Top Banner
LoQAtE—Lo calization and Q uantitation AT las of the yeast proteomE . A new tool for multiparametric dissection of single-protein behavior in response to biological perturbations in yeast Michal Breker 1 , Melissa Gymrek 2 , Ofer Moldavski 1 and Maya Schuldiner 1, * 1 Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel and 2 Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA Received August 13, 2013; Revised and Accepted September 23, 2013 ABSTRACT Living organisms change their proteome dramatically to sustain a stable internal milieu in fluctuating envir- onments. To study the dynamics of proteins during stress, we measured the localization and abundance of the Saccharomyces cerevisiae proteome under various growth conditions and genetic backgrounds using the GFP collection. We created a database (DB) called ‘LoQAtE’ (Lo calizaiton and Q uantitation At las of the yeast proteomE ), available online at http:// www.weizmann.ac.il/molgen/loqate/, to provide easy access to these data. Using LoQAtE DB, users can get a profile of changes for proteins of interest as well as querying advanced intersections by either abundance changes, primary localization or localiza- tion shifts over the tested conditions. Currently, the DB hosts information on 5330 yeast proteins under three external perturbations (DTT, H 2 O 2 and nitrogen starvation) and two genetic mutations [in the chap- eronin containing TCP1 (CCT) complex and in the proteasome]. Additional conditions will be uploaded regularly. The data demonstrate hundreds of local- ization and abundance changes, many of which were not detected at the level of mRNA. LoQAtE is designed to allow easy navigation for non-experts in high-content microscopy and data are available for download. These data should open up new perspec- tives on the significant role of proteins while combat- ing external and internal fluctuations. INTRODUCTION The budding yeast Saccharomyces cerevisiae robustly adapts to a variety of fluctuations such as extreme environ- ments, genetic mutations and life phases. Many cellular components within the cell (such as DNA, RNA, proteins and lipids) rearrange dynamically to promote survival (1–17). However, although vast amounts of data have been collected on chromatin modifications and transcrip- tional responses under changing conditions, only a handful of pioneering studies describe the dynamics of proteins (2,9–11). To enable a broader view of the proteomic response under varying conditions, we constructed an auto- mated microscopy setup that allows reproducible, accurate and sensitive measurements of the localization and abun- dance of fluorescently tagged proteins at single-cell reso- lution. Using this setup, we visualized the yeast Green Fluorescence Protein (GFP) library in which nearly 5500 yeast proteins are tagged with a C’ terminal GFP under their natural promoter (18). Visualizing all strains under various internal and external fluctuations, we found >100 proteins that could be detected for the first time and determined their subcellular localization (4). We then tracked proteome-level changes using two methods: first, we classified proteins into 13 localization categories and detected hundreds of proteins that shift between different cellular locals under stress (4,19). We then computed abun- dance based on the fluorescence intensity of the GFP signal and found all proteins that change their abundance (4,19). Comparison with transcriptome data (14) demonstrated that up to 60% of proteomic changes could not be pre- dicted from the dynamics of their transcripts, therefore, emphasizing the importance of studying proteins directly (20,21). To easily browse and download all aspects of these data, we built the LOcalization and Quantitation ATlas of the yeast proteomE (LoQAtE) database (DB). The current article presents how ‘LoQAtE’ can be easily used for uncovering new biological phenomena. DATABASE DESIGN AND IMPLEMENTATION The DB is constructed such that both browsing for a protein of interest and advanced intersections of various *To whom correspondence should be addressed. Tel: +972 8 9346346; Fax:+972 8 9346373; Email: [email protected] Nucleic Acids Research, 2013, 1–5 doi:10.1093/nar/gkt933 ß The Author(s) 2013. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/3.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected] Nucleic Acids Research Advance Access published October 22, 2013 at Weizmann Institute of Science on October 23, 2013 http://nar.oxfordjournals.org/ Downloaded from at Weizmann Institute of Science on October 23, 2013 http://nar.oxfordjournals.org/ Downloaded from at Weizmann Institute of Science on October 23, 2013 http://nar.oxfordjournals.org/ Downloaded from at Weizmann Institute of Science on October 23, 2013 http://nar.oxfordjournals.org/ Downloaded from at Weizmann Institute of Science on October 23, 2013 http://nar.oxfordjournals.org/ Downloaded from
5

LoQAtE—Localization and Quantitation ATlas of the yeast ... · In addition, the results of each search can be downloaded in an excel table or as raw images. ‘Search by movement’

Aug 29, 2019

Download

Documents

dangnguyet
Welcome message from author
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
Page 1: LoQAtE—Localization and Quantitation ATlas of the yeast ... · In addition, the results of each search can be downloaded in an excel table or as raw images. ‘Search by movement’

LoQAtEmdashLocalization and Quantitation ATlas of theyeast proteomE A new tool for multiparametricdissection of single-protein behavior in responseto biological perturbations in yeastMichal Breker1 Melissa Gymrek2 Ofer Moldavski1 and Maya Schuldiner1

1Department of Molecular Genetics Weizmann Institute of Science Rehovot 7610001 Israel and 2WhiteheadInstitute for Biomedical Research Nine Cambridge Center Cambridge MA 02142 USA

