proteins of indicator an is domains protein fluorescent two the from intensities fluorescence of ratio the whereby kinetics, a as function fluore can as fusion protein kinetics fluorescent tandem A characteristic matures. it with fluorescent becomes protein each ( spectra emission well-separated having proteins fluorescent We tFTs of single-color pairs designed by fusing timers protein Tandem fluorescent RESULTS point. time single a at collected data using screens high-throughput with degradation protein of regulators identify to and complexes pore nuclear of ing of versatility this approach, we used tFTs to study traffick subcellular time spans ranging from ~10 min to many hours. To the demonstrate measure to designed be can timers The limitations. these overcome plot. each in maximum the to normalized sfGFP. of are Ratios brightness the to normalized are curves intensity Fluorescence maturation. mCherry ( ( constants rate maturation the with (gray, green) sfGFP and red) (black, mCherry of fusion a tandem of ( constants rate ( 1 Figure s e l c i rt A  SPB new the is retained whereas (bud), cell into regated daughter the Supplementary Methods Supplementary a

) Illustration of a conventional fluorescent timer (FT) with maturation maturation with (FT) timer fluorescent a conventional of ) Illustration To demonstrate this approach, we combined the monomeric red red monomeric the combined we approach, this Todemonstrate that (tFTs) timers protein fluorescent tandem describe we Here a Intensity (a.u.) S. cerevisiae S. s 0.2 0.4 0.6 0.8 cent timer if the two fluorophores mature with different different with mature fluorophores two the if timer cent

0 1 1 Tandem fluorescent protein fusions as fluorescent timers. timers. fluorescent as fusions protein Tandem fluorescent 8 6 4 2 0 1 7 8 FT blueform , with a monomeric variant of the superfolder green fluo green superfolder the of variant monomeric a with , . A pool of mCherry-sfGFP molecules should be mostly mostly be should molecules mCherry-sfGFP of pool A . Time afterproduction(h) Fig. 1 Fig. m m , , FT redform n (Online Methods and and Methods (Online ) of the fluorescent timer ‘fast-FT’ timer fluorescent the ) of Supplementary Note 1 b ). The broad range of readily available single- available readily of range broad The ). FT red/blue ). For simplicity, a one-step process represents represents process a one-step simplicity, For ). n ratio 10 m 1 , , b m Intensity (a.u.) 2 Supplementary Fig. 1 Fig. Supplementary 0.2 0.4 0.6 0.8 ) determined in this study study this in ) determined 0 1 Fig. 1 Fig. 3 2 1 0 ). In particular, the matura Supplementary Table 1 Table Supplementary Fig. 1 Fig. Time afterproduction(h) m 1 m b 2 ). After translation, translation, After ). b 1 5 ). . ( ratio mCherry/sfGFP b ) Behavior ) Behavior m mCherry 2 m sfGFP 1 ).