Received August 13 2013 Revised and Accepted September 23 2013

ABSTRACT

Living organisms change their proteome dramaticallyto sustain a stable internal milieu in fluctuating envir-onments To study the dynamics of proteins duringstress we measured the localization and abundanceof the Saccharomyces cerevisiae proteome undervarious growth conditions and genetic backgroundsusing the GFP collection We created a database (DB)called lsquoLoQAtErsquo (Localizaiton and Quantitation Atlasof the yeast proteomE) available online at httpwwwweizmannacilmolgenloqate to provideeasy access to these data Using LoQAtE DB userscan get a profile of changes for proteins of interest aswell as querying advanced intersections by eitherabundance changes primary localization or localiza-tion shifts over the tested conditions Currently theDB hosts information on 5330 yeast proteins underthree external perturbations (DTT H2O2 and nitrogenstarvation) and two genetic mutations [in the chap-eronin containing TCP1 (CCT) complex and in theproteasome] Additional conditions will be uploadedregularly The data demonstrate hundreds of local-ization and abundance changes many of whichwere not detected at the level of mRNA LoQAtE isdesigned to allow easy navigation for non-experts inhigh-content microscopy and data are available fordownload These data should open up new perspec-tives on the significant role of proteins while combat-ing external and internal fluctuations

INTRODUCTION

The budding yeast Saccharomyces cerevisiae robustlyadapts to a variety of fluctuations such as extreme environ-ments genetic mutations and life phases Many cellular

components within the cell (such as DNA RNA proteinsand lipids) rearrange dynamically to promote survival(1ndash17) However although vast amounts of data havebeen collected on chromatin modifications and transcrip-tional responses under changing conditions only a handfulof pioneering studies describe the dynamics of proteins(29ndash11) To enable a broader view of the proteomicresponse under varying conditions we constructed an auto-mated microscopy setup that allows reproducible accurateand sensitive measurements of the localization and abun-dance of fluorescently tagged proteins at single-cell reso-lution Using this setup we visualized the yeast GreenFluorescence Protein (GFP) library in which nearly 5500yeast proteins are tagged with a Crsquo terminal GFP undertheir natural promoter (18) Visualizing all strains undervarious internal and external fluctuations we found gt100proteins that could be detected for the first time anddetermined their subcellular localization (4) We thentracked proteome-level changes using two methods firstwe classified proteins into 13 localization categories anddetected hundreds of proteins that shift between differentcellular locals under stress (419) We then computed abun-dance based on the fluorescence intensity of the GFP signaland found all proteins that change their abundance (419)Comparison with transcriptome data (14) demonstratedthat up to 60 of proteomic changes could not be pre-dicted from the dynamics of their transcripts thereforeemphasizing the importance of studying proteins directly(2021) To easily browse and download all aspects ofthese data we built the LOcalization and QuantitationATlas of the yeast proteomE (LoQAtE) database (DB)The current article presents how lsquoLoQAtErsquo can be easilyused for uncovering new biological phenomena

DATABASE DESIGN AND IMPLEMENTATION

The DB is constructed such that both browsing for aprotein of interest and advanced intersections of various

To whom correspondence should be addressed Tel +972 8 9346346 Fax +972 8 9346373 Email mayaschuldinerweizmannacil

Nucleic Acids Research 2013 1ndash5doi101093nargkt933

The Author(s) 2013 Published by Oxford University PressThis is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (httpcreativecommonsorglicensesby-nc30) which permits non-commercial re-use distribution and reproduction in any medium provided the original work is properly cited For commercialre-use please contact journalspermissionsoupcom

Nucleic Acids Research Advance Access published October 22 2013 at W

eizmann Institute of Science on O

ctober 23 2013httpnaroxfordjournalsorg

Dow

nloaded from

at Weizm

ann Institute of Science on October 23 2013

httpnaroxfordjournalsorgD

ownloaded from

at W

eizmann Institute of Science on O

ctober 23 2013httpnaroxfordjournalsorg

Dow

nloaded from

at Weizm

ann Institute of Science on October 23 2013

httpnaroxfordjournalsorgD

ownloaded from

at W

eizmann Institute of Science on O

ctober 23 2013httpnaroxfordjournalsorg

Dow

nloaded from

parameters can be performed easily LoQAtE holds infor-mation on 328 localization shifts of proteins from oneorganelle to another and 1400 abundance change eventsEach change may contribute directly to the ability of thecell to respond to the perturbation studied or it can serveas a marker for cellular changes if it is a downstream effectof the stress Using LoQAtE such changes can beuncovered and studied in several methods

Studying a specific protein

Quick searchThe most simple utility of the DB is to insert a single nameor a tab-delimited list of open reading frames (ORFs)genenames (for an example of the results screen see Figure 1A)and receive a lsquoquick searchrsquo result This result presents thelocalization and abundance of all proteins of interestunder all conditions tested as well as the lsquofold changersquo ofprotein abundance in each condition relative to a controlbaseline condition (synthetic defined medium) and a stat-istical assessment of the significance of this change Fullresults including the microscopic images are presented forup to 100 proteins (to enable rapid display of the searchresults) At any moment the full list of results can bedownloaded either as a csv file summarizing the data oras a folder of the raw images For ease of utility the defaultchannel shown in the results screen is the GFP channelhowever the bright field image is available in a clickablefashion

Studying a group of proteins

The ability to search by more advanced criteria enablesusers to uncover patterns in protein behavior that may nothave been revealed by examining a single protein Forexample categorizing groups of proteins that shift theirlocalization from the same origin to the same target or-ganelle may allow a unique view on the functional changesin each organelle provide a basis for analysis of thefactors mediating these movements or may uncover ajoint function In another example regulatory elementsmay be found for groups of proteins that are downupregulated concordantly We have therefore builtseveral interfaces to enable advanced queries of the dataand to enable such grouping

Advanced search

1 Search by localization It allows the user to choose allproteins localized to a specific organelles in a definedcondition In each query one condition can be chosenand as many as 13 localization categories (or lsquobelowthresholdrsquo that represents no expression) can be pickedResults are presented and can be downloaded as describedearlier in text

2 Search by abundance It allows the user to choose a setof proteins that changed abundance concordantly for aspecific condition or for several conditions in an lsquoANDrsquorelation To use this option one or more growth condi-tions should be chosen and for each the type of abun-dance change (upregulated no change or downregulated)should be defined Additionally the user can filter results

to a specific organelle Results are presented and can bedownloaded as described earlier in text

3 Search by movement A schematic diagram depictingthe types and numbers of changes in cellular localizationobserved during yeast growth in the three environmentalstresses is available In a clickable fashion the user canchoose one arrow (representative of one type of localiza-tion shift in a specific condition) and click the searchbutton Results provide the entire list of proteins classifiedas undergoing this shift and can be downloaded asdescribed earlier in text (Figure 1B)

Using the advanced search to study transcriptionalnetworks

Using the data in LoQAtE provides the opportunity touncover important layers of cellular function that couldnot have been uncovered by mRNA levels aloneUncovering the extent of post-transcriptional regulationcan be easily done by comparing all changes observedunder a specific condition with the transcriptionalresponse under this condition (14) These types of datamay potentially be further characterized to definemRNA motifs or secondary structures affecting transla-tion efficiency or mRNA stability as well as protein levelsignals such as stress-dependent degrons or binding motifsthat affect protein abundance in the absence oftranscription

To demonstrate one such utility we have used thelsquoSearch by Abundancersquo and lsquoSearch by Movementrsquo toretrieve the entire group of transcription factors (TFs)that change abundance or localization during nitrogenstarvation out of a list of all predicted and known TFsin yeast (courtesy of Professor Eran Segal) (see Figure 2Aand B) Interestingly comparing these 33 proteins withtheir mRNA levels under the exact same conditions un-covered that the transcripts of 48 of them had no de-tectable change (14) To uncover the potential affect ofthese post-transcriptional changes on adaptation to star-vation we used YEASTRACT (22ndash24) to predict theirpotential targets Then we calculated the percentage ofabundance changes discovered in LoQAtE that can beexplained by these TFs (see Figure 2C) Intriguingly anastounding fraction of changes in protein levels measuredunder nitrogen starvation could be explained by the post-transcriptional changes of this handful of TFs As theseconditions have been extensively studied from the tran-scriptional perspective (14) without the post-transcrip-tional knowledge this means that the conclusionsreached might have been incomplete

SIMILAR DATABASES

LoQAtE DB is the first ever resource that particularlyconcentrates on the dynamic characteristics of the yeastproteome The growing interest in directly measuringproteomic features has brought about several additionalDBs presenting systematic proteomic data The pioneeringyeast localization DB (httpyeastgfpyeastgenomeorg)provides information about the localization and

2 Nucleic Acids Research 2013

Figure 1 Screen shots of two result windows in a LoQAtE search The LoQAtE allows various search functions to be performed Both quick searchand advanced search (Search by abundance or localization) (A) enables querying for large groups of proteins and results in the entire localization andabundance data for each protein being presented In addition the results of each search can be downloaded in an excel table or as raw imageslsquoSearch by movementrsquo (B) allows the user to choose a movement of interest represented as an arrow on a schematic representation of the cell andreceive information on all proteins that are assigned to this category

Nucleic Acids Research 2013 3

abundance of the majority of yeast proteins understandard laboratory growth conditions based on the con-struction of the GFP collection (18) Additionally re-analysis of the GFP collection and additional proteinsinvolved in lipid metabolism was performed by high-resolution confocal imaging (25) and is available atthe YPL+DB (httpyplpuni-grazatindexphp) (2627)For a broader range of proteomic data the YeastResource Center (YRC) (httpdeptswashingtoneduyeastrc) is a collaborative web site that gathers severalcore technologies and provides a breadth of informationsuch as quantitative mass-spectrometry sequence-function relationships microscopy structure and compu-tational strategies for structure-function predictionsFinally organelle DB (httporganelledblsiumicheduindexphp) compiles protein localization data that areorganized into gt50 organelles subcellular structures andprotein complexes (2829) The data set includes 138 or-ganisms spanning the eukaryotic kingdom and incorpor-ates ongoing results from large-scale studies of proteinlocalization in yeast S cerevisiaeA similar effort in human cell lines is based on the

library of annotated reporter cell-clones (LARC) inwhich each strain (out of 2180) contains an endogenousprotein fused to yellow fluorescent protein (YFP) (30)This DB (httpwwwweizmannacilmcbUriAlonDynamProt) provides the expression and localization ofeach protein over time in response to various drugs andis regularly updated with newly created clones (30) Amore comprehensive human proteome atlas has recentlybecome available and harbors expression data for thou-sands of human proteins from a large number of healthytissues tumors cell lines and subcellular localizations allbased on antibody staining (httpwwwproteinatlasorg)(31) A principally similar DB called lsquoPeptideAtlasrsquo(httpwwwpeptideatlasorg) harbor growing data onproteins expressed under various conditions for a wide

variety of organisms from yeast to human collected bytandem mass spectrometry (MSMS) (32)

OUTLOOK

The LoQAtE DB in its first version contains easily ac-cessible and user-friendly information about subcellularlocalization and levels for 5330 yeast proteins underthree environmental stress conditions and two genetic per-turbations The richness of post-translational events docu-mented in this DB underlines the significance of studyingthe level of proteins to capture a true picture of a cellrsquosresponse to stress Starting from behavior of individualproteins and up to a birdrsquos eye view on the entire cell asa holistic functional unit this DB enables scientists to gaina new insight on cellular dynamics As more screens areperformed in our laboratory they will be added into thispublicly available atlas This rich resource should serve formultiparametric searches and discovery of new proteinfunctions illuminated only by the combination of suchvast amounts of data