). ). - - - -

( nuclear envelope in the bud than in the mother for most mobility. protein intracellular on tFTs that inform can indicates ( cytoplasm the with exchanged not are nucleoporins its and stable is NPC of core the the that notion the ( cytoplasm the in than envelope nuclear the at ratios intensity sfGFP mCherry/ higher substantially exhibited components, NPC scaffold and Methods, (Online mitosis yeast during NPCs of segregation age-dependent of possibility the investigate to sfGFP mCherry- with tagged nucleoporins expressing strains of library a organism in this asymmetric is inherently As division cell sfGFP intensity ratio of Rax2-mCherry-sfGFP correlated positively positively correlated Rax2-mCherry-sfGFP of ratio intensity sfGFP mark sites of which divisions cell previous sion (bud neck), where it forms stable rings with associated bud scars, divi cell of site the to relocalizes and buds emerging of cortex at the mean 0.43 of ( mCherry-sfGFP with tagged proteins both bud for the in than cell mother the in membrane plasma the at higher tides mother the in polypep retained synthesized newly receives bud the are whereas ­compartment, Pma1 and Hxt1 proteins membrane kar9 cells of the ~40% in bud into the SPB new that the is such segregated SPB inheritance impairs micr cytoplasmic between interactions the of Deletion the two between with SPBs cell cycle progression decreased ( ( ( bud the in SPB the at ratio intensity mCherry/sfGFP a higher displayed Spc42-mCherry-sfGFP cell mother the in goes closed mitosis, and cells its mitosis, are in NPCs stable dividing closed also goes cells aging in barrier permeability damage over accumulate the disrupts nucleocytoplasmic time, which can NPCs Stable cells. nondividing in stable are but division cell ing dur down breaks envelope nuclear the when disassemble NPCs sis, organization nuclear and pores in the nuclear envelope and control nucleocytoplasmic transport NPC comprises ~30 different proteins, nucleoporins, called that form Each components. (NPC) complex pore nuclear of inheritance and ( variability this to contribute tic nature of protein fluorescent maturation cell and histories distinct intensity ratios of similar structures is apparent ( ( However,cell significant cell-to-cell variation in individual absolute mCherry/sfGFP every nearly in accurately structures determined different was of age relative the as cells single within robust are timer this with measurements timer. Comparative a as fusion functions mCherry-sfGFP tandem the that demonstrate experiments ( time third the for dividing ratio cells in intensity values their by distinguished be could generations four of increased as cells progressed through successive cell cycles. Structures sured at birth scars, the sites of the first cell division, the intensity ratio mea When scars. bud at than necks bud or buds emerging at lower ( structures labeled of age the with Fig. Fig. 3 Fig. 3 Fig. Fig. 2a Fig. Notably, the mCherry/sfGFP intensity ratio was higher at the the at higher was ratio intensity mCherry/sfGFP the Notably, Finally, we examined structures marked with Rax2. Rax2 appears appears Rax2 Rax2. with marked structures examined we Finally, plasma integral the of pools preexisting SPBs, to contrast In Next, we applied the mCherry-sfGFP Next, timer to we analyze the mobility applied the mCherry-sfGFP Supplementary Table 1 Table Supplementary ∆ 20 cells showed showed cells ± c , b 21 s.d., s.d., ; ; the advance online publication online advance – ± and and . Accordingly, the mCherry/sfGFP intensity ratio ratio intensity mCherry/sfGFP the Accordingly, . c 0.07 and 0.37 0.37 and 0.07 ). The The ). R n Supplementary Fig. 3 Fig. Supplementary b = 40). = / R KAR9 R m b ratio varied with nucleoporin stability as expected; 1 / 9 R R . Accordingly, cells expressing the SPB marker marker SPB the expressing cells Accordingly, . b m / gene, which encodes a factor that mediates mediates that factor a encodes which gene, R ratio decreased as the relative age difference difference age relative the as decreased ratio 1 m 9 2 ± . Consistently, a corresponding fraction of fraction a corresponding . Consistently, 3 ratios below one ( one below ratios 0.09 for Hxt1 and Pma1, respectively; respectively; Pma1, and Hxt1 for 0.09 ( R Fig. 3 Fig. Supplementary Note 2 Supplementary ). Several nucleoporins, in particular particular in nucleoporins, Several ). b ) than at the SPB in the mother ( mother the in SPB the at than ) 2 a 4 Fig. 2 Fig. ). In organisms with open mito open with organisms In ). . In contrast, . In contrast, ). This result is consistent with with consistent is result This ). Supplementary Note 3 Note Supplementary o

tubules and the bud cortex, cortex, bud the and tubules nature biotechnology nature f 2 ). The intensity ratio was was ratio intensity The ). 2 ( Fig. 2 Fig. Fig. 2 Fig. Fig. Fig. 2 Supplementary Fig. 2 Fig. Supplementary Fig. Fig. 2 Fig. 2 Fig. S. cerevisiae S. f ). Together, these these Together, ). e c ). ). The mCherry/ ). f ). ). ). The stochas d nucleoporins nucleoporins ­ , , R 2 b 7 / , we used , we used Fig. Fig. 2 R ), and it it and ), Fig. Fig. under m R ratio ratio was was 25 R , b 2 m 26 ). ). ). ). ------­ ) .

© 2012 Nature America, Inc. All rights reserved. partitioning of such NPCs in the context of an overall asymmetric asymmetric overall an of context the in NPCs equal such of to ­partitioning contribute could NPCs mature more or older and somes chromo between interaction physical mother Preferential between nuclei. bud NPCs and existing of partitioning age-dependent by P between mother and bud NPC densities is statistically significant with 1.53 in a results and bud nuclei mother of between old NPCs partitioning 50 division, of nuclear end the that old the into possibility NPCs are the transported bud. actively At into account that the bud receives only ~38% of all NPCs all of ~38% only receives bud the that account into of labeling tial old and new NPCs ( exchange tag recombination-induced with confirmed s.d., ( envelope nuclear of in late the anaphase when two by nuclei were a connected tubule thin Individual Nup2-DsRed1 dots also moved from the mother to the bud the bud neck in early anaphase ( bud with the together nucleus, as the nuclear envelope was traversing ( h NPCs oldest the only ~10 labels Nup2-DsRed1 of half-time a with maturation, slow extremely undergoes DsRed1 DsRed1. protein fluorescent red the with tagged Nup2 nucleoporin the using NPCs old of segregation the examined anaphase during limited is NPCs As new of possibilities. assembly two these between distinguish to sought We bud. the into NPCs old of segregation biased or cell mother the in new NPCs of assembly preferential age: NPC of distribution asymmetric the for account can Two mother. the mechanisms in than bud the in Fig. Supplementary 4 cell. mother the of age the to according ordered and grouped cells, individual for plotted are cartoons the in 5 bar, indicated Scale structures indicated. as color-coded are images mCherry/sfGFP Ratiometric cycle). cell third cyan, cycle; cell second green, cycle; cell first orange, birth; (red, origin to according arrowheads with marked are structures Different stage. cycle cell and age cell to according ordered cells diploid representative ( Rax2-mCherry-sfGFP. with marked structures of ratios intensity 5 bar, Scale indicated. as color-coded are images mCherry/sfGFP Ratiometric cells. (b) bud and (m) mother in (s) compartments secretory and (v) vacuoles (p), membrane plasma the at fusions Pma1 and Hxt1 of ratios ( anaphase. and type wild in ( anaphase of ( metaphase in analyzed cell single a to corresponds point Each microscopy. time-lapse with followed 3 ratio intensity and (Int) intensity sfGFP to according color-coded are panel) (bottom images ( bud the in or toward ( mother the in retained SPBs at Spc42-mCherry-sfGFP of ( timer. mCherry-sfGFP the with structures cellular of 2 Figure nature biotechnology nature < 10 < µ We conclude that asymmetric distribution of NPC age is achieved achieved is age of NPC distribution We asymmetric that conclude Time-lapse imaging revealed Nup2-DsRed1 dots moving into the the into moving dots Nup2-DsRed1 revealed imaging Time-lapse m. ( m. n ± = 83 cells) were found in the bud. This result was independently 0.13 fold higher density of density old in fold NPCs 0.13 higher the bud difference (the −6 a b

– ) ) in a paired paired a in Analysis of differential inheritance inheritance differential of Analysis c R ) mCherry/sfGFP intensity ratios ratios intensity mCherry/sfGFP ) d b / µ ) mCherry/sfGFP intensity intensity mCherry/sfGFP ) R t m. ( m. = 30 min). ( min). 30 = R m t R = 0 min) and at the end end the at and min) 0 = , as indicated. Scale bar, bar, Scale indicated. as , ratios in cells from from cells in ratios m kar9 ) and at SPBs directed directed SPBs at and ) e , f ) mCherry/sfGFP mCherry/sfGFP ) t ∆ R -test) ( -test) Fig. 3 Fig. cells at the end of of end the at cells ). ). This indicates that NPCs are on average older b ). ( ). a c e

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0 Ratio mCherry sfGFP mCherry-sfGFP Supplementary Note 4 Note Supplementary Emerging b m sfGF p v e bud . mCherry/sfGFP intensity ratios measured at the the at measured ratios intensity mCherry/sfGFP . v v v Birt scar P m b o h mCherry cytoplasmic cytoplasmic transport and nuclear organization. 3 3 Neck . Individual cellsgroupedbyage ). Upon translation, the N-terminal ubi N-terminal the ). Upon translation, Ratio 3 2 In 2 . Instead, age-dependent segrega age-dependent . Instead, 7 t , , our results imply that old NPCs and and R sca Bu Pma1- d r mCherry-sfGFP b Supplementary Theory Supplementary sfGF v v v b v m P s m p mCherry ticles e l c i rt A Ratio Ag e Fig. In In t t

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© 2012 Nature America, Inc. All rights reserved. lysis of a strain with heterogeneous protein expression levels caused caused levels expression protein heterogeneous with strain a of lysis ana single-cell with as demonstrated rate, production protein of the ( constructs of overexpression of of function ( a stability as construct increased constructs different expressing strains m of focus; arrowhead indicates initial direction of movement. Mother (m) and bud (b) compartments are indicated. Scale bars, 2 mother nucleus (green arrowheads) into the bud. Tracks of NPC movement are color coded according to duration; tracking stopped when the NPCs movedDsRed1 out dots in early anaphase relative to the bud neck (white arrowheads). (iii) A single Nup2-DsRed1 dot (red arrowheads) moves from the base of the The difference between cytoplasm. Symmetrically localized nucleoporins with phenylalanine-glycine (FG) repeats (FG Nups) are colored in green/brown. Error bars indicate s.e.m. measured in mother ( (s.e.m.) are smaller than the symbols representing the data points. ( cytoplasm. Colors indicate the structural assignments according to ( Figure 3 s e l c i rt A  b c a