ACKNOWLEDGEMENTS

The authors would like to thank Jonathan Weissman forproviding them with the full GFP library and AmnonHorovitz and Michal Nadler-Holly for their fruitful col-laboration studying the CCT complex They would like tothank Keren Katzav Genia Brodsky and Orit Bechar forthe graphical designs of the database and Rachel Baumanand Anastasia Zarankin from the internet services sectionof the Weizmann Institute for constructing the LoQAtEdatabase

FUNDING

Isreali Ministry of Science the European ResearchCouncil (ERC) Starting Grant [StG 260395] and

Figure 2 Application of LoQAtE to study regulatory networks Two advanced search options were used to put-together a list of all yeast TFs thatchanged abundance (search by abundance) or localization (search by movement) (A) Example of change in localization during nitrogen starvationScale bar represents 5 m (B) The list of 33 TFs was compared with microarray data on expression changes under the same stress condition (14) touncover that 16 of them were not documented to have any change in their transcripts under the exact same experimental conditions (C) Using theDB for regulatory motifs (YEASTRACT) we extracted the targets for these 16 TFs and found that an enormous fraction of all changes in proteinlevels found in LoQAtE during nitrogen starvation (using the Search by Abundance option) (22ndash24) can be explained by 10 of the TFs

4 Nucleic Acids Research 2013

reintegration grant [IRG 239224] EMBO YoungInvestigator Fellow (to MS) Recipient of an MITInternational Science and Technology Initiative (MISTI)(to MS) Funding for open access charge ERC StartingGrant [260395]

Conflict of interest statement None declared

REFERENCES

1 Frenkel-MorgensternM CohenAA Geva-ZatorskyNEdenE PriluskyJ IssaevaI SigalA Cohen-SaidonCLironY CohenL et al (2010) Dynamic Proteomics a databasefor dynamics and localizations of endogenous fluorescently-taggedproteins in living human cells Nucleic Acids Res 38D508ndashD512

2 AragonT van AnkenE PincusD SerafimovaIMKorennykhAV RubioCA and WalterP (2009) MessengerRNA targeting to endoplasmic reticulum stress signalling sitesNature 457 736ndash740

3 BeyerA HollunderJ NasheuerHP and WilhelmT (2004)Post-transcriptional expression regulation in the yeastSaccharomyces cerevisiae on a genomic scale Mol CellProteomics 3 1083ndash1092

4 BrekerM GymrekM and SchuldinerM (2013) A novel single-cell screening platform reveals proteome plasticity during yeaststress responses J Cell Biol 200 839ndash850

5 EdenE Geva-ZatorskyN IssaevaI CohenA DekelEDanonT CohenL MayoA and AlonU (2011) Proteomehalf-life dynamics in living human cells Science 331 764ndash768

6 ErjavecN LarssonL GranthamJ and NystromT (2007)Accelerated aging and failure to segregate damaged proteins inSir2 mutants can be suppressed by overproducing the proteinaggregation-remodeling factor Hsp104p Genes Dev 212410ndash2421

7 HedbackerK TownleyR and CarlsonM (2004) Cyclic AMP-dependent protein kinase regulates the subcellular localization ofSnf1-Sip1 protein kinase Mol Cell Biol 24 1836ndash1843

8 LeeMV TopperSE HublerSL HoseJ WengerCDCoonJJ and GaschAP (2011) A dynamic model of proteomechanges reveals new roles for transcript alteration in yeast MolSyst Biol 7 514

9 SigalA MiloR CohenA Geva-ZatorskyN KleinYAlalufI SwerdlinN PerzovN DanonT LironY et al (2006)Dynamic proteomics in individual human cells uncoverswidespread cell-cycle dependence of nuclear proteins NatMethods 3 525ndash531

10 TkachJM YimitA LeeAY RiffleM CostanzoMJaschobD HendryJA OuJ MoffatJ BooneC et al (2012)Dissecting DNA damage response pathways by analysing proteinlocalization and abundance changes during DNA replicationstress Nat Cell Biol 14 966ndash976

11 NewmanJR GhaemmaghamiS IhmelsJ BreslowDKNobleM DeRisiJL and WeissmanJS (2006) Single-cellproteomic analysis of S cerevisiae reveals the architecture ofbiological noise Nature 441 840ndash846

12 Stathopoulos-GerontidesA GuoJJ and CyertMS (1999)Yeast calcineurin regulates nuclear localization of the Crz1ptranscription factor through dephosphorylation Genes Dev 13798ndash803

13 ToulmayA and PrinzWA Direct imaging reveals stablemicrometer-scale lipid domains that segregate proteins in livecells J Cell Biol 202 35ndash44

14 GaschAP SpellmanPT KaoCM Carmel-HarelOEisenMB StorzG BotsteinD and BrownPO (2000) Genomicexpression programs in the response of yeast cells to environmentalchanges Mol Biol Cell 11 4241ndash4257

15 HughesTR MartonMJ JonesAR RobertsCJStoughtonR ArmourCD BennettHA CoffeyE DaiHHeYD et al (2000) Functional discovery via a compendiumof expression profiles Cell 102 109ndash126

16 CaustonHC RenB KohSS HarbisonCT KaninEJenningsEG LeeTI TrueHL LanderES and YoungRA(2001) Remodeling of yeast genome expression in response toenvironmental changes Mol Biol Cell 12 323ndash337