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According to our experimental estimates of mCherry and sfGFP sfGFP and mCherry of estimates experimental our to According Nup2-DsRed1 Nup2-DsRed1 NLS-eGFP NLS-eGFP GAL1 7 iii i 4 sperfram 40 sperframe . tFTs with red fluorescent proteins that mature more slowly slowly more mature that proteins fluorescent red with tFTs . and and ), the mCherry-sfGFP timer should be suitable to study the the study to suitable be should timer mCherry-sfGFP the ),

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© 2012 Nature America, Inc. All rights reserved. marked for degradation by Ubr1 in combination with the E2 E2 the with combination in are Ubr1 by degradation (Ubi-K/W/N/Q-mCherry-sfGFP) for N-degron marked the of residue N the as glutamine or asparagine tryptophan, Constructs lysine, pathway. with rule N-end the of branch specific a for motif destabilizing a with N-degron an by followed pro-N-degrons, Ubi of composed five each to fused was timer mCherry-sfGFP The tion. degrada protein of pathway rule N-end the of components identify in turnover protein of regulators for screen to tFTs on based assay throughput challenging is proteins specific of dation degra for responsible factors identifying advances, recent Despite turnover protein of regulators for screening High-throughput ( cells HeLa in expressed fusions different of turnover the evaluate to sufficient was of range mCherry-sfGFP time the Nevertheless, cases. with strains ( value color-coded a single by represented ( red. in highlighted ( pro-N-degron). the in XZ (residues Doa10 by recognized N-degron the define residues acid amino N-terminal two The pathway. rule N-end the of ( T). (S, perturbations genetic two for exemplified is ratios intensity mCherry/sfGFP of analysis The turnover. 5 Figure nature biotechnology nature at the dipeptide a with cysteine-leucine construct The Rad6. enzyme b a c Supplementary Fig. 10 Fig. Supplementary – e interest (FOI) ) Identification of gene deletions that stabilize the indicated N-degrons. Only gene deletions with positive positive with deletions gene Only N-degrons. indicated the stabilize that deletions gene of ) Identification XZ = Frequency Fusion of Ratio using SG Prom

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Fig. 5 Fig. ubx4∆ doa10 3 ubr1 3 rpn9∆ 20 . A nondegradable mCherry-sfGFP fusion fusion mCherry-sfGFP nondegradable A .

Supplementary Fig. 11 Fig. Supplementary rpn10∆ ∆ irc25∆ ∆ b b ) N-degrons used to screen for components components for screen to used ) N-degrons ). Each construct was introduced into into introduced was construct Each ). rad23∆ ∆ -scores are shown. Expected hits are are hits Expected shown. are -scores

33) ( 33) ufd4∆ e 0 Abundance andstability ufd5∆ ∆-score mCherry/sfGFP ratio ∆-score mCherry/sfGFP ratio 120

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ufd3∆ ∆ 15 ∆ ∆ UPS UFD pathway DOA10 UBR1 3 ufd3 3 0 ubx4 doa4 5 nta1 using syn using ate1 ubp6 branch ufd5 ∆ 20 branch ∆ ∆ ∆ ∆ ump1 ∆ hul5 ubr1 ∆ ufd4 25 ∆ 0 ∆ ∆ ∆  - -

© 2012 Nature America, Inc. All rights reserved. generate a palette of tFTs suitable for studying processes on vastly vastly on processes studying for suitable tFTs of palette a generate to kinetics maturation different with proteins fluorescent red with cells on time scales from ~10 min to several hours. sfGFP can be fused living in fusions protein expressed endogenously of inheritance and The bright mCherry-sfGFP timer allows measuring turnover, mobility rescent proteins for sensitive measurements of intracellular dynamics. fluo single-color provides to an exploit state-of-the-art, opportunity nature of The modular tFTsapproaches. high-throughput with stasis homeo proteome deciphering for avenues new up opening thereby scales, population to subcellular from cells living in trafficking and turnover protein of study the unify that reporters versatile tFTsare DISCUSSION pathways. degradation of of and redundancy tFTsapplications to potential cooperativity study suggests and pathway UFD the in Ufd4 and Ubr1 between eration ( mutant the in P-mCherry-sfGFP Ubi- of stabilization more observed we colonies, whole of imaging detected be not could cooperation this of the UFD pathway, of in ubiquitination UFD substrates the in mCherry-sfGFP Ubi-P- of degradation the restored partially ubiquitin of expression pool ubiquitin free the of regeneration the to contributes and proteasome the at substrates from chains ubiquitin Ubi- Note 6 Supplementary stabilized ( reproducibly P-mCherry-sfGFP Ubp6, protease ubiquitin-specific ( several other ubiquitin-proteasome expected system components, as including the pathway, UFD the of ponents and ( experiments chase cycloheximide with confirmed and components system ubiquitin-proteasome ous was mCherry-sfGFP reproducibly observed in deletion strains of vari nuclear the envelope, where resides Doa10 and reticulum endoplasmic the to Ubc7 recruits Cue1 ( pathway rule N-end the of identified screen effect; stabilizing its for ubc6 expected as which from strains in stabilized was 3 Table Supplementary glutamine or asparagine ( N-terminal with of degradation constructs for required specifically transferase tRNA-protein Ate1 the and amidase Nta1 the yielded screens the instance, branch in screens with Ubi-K/W/N/Q-mCherry-sfGFP constructs. For abundance. increased intensity, indicating sfGFP the for and stability, increased indicating high their by identified were construct particular a of degradation impaired with ( fusion mCherry-sfGFP nondegradable the with performed a control screen sfGFP intensity ratio) for mCherry/ each colony and values from corresponding the and intensity sfGFP normalized (the measurements ( differences the ­calculated s e l c i rt A  from bursts) a time different scales, few minutes transcriptional (e.g., i. 5c Fig.