17 RohTY and ZhaoK (2008) High-resolution genome-widemapping of chromatin modifications by GMAT Methods MolBiol 387 95ndash108

18 HuhWK FalvoJV GerkeLC CarrollAS HowsonRWWeissmanJS and OrsquoSheaEK (2003) Global analysis of proteinlocalization in budding yeast Nature 425 686ndash691

19 Nadler-HollyM BrekerM GruberR AziaA GymrekMEisensteinM WillisonKR SchuldinerM and HorovitzAInteractions of subunit CCT3 in the yeast chaperonin CCTTRiCwith QN-rich proteins revealed by high-throughput microscopyanalysis Proc Natl Acad Sci USA 109 18833ndash18838

20 PicottiP Clement-ZizaM LamH CampbellDS SchmidtADeutschEW RostH SunZ RinnerO ReiterL et alA complete mass-spectrometric map of the yeast proteomeapplied to quantitative trait analysis Nature 494 266ndash270

21 de GodoyLM OlsenJV CoxJ NielsenML HubnerNCFrohlichF WaltherTC and MannM (2008) Comprehensivemass-spectrometry-based proteome quantification of haploidversus diploid yeast Nature 455 1251ndash1254

22 TeixeiraMC MonteiroP JainP TenreiroS FernandesARMiraNP AlenquerM FreitasAT OliveiraAL and Sa-CorreiaI (2006) The YEASTRACT database a tool for theanalysis of transcription regulatory associations in Saccharomycescerevisiae Nucleic Acids Res 34 D446ndashD451

23 MonteiroPT MendesND TeixeiraMC drsquoOreySTenreiroS MiraNP PaisH FranciscoAP CarvalhoAMLourencoAB et al (2008) YEASTRACT-DISCOVERER newtools to improve the analysis of transcriptional regulatoryassociations in Saccharomyces cerevisiae Nucleic Acids Res 36D132ndashD136

24 AbdulrehmanD MonteiroPT TeixeiraMC MiraNPLourencoAB dos SantosSC CabritoTR FranciscoAPMadeiraSC AiresRS et al (2010) YEASTRACT providing aprogrammatic access to curated transcriptional regulatoryassociations in Saccharomyces cerevisiae through a web servicesinterface Nucleic Acids Res 39 D136ndashD140

25 NatterK LeitnerP FaschingerA WolinskiH McCraithSFieldsS and KohlweinSD (2005) The spatial organization oflipid synthesis in the yeast Saccharomyces cerevisiae derived fromlarge scale green fluorescent protein tagging and high resolutionmicroscopy Mol Cell Proteomics 4 662ndash672

26 HabelerG NatterK ThallingerGG CrawfordMEKohlweinSD and TrajanoskiZ (2002) YPLdb the yeastprotein localization database Nucleic Acids Res 30 80ndash83

27 KalsM NatterK ThallingerGG TrajanoskiZ andKohlweinSD (2005) YPLdb2 the yeast protein localizationdatabase version 20 Yeast 22 213ndash218

28 WiwatwattanaN and KumarA (2005) Organelle DB a cross-species database of protein localization and function NucleicAcids Res 33 D598ndash604

29 WiwatwattanaN LandauCM CopeGJ HarpGA andKumarA (2007) Organelle DB an updated resource ofeukaryotic protein localization and function Nucleic Acids Res35 D810ndashD814

30 Frenkel-MorgensternM CohenAA Geva-ZatorskyNEdenE PriluskyJ IssaevaI SigalA Cohen-SaidonCLironY CohenL et al (2010) Dynamic Proteomics a databasefor dynamics and localizations of endogenous fluorescently-taggedproteins in living human cells Nucleic Acids Res 38D508ndashD512

31 UhlenM OksvoldP FagerbergL LundbergE JonassonKForsbergM ZwahlenM KampfC WesterK HoberS et al(2010) Towards a knowledge-based Human Protein Atlas NatBiotechnol 28 1248ndash1250

32 DesiereF DeutschEW NesvizhskiiAI MallickP KingNLEngJK AderemA BoyleR BrunnerE DonohoeS et al(2005) Integration with the human genome of peptidesequences obtained by high-throughput mass spectrometryGenome Biol 6 R9

Nucleic Acids Research 2013 5

Page 2: LoQAtE—Localization and Quantitation ATlas of the yeast ... · In addition, the results of each search can be downloaded in an excel table or as raw images. ‘Search by movement’

parameters can be performed easily LoQAtE holds infor-mation on 328 localization shifts of proteins from oneorganelle to another and 1400 abundance change eventsEach change may contribute directly to the ability of thecell to respond to the perturbation studied or it can serveas a marker for cellular changes if it is a downstream effectof the stress Using LoQAtE such changes can beuncovered and studied in several methods

Studying a specific protein

Quick searchThe most simple utility of the DB is to insert a single nameor a tab-delimited list of open reading frames (ORFs)genenames (for an example of the results screen see Figure 1A)and receive a lsquoquick searchrsquo result This result presents thelocalization and abundance of all proteins of interestunder all conditions tested as well as the lsquofold changersquo ofprotein abundance in each condition relative to a controlbaseline condition (synthetic defined medium) and a stat-istical assessment of the significance of this change Fullresults including the microscopic images are presented forup to 100 proteins (to enable rapid display of the searchresults) At any moment the full list of results can bedownloaded either as a csv file summarizing the data oras a folder of the raw images For ease of utility the defaultchannel shown in the results screen is the GFP channelhowever the bright field image is available in a clickablefashion

Studying a group of proteins

The ability to search by more advanced criteria enablesusers to uncover patterns in protein behavior that may nothave been revealed by examining a single protein Forexample categorizing groups of proteins that shift theirlocalization from the same origin to the same target or-ganelle may allow a unique view on the functional changesin each organelle provide a basis for analysis of thefactors mediating these movements or may uncover ajoint function In another example regulatory elementsmay be found for groups of proteins that are downupregulated concordantly We have therefore builtseveral interfaces to enable advanced queries of the dataand to enable such grouping

Advanced search

1 Search by localization It allows the user to choose allproteins localized to a specific organelles in a definedcondition In each query one condition can be chosenand as many as 13 localization categories (or lsquobelowthresholdrsquo that represents no expression) can be pickedResults are presented and can be downloaded as describedearlier in text

2 Search by abundance It allows the user to choose a setof proteins that changed abundance concordantly for aspecific condition or for several conditions in an lsquoANDrsquorelation To use this option one or more growth condi-tions should be chosen and for each the type of abun-dance change (upregulated no change or downregulated)should be defined Additionally the user can filter results

to a specific organelle Results are presented and can bedownloaded as described earlier in text

3 Search by movement A schematic diagram depictingthe types and numbers of changes in cellular localizationobserved during yeast growth in the three environmentalstresses is available In a clickable fashion the user canchoose one arrow (representative of one type of localiza-tion shift in a specific condition) and click the searchbutton Results provide the entire list of proteins classifiedas undergoing this shift and can be downloaded asdescribed earlier in text (Figure 1B)

Using the advanced search to study transcriptionalnetworks

Using the data in LoQAtE provides the opportunity touncover important layers of cellular function that couldnot have been uncovered by mRNA levels aloneUncovering the extent of post-transcriptional regulationcan be easily done by comparing all changes observedunder a specific condition with the transcriptionalresponse under this condition (14) These types of datamay potentially be further characterized to definemRNA motifs or secondary structures affecting transla-tion efficiency or mRNA stability as well as protein levelsignals such as stress-dependent degrons or binding motifsthat affect protein abundance in the absence oftranscription

To demonstrate one such utility we have used thelsquoSearch by Abundancersquo and lsquoSearch by Movementrsquo toretrieve the entire group of transcription factors (TFs)that change abundance or localization during nitrogenstarvation out of a list of all predicted and known TFsin yeast (courtesy of Professor Eran Segal) (see Figure 2Aand B) Interestingly comparing these 33 proteins withtheir mRNA levels under the exact same conditions un-covered that the transcripts of 48 of them had no de-tectable change (14) To uncover the potential affect ofthese post-transcriptional changes on adaptation to star-vation we used YEASTRACT (22ndash24) to predict theirpotential targets Then we calculated the percentage ofabundance changes discovered in LoQAtE that can beexplained by these TFs (see Figure 2C) Intriguingly anastounding fraction of changes in protein levels measuredunder nitrogen starvation could be explained by the post-transcriptional changes of this handful of TFs As theseconditions have been extensively studied from the tran-scriptional perspective (14) without the post-transcrip-tional knowledge this means that the conclusionsreached might have been incomplete

SIMILAR DATABASES

LoQAtE DB is the first ever resource that particularlyconcentrates on the dynamic characteristics of the yeastproteome The growing interest in directly measuringproteomic features has brought about several additionalDBs presenting systematic proteomic data The pioneeringyeast localization DB (httpyeastgfpyeastgenomeorg)provides information about the localization and

2 Nucleic Acids Research 2013

Figure 1 Screen shots of two result windows in a LoQAtE search The LoQAtE allows various search functions to be performed Both quick searchand advanced search (Search by abundance or localization) (A) enables querying for large groups of proteins and results in the entire localization andabundance data for each protein being presented In addition the results of each search can be downloaded in an excel table or as raw imageslsquoSearch by movementrsquo (B) allows the user to choose a movement of interest represented as an arrow on a schematic representation of the cell andreceive information on all proteins that are assigned to this category

Nucleic Acids Research 2013 3

abundance of the majority of yeast proteins understandard laboratory growth conditions based on the con-struction of the GFP collection (18) Additionally re-analysis of the GFP collection and additional proteinsinvolved in lipid metabolism was performed by high-resolution confocal imaging (25) and is available atthe YPL+DB (httpyplpuni-grazatindexphp) (2627)For a broader range of proteomic data the YeastResource Center (YRC) (httpdeptswashingtoneduyeastrc) is a collaborative web site that gathers severalcore technologies and provides a breadth of informationsuch as quantitative mass-spectrometry sequence-function relationships microscopy structure and compu-tational strategies for structure-function predictionsFinally organelle DB (httporganelledblsiumicheduindexphp) compiles protein localization data that areorganized into gt50 organelles subcellular structures andprotein complexes (2829) The data set includes 138 or-ganisms spanning the eukaryotic kingdom and incorpor-ates ongoing results from large-scale studies of proteinlocalization in yeast S cerevisiaeA similar effort in human cell lines is based on the

library of annotated reporter cell-clones (LARC) inwhich each strain (out of 2180) contains an endogenousprotein fused to yellow fluorescent protein (YFP) (30)This DB (httpwwwweizmannacilmcbUriAlonDynamProt) provides the expression and localization ofeach protein over time in response to various drugs andis regularly updated with newly created clones (30) Amore comprehensive human proteome atlas has recentlybecome available and harbors expression data for thou-sands of human proteins from a large number of healthytissues tumors cell lines and subcellular localizations allbased on antibody staining (httpwwwproteinatlasorg)(31) A principally similar DB called lsquoPeptideAtlasrsquo(httpwwwpeptideatlasorg) harbor growing data onproteins expressed under various conditions for a wide

variety of organisms from yeast to human collected bytandem mass spectrometry (MSMS) (32)