Ubr1 was recently shown to cooperate with Ufd4, the E3 enzyme enzyme E3 the Ufd4, with cooperate to shown recently was Ubr1 com lacking strains in stabilized was Ubi-P-mCherry-sfGFP the of components known all identified reproducibly We 14 ∆ strain was not present in the library (but subsequently validated , , Supplementary Fig. 12 Fig. Supplementary Supplementary Note 6 Supplementary , Supplementary Fig. 16 Fig. Supplementary f , , upeetr Fg 13 Fig. Supplementary 3 7 ( ∆ Fig. 5d Fig. -scores both for the mCherry/sfGFP intensity ratio, ratio, intensity mCherry/sfGFP the for both -scores CUE1 i. 5e Fig. , and ubp6 f as a new component of the the of component new a as and and Supplementary Fig. 14 Fig. Supplementary ). The Ubi-CL-mCherry-sfGFP construct construct Ubi-CL-mCherry-sfGFP The ). ufd4 ∆ Supplementary Table Supplementary 3 ∆ , -scores, -scores, Fig. 5 Fig. f Supplementary Fig. 13 Fig. Supplementary strain ( strain ∆ , , 3 and 8 and and

ubr1 . Significant stabilization of . stabilization Significant Ubi-CL- Supplementary Figs. 13 13 Figs. Supplementary ). This demonstrates demonstrates This ). Fig. 5 Fig. d Supplementary Note 6 Supplementary Supplementary Table 3 Supplementary , , ∆ and and DOA10 Supplementary Fig. 15 Fig. Supplementary Fig. 5 Fig. strain compared to the the to compared strain upeetr Nt 6 Note Supplementary in vivo in f , , Supplementary Fig. 14 Fig. Supplementary Supplementary Figs. 13 13 Figs. Supplementary a ) between fluorescence fluorescence between ) or or i. 5 Fig. 4 3 0 9 . Using fluorescence fluorescence Using . . Accordingly, over Accordingly, . UBC7 ). In addition, this this addition, In ). ). ). Ubp6 removes e DOA10 ). Deletions of of Deletions ). ), whereas the the whereas ), were deleted, deleted, were in vivo in in in vitro

residues ). Strains ). Strains

and arginyl- ). branch branch ). coop ufd4 UBR1 and and , , but 14 3 ∆ ). ). 3 - - - - - ­ ,

diseases and diseases allow the of identification relevant clinically compounds tFTs should facilitate the investigation of cellular mechanisms of such homeostasis protein in deficiencies with to cancer, are associated infection from disorders, Many disease. and health in homeostasis by ectopic expression of protein fusions tFTs in whole organisms, thus commonly minimizing artifacts caused with dynamics protein of studies systematic for technologies such of genome editing in other model systems are a promisingnucleases for activator-like tool tion effector targeted cells stem embryonic mouse in possible is targeting yeast in tagging protein endogenous for for genome manipulation with hom across environmental and genetic perturbations. Standard approaches homeostasis proteome of characterization systematic enable should be readily identified with tFTs. Genome-wide protein tagging with tFTs specific proteins or components of individual of degradationdegradation in involved factors pathways genetics, yeast automated and can ing require time-course measurements. Using snapshot fluorescence imag scales. time accessible of spectrum the evolution protein fluorescent for strategies Established and morphogenesis). days differentiation (e.g., to several 5. 4. 3. 2. 1. reprints/index.html. R Published online at The authors declare no competing interests.financial the resultsdiscussed and commented on the manuscript. M.K., A.Kh. and P.J.K. wrote the manuscript with input from E.S. All authors and M.K. developed theory. P.J.K., J.D.B. and A.Kh. developed analytical tools. mammalian cell work. G.P. contributed reagents. P.J.K., J.D.B., W.H.,A.Kh., M.W. M.M., A.Kh., A.B., A.Ka., S.T. and P.J.K. did all yeast experiments. B.R.M. did M.K. conceived and, together with E.S., A.Kh. and P.J.K., the designed project. and A. Kinkhabwala for discussions. and the Novartis Stiftung for funding to A.Ka. We thank P.I. D.Bastiaens, Gilmour Network of Excellence in Systems Microscopy for funding to J.D.B. and W.H., OrganizationBiology for funding to ALTFA.Kh. (EMBO the 1124-2010), EU-FP7 the Howard Hughes Medical Institute for funding to P.J.K., the European Molecular funding to M.K., the German Research Foundation for funding to E.S. (SFB638), FACS Facility of EMBL for analyses; the flow CellNetworks cytometry cluster for Facility of EMBL for support with A. microscopy; Riddell, A.P. andGonzalez the We are grateful to Y. Belyaev, S. Terjung and the Advanced Light Microscopy Note: Supplementary information is available in the the pape the of in version available are references associated any and Methods M proteins. critical of stability the or affect pathways degradation specific modulate that CO Au Ac eprints and permissions information is available online at http://www.nature.com/ at online available is information permissions and eprints