OUTLOOK

The LoQAtE DB in its first version contains easily ac-cessible and user-friendly information about subcellularlocalization and levels for 5330 yeast proteins underthree environmental stress conditions and two genetic per-turbations The richness of post-translational events docu-mented in this DB underlines the significance of studyingthe level of proteins to capture a true picture of a cellrsquosresponse to stress Starting from behavior of individualproteins and up to a birdrsquos eye view on the entire cell asa holistic functional unit this DB enables scientists to gaina new insight on cellular dynamics As more screens areperformed in our laboratory they will be added into thispublicly available atlas This rich resource should serve formultiparametric searches and discovery of new proteinfunctions illuminated only by the combination of suchvast amounts of data

ACKNOWLEDGEMENTS

The authors would like to thank Jonathan Weissman forproviding them with the full GFP library and AmnonHorovitz and Michal Nadler-Holly for their fruitful col-laboration studying the CCT complex They would like tothank Keren Katzav Genia Brodsky and Orit Bechar forthe graphical designs of the database and Rachel Baumanand Anastasia Zarankin from the internet services sectionof the Weizmann Institute for constructing the LoQAtEdatabase

FUNDING

Isreali Ministry of Science the European ResearchCouncil (ERC) Starting Grant [StG 260395] and

Figure 2 Application of LoQAtE to study regulatory networks Two advanced search options were used to put-together a list of all yeast TFs thatchanged abundance (search by abundance) or localization (search by movement) (A) Example of change in localization during nitrogen starvationScale bar represents 5 m (B) The list of 33 TFs was compared with microarray data on expression changes under the same stress condition (14) touncover that 16 of them were not documented to have any change in their transcripts under the exact same experimental conditions (C) Using theDB for regulatory motifs (YEASTRACT) we extracted the targets for these 16 TFs and found that an enormous fraction of all changes in proteinlevels found in LoQAtE during nitrogen starvation (using the Search by Abundance option) (22ndash24) can be explained by 10 of the TFs

4 Nucleic Acids Research 2013

reintegration grant [IRG 239224] EMBO YoungInvestigator Fellow (to MS) Recipient of an MITInternational Science and Technology Initiative (MISTI)(to MS) Funding for open access charge ERC StartingGrant [260395]

Conflict of interest statement None declared

REFERENCES

1 Frenkel-MorgensternM CohenAA Geva-ZatorskyNEdenE PriluskyJ IssaevaI SigalA Cohen-SaidonCLironY CohenL et al (2010) Dynamic Proteomics a databasefor dynamics and localizations of endogenous fluorescently-taggedproteins in living human cells Nucleic Acids Res 38D508ndashD512

2 AragonT van AnkenE PincusD SerafimovaIMKorennykhAV RubioCA and WalterP (2009) MessengerRNA targeting to endoplasmic reticulum stress signalling sitesNature 457 736ndash740

3 BeyerA HollunderJ NasheuerHP and WilhelmT (2004)Post-transcriptional expression regulation in the yeastSaccharomyces cerevisiae on a genomic scale Mol CellProteomics 3 1083ndash1092

4 BrekerM GymrekM and SchuldinerM (2013) A novel single-cell screening platform reveals proteome plasticity during yeaststress responses J Cell Biol 200 839ndash850

5 EdenE Geva-ZatorskyN IssaevaI CohenA DekelEDanonT CohenL MayoA and AlonU (2011) Proteomehalf-life dynamics in living human cells Science 331 764ndash768

6 ErjavecN LarssonL GranthamJ and NystromT (2007)Accelerated aging and failure to segregate damaged proteins inSir2 mutants can be suppressed by overproducing the proteinaggregation-remodeling factor Hsp104p Genes Dev 212410ndash2421

7 HedbackerK TownleyR and CarlsonM (2004) Cyclic AMP-dependent protein kinase regulates the subcellular localization ofSnf1-Sip1 protein kinase Mol Cell Biol 24 1836ndash1843

8 LeeMV TopperSE HublerSL HoseJ WengerCDCoonJJ and GaschAP (2011) A dynamic model of proteomechanges reveals new roles for transcript alteration in yeast MolSyst Biol 7 514

9 SigalA MiloR CohenA Geva-ZatorskyN KleinYAlalufI SwerdlinN PerzovN DanonT LironY et al (2006)Dynamic proteomics in individual human cells uncoverswidespread cell-cycle dependence of nuclear proteins NatMethods 3 525ndash531

10 TkachJM YimitA LeeAY RiffleM CostanzoMJaschobD HendryJA OuJ MoffatJ BooneC et al (2012)Dissecting DNA damage response pathways by analysing proteinlocalization and abundance changes during DNA replicationstress Nat Cell Biol 14 966ndash976

11 NewmanJR GhaemmaghamiS IhmelsJ BreslowDKNobleM DeRisiJL and WeissmanJS (2006) Single-cellproteomic analysis of S cerevisiae reveals the architecture ofbiological noise Nature 441 840ndash846

12 Stathopoulos-GerontidesA GuoJJ and CyertMS (1999)Yeast calcineurin regulates nuclear localization of the Crz1ptranscription factor through dephosphorylation Genes Dev 13798ndash803

13 ToulmayA and PrinzWA Direct imaging reveals stablemicrometer-scale lipid domains that segregate proteins in livecells J Cell Biol 202 35ndash44