ethods Analysis of protein dynamics is key for understanding proteome proteome understanding for key is dynamics protein of Analysis Notably, analysis of steady-state protein turnover with tFTs does not k th MPET oioo RI Poetxc tes n idcbe hprn ntok in networks chaperone inducible and Balch, W.E., Morimoto, R.I., Dillin, stressA. & Kelly, J.W. Adapting proteostasis for disease Proteotoxic R.I. to response cellular Morimoto, a Aggresomes: R.R. Kopito, & C.L. Ward, J.A., Johnston, cancer. and control cell-cycle ligases: Ubiquitin K. Nakayama, & K.I. Nakayama, R. Schoenheimer, intervention. aging. and disease neurodegenerative proteins. misfolded Cancer Rev. Nat. 1942). MA, Cambridge, Press, no o w r advance online publication online advance IN con l edgme G G F Science tr IN http://www.nature.com/doifinder/10.1038/nbt.228 A i

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© 2012 Nature America, Inc. All rights reserved. consisting of an inverted epifluorescence microscope (IX70, Olympus) Olympus) (IX70, microscope NA a 100× Technology), 1.4 epifluorescence (Chroma filters and TRITC FITC with equipped inverted an of consisting with immobilization. washing after min 90 by within done was water. Imaging removed was A (C2010, concanavalin A excess and concanavalin water in 6% Sigma) with min 5 for coated were dishes/chambers The imaging. before min 30 for Nunc) 155409, (Lab-Tek chambers 8-well or MatTek) (P35G-1.5-14-C, dishes bottom glass in immobilized and °C 30 at microscopy. Fluorescence intensities. population median from determined were sample each of ratios intensity mCherry/sfGFP cells. wild-type of autofluorescence using background-corrected were intensities fluorescence standardized The measurements. intensity fluorescence on fluctuations instrumental of ence influ the eliminating protocol, manufacturer’s to according levels intensity cell standardize to used were sample each in beads reference of species six of in MATLAB intensities (MathWorks).were lysis performed The fluorescence ana and statistical correction standardization, data Subsequent sample. each fluid. sheath as FACSFlow BD used was light. laser scattered of detection prevented path optical the in placed nm filters Notch 568 and Holographic nm 488 respectively. mCherry, and sfGFP for filters bandpass 630/40 and 512/15 with detected was Fluorescence respectively. nm), (568.2 mCherry and nm) (488 sfGFP of excitation for used were (Coherent) lasers measured in were a Moflo Argon Coulter). cell sorter (Beckman and Samples Krypton Sabre reference. internal an as Biosciences) BD #556286, peaks), (6 (3.0–3.4 beads multi-spectral with cytometry. Flow vectors. expression mCherry-GFP CyclinB and B-mCherry-sfGFP Cyclin construct to This used was (Invitrogen). plasmid pCDNA5/FRT/TO into cloned was synthesis, full-gene Ubi-X- described previously cassettes described previously with compatible sites annealing primer S2/S3 contain to designed gene tagging with and mCherry-sfGFP sfGFP-mCherry by PCR targeting were tion sfGFP Table Supplementary 2 cassettes. tagging and Plasmids (Invitrogen). manufacturer by the described as 2000 Lipofectamine using chambers 4-well in constructs sfGFP 18 h before imaging, cells were transiently transfected with different mCherry- 5% CO with atmosphere in a humidified and 2 FBS mM glutamine, anaphase. entered cells as arrest, G1 from release after min 60 started was Imaging buds. from shmoo) or projection mating a incubation with 10 0.3 ( bud compartments with incubated and medium β SC-glucose in grown were cells the ( exchange tag For recombination-induced galactose. of control the under ( fusions mCherry-sfGFP ing express Strains otherwise. indicated unless glucose), w/v 2% with medium appropriate. when microscopy, fluorescence with confirmed and immunoblotting using validated was fusions of protein Expression PCR. by validated were tagging gene and deletion Gene (Finnzymes). polymerase as targeting, PCR described on based procedures standard using done were deletion in listed are conditions. growth and construction Strain METHODS ONLINE nature biotechnology nature camera HQ CCD a CoolSNAP Olympus), (PlanApo, objective immersion oil i. 4 Fig. -estradiol (E8875, Sigma) for 3 h. To compare protein age between mother and All images were recorded on a DeltaVision RT system (Applied Precision) Precision) (Applied RT system DeltaVision a on recorded were images All of tables data raw in defined were beads reference and cells of Populations by generated sequence, mCherry-sfGFP codon-optimized human A 10% inactivated heat with medium DMEM in cultured were cells HeLa Yeast strains were grown at 30 °C in SC-glucose medium (synthetic complete 4 9 was introduced into the sequence of sfGFP. Cassettes for endogenous endogenous for sfGFP. Cassettes of sequence the into introduced was 1 d 8 were obtained by full gene synthesis. The monomeric V206R muta V206R monomeric The synthesis. gene full by obtained were ) were grown in SC medium with 3% w/v raffinose and 2% w/v w/v 2% and raffinose w/v 3% with medium SC in grown were ) 4 7 . Cassettes for PCR targeting were amplified with Phusion DNA Phusion with amplified were targeting PCR for Cassettes . Supplementary Table 1 Table Supplementary Yeast strains in the exponential growth phase were mixed mixed were phase growth exponential the in Yeaststrains µ 4 g/ml g/ml Figs. Figs. 7 . All Ubi-X-mCherry-sfGFP fusions are based on on based are fusions Ubi-X-mCherry-sfGFP All . . Yeast codon-optimized sequences . of YeastmCherry sequences codon-optimized α 2 -factor) to unequivocally distinguish mothers (with d Yeast strains were grown in SC-glucose medium medium SC-glucose in grown were Yeaststrains and β -galactosidase and Ubi-XZ-Ura3 fusions Ubi-XZ-Ura3 and -galactosidase 3 Plasmids used in this study are listed in in listed are study this in used Plasmids c µ ), strains were synchronized in ), G1 were h strains (~2.5 synchronized m Sphero Rainbow Calibration Particles Particles Calibration Rainbow m Sphero . Chromosomal gene tagging and gene gene and tagging gene Chromosomal . Yeast strains used in this study study this in Yeast used strains Supplementary Fig. 6 Fig. Supplementary GAL1 promoter promoter 2 at 37 °C. µ 37 4 8 R42A g/ml g/ml , and 50 . ), ), ­ - - - -

Further details on the characterization of mCherry-sfGFP are provided in in provided Methods are Supplementary mCherry-sfGFP of characterization the on details Further curation. track manual by followed tracking cell for used was (MathWorks) and ( sfGFP mCherry of analysis of the the maturation kinetics to generated determine series For induction time-lapse object. segmented each for stored were pixels tributing and of ratio number the con intensity resulting the final s.d., their levels, intensity The autofluorescence. cellular for corrected subsequently were ments measure All mask. segmentation each in levels intensity the to applied was correction field Flat regions. cytoplasmic and envelope nuclear of separately, and, cells whole of signals nucleoporin identify to thresholding itera intensity tive and adaptive to subjected were nucleoporins tagged expressing autofluorescence. Cells cellular of levels iterative the and determine adaptive to to thresholding intensity subjected were cells Wild-type fluctuations. sity and for lamp inten for background corrected were (MathWorks). images All system. DeltaVision the in sensor a using acquisition image during recorded was source light the of level intensity time-dependent The session. imaging and slide were (for without in correction) acquired flat illumination field each of wild-type cells (cellular background), of a uniformly fluorescent microscope Images acquired. of an empty of position the chamber (medium background), ( nm of 300–400 size step 3b ( images Single-plane chamber. temperature-controlled a and (Photometrics), a motorized stage (NanoMotion II Control, Applied Precision) screen functions mitochondrial analysis. the from omitted and screen control nondegradable to the in ion related mostly genes ( lacking strains ~250 where B-score normalized a yielding measurements, corrected of (MAD) deviation absolute by by median the per-plate dividing normalized were measurements corrected the plates/screens, different between com For parison measurements. mCherry/sfGFP and sfGFP the to independently method B-score the with for plate effects edge corrected package Bioconductor cellHTS2 the with performed were package Bioconductor the EBImage using extracted were colony each of intensities fluorescence sfGFP and mCherry mean the and segmented All field–corrected, flat correction. were images field sample flat for acquired were plate fluorescent uniformly a of Images 3). and 2 (replicates imaging emission) nm 628/40 excitation, nm 23 (580/ mCherry and emission) nm 520/35 excitation, nm (465/30 sfGFP for filters and (QImaging) camera Retiga4000DC a system, illumination based field Decon imaging station (Decon wide Science Tec, a Germany) equipped with with an or LED- 1) (replicate for imaging filters fluorescence and mCherry and camera sfGFP CCD 4-megapixel a with equipped (Kodak) imager imaging. before h 24–48 for format 1,536-colony in grown were deletion gene specific a and proto SGA standard cols using done haploids—were of selection sporulation, diploids, of manipulations—selection library subsequent All Instruments). spores were mated with the strains query using a RoToR pinning robot (Singer resulting the and sporulated was library The positions. plate empty eliminate to condensed was library the and removed were genes essentials of deletions yeast gene deletions with heterozygous strains diploid of library genome-wide a with triplicate in crossed was strain (ref. Y8205 strain query SGA the into introduced USA). Health, of Institutes in ImageJ (National segmentation manual using were quantified cells tiguous con of groups or cells single of intensities fluorescence mean and images all to applied were corrections background and field Flat Olympus). (PlanApo, Supplementary Fig. 12 Supplementary , HeLa cells were imaged at were imaged HeLa cells 37 °C a with 60× NA objective 1.4 oil immersion MATLAB in written software custom with processed were Images A gene deletion was considered to stabilize an mCherry-sfGFP fusion in in fusion mCherry-sfGFP an stabilize to considered was deletion gene A in affected strongly were constructs all of B-scores normalized The were ratios intensity mCherry/sfGFP and intensities fluorescence sfGFP fluorescence IS4000MM-Pro field wide a with imaged were plates The ( degrons to different fused mCherry-sfGFP with Constructs c 3 and and 6 . The resulting haploid strains each carrying an mCherry-sfGFP fusion fusion mCherry-sfGFP an carrying each strains haploid resulting . The i s , , if the respective colony showed increased sfGFP intensity and increased j and and Supplementary Figs. 2 Figs. Supplementary p reference the colony row, column and plate, respectively. row,plate, and colony the column reference Supplementary Fig. 8a Supplementary ). These strains were identified in an were ). strains automated These identified fash Fig. 2a Fig. . – 5 – 1 c 4 4 . Further data normalization and analysis analysis and normalization data Further . , e and and and 8 3 5 Supplementary Figs. 5 Figs. Supplementary ) or stacks spanning 5–6 5–6 spanning stacks or ) . . For 2 replicates and 3, with strains ), ), a in written MATLABmodule 36). Each resulting query query resulting Each 36). 5 doi:10.1038/nbt.2281 3 B ˆ , which , was applied which 5 ij 2 p . for each colony, each for Fig. 5 Fig. Figs. 2d 2d Figs. and µ m with a with m 6 b ) ) were ) ) were and ------

© 2012 Nature America, Inc. All rights reserved. screen replicates. screen in listed are positive with deletions gene All replicates. across median the taking by obtained was deletion gene in included were replicate screen one least at in scores. negative avoid to added was of unity an offset and tion, employed to was obtain a reasonable spread in the scores for visualiza subsequent transformation logarithmic natural The hits. stronger considered were where hits, positive of cloud the of center ( ined and with deletions gene Only channels. ( screen control the in colony corresponding the to compared as ratio, intensity mCherry/sfGFP doi:10.1038/nbt.2281 ∆ ij s Each Each screen replicate was analyzed independently. Gene deletions with p ∆ − = -score mCherry/sfGFP ratio > -score 0 stability) were mCherry/sfGFP exam (increased further Fig. 5c Fig. B B ˆ ij s p – ˆ Supplementary Table 3 Table Supplementary ij c f p ). Gene deletions at a larger distance distance larger a at deletions Gene ). ) calculated independently for ) independently calculated the sfGFP and mCherry/sfGFP c ( Fig. 5 Fig. ∆ -score sfGFP and and sfGFP -score a ). This comparison was quantified by the by the quantified was comparison This ). ∆ -score sfGFP > 0 (increased abundance) abundance) > 0 (increased sfGFP -score , ranked by the median median the by ranked , M ∆ is the Mahalanobis distance, distance, Mahalanobis the is -score mCherry/sfGFP ratio ratio mCherry/sfGFP -score Figure 5 Figure d = log(1 + + log(1 = f . A final final A . d of the three three the of M ) from the the from ) d of each each of ∆ -score -score d

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