14 GaschAP SpellmanPT KaoCM Carmel-HarelOEisenMB StorzG BotsteinD and BrownPO (2000) Genomicexpression programs in the response of yeast cells to environmentalchanges Mol Biol Cell 11 4241ndash4257

15 HughesTR MartonMJ JonesAR RobertsCJStoughtonR ArmourCD BennettHA CoffeyE DaiHHeYD et al (2000) Functional discovery via a compendiumof expression profiles Cell 102 109ndash126

16 CaustonHC RenB KohSS HarbisonCT KaninEJenningsEG LeeTI TrueHL LanderES and YoungRA(2001) Remodeling of yeast genome expression in response toenvironmental changes Mol Biol Cell 12 323ndash337

17 RohTY and ZhaoK (2008) High-resolution genome-widemapping of chromatin modifications by GMAT Methods MolBiol 387 95ndash108

18 HuhWK FalvoJV GerkeLC CarrollAS HowsonRWWeissmanJS and OrsquoSheaEK (2003) Global analysis of proteinlocalization in budding yeast Nature 425 686ndash691

19 Nadler-HollyM BrekerM GruberR AziaA GymrekMEisensteinM WillisonKR SchuldinerM and HorovitzAInteractions of subunit CCT3 in the yeast chaperonin CCTTRiCwith QN-rich proteins revealed by high-throughput microscopyanalysis Proc Natl Acad Sci USA 109 18833ndash18838

20 PicottiP Clement-ZizaM LamH CampbellDS SchmidtADeutschEW RostH SunZ RinnerO ReiterL et alA complete mass-spectrometric map of the yeast proteomeapplied to quantitative trait analysis Nature 494 266ndash270

21 de GodoyLM OlsenJV CoxJ NielsenML HubnerNCFrohlichF WaltherTC and MannM (2008) Comprehensivemass-spectrometry-based proteome quantification of haploidversus diploid yeast Nature 455 1251ndash1254

22 TeixeiraMC MonteiroP JainP TenreiroS FernandesARMiraNP AlenquerM FreitasAT OliveiraAL and Sa-CorreiaI (2006) The YEASTRACT database a tool for theanalysis of transcription regulatory associations in Saccharomycescerevisiae Nucleic Acids Res 34 D446ndashD451

23 MonteiroPT MendesND TeixeiraMC drsquoOreySTenreiroS MiraNP PaisH FranciscoAP CarvalhoAMLourencoAB et al (2008) YEASTRACT-DISCOVERER newtools to improve the analysis of transcriptional regulatoryassociations in Saccharomyces cerevisiae Nucleic Acids Res 36D132ndashD136

24 AbdulrehmanD MonteiroPT TeixeiraMC MiraNPLourencoAB dos SantosSC CabritoTR FranciscoAPMadeiraSC AiresRS et al (2010) YEASTRACT providing aprogrammatic access to curated transcriptional regulatoryassociations in Saccharomyces cerevisiae through a web servicesinterface Nucleic Acids Res 39 D136ndashD140

25 NatterK LeitnerP FaschingerA WolinskiH McCraithSFieldsS and KohlweinSD (2005) The spatial organization oflipid synthesis in the yeast Saccharomyces cerevisiae derived fromlarge scale green fluorescent protein tagging and high resolutionmicroscopy Mol Cell Proteomics 4 662ndash672

26 HabelerG NatterK ThallingerGG CrawfordMEKohlweinSD and TrajanoskiZ (2002) YPLdb the yeastprotein localization database Nucleic Acids Res 30 80ndash83

27 KalsM NatterK ThallingerGG TrajanoskiZ andKohlweinSD (2005) YPLdb2 the yeast protein localizationdatabase version 20 Yeast 22 213ndash218

28 WiwatwattanaN and KumarA (2005) Organelle DB a cross-species database of protein localization and function NucleicAcids Res 33 D598ndash604

29 WiwatwattanaN LandauCM CopeGJ HarpGA andKumarA (2007) Organelle DB an updated resource ofeukaryotic protein localization and function Nucleic Acids Res35 D810ndashD814

30 Frenkel-MorgensternM CohenAA Geva-ZatorskyNEdenE PriluskyJ IssaevaI SigalA Cohen-SaidonCLironY CohenL et al (2010) Dynamic Proteomics a databasefor dynamics and localizations of endogenous fluorescently-taggedproteins in living human cells Nucleic Acids Res 38D508ndashD512

31 UhlenM OksvoldP FagerbergL LundbergE JonassonKForsbergM ZwahlenM KampfC WesterK HoberS et al(2010) Towards a knowledge-based Human Protein Atlas NatBiotechnol 28 1248ndash1250

32 DesiereF DeutschEW NesvizhskiiAI MallickP KingNLEngJK AderemA BoyleR BrunnerE DonohoeS et al(2005) Integration with the human genome of peptidesequences obtained by high-throughput mass spectrometryGenome Biol 6 R9

Nucleic Acids Research 2013 5

Page 3: LoQAtE—Localization and Quantitation ATlas of the yeast ... · In addition, the results of each search can be downloaded in an excel table or as raw images. ‘Search by movement’

Figure 1 Screen shots of two result windows in a LoQAtE search The LoQAtE allows various search functions to be performed Both quick searchand advanced search (Search by abundance or localization) (A) enables querying for large groups of proteins and results in the entire localization andabundance data for each protein being presented In addition the results of each search can be downloaded in an excel table or as raw imageslsquoSearch by movementrsquo (B) allows the user to choose a movement of interest represented as an arrow on a schematic representation of the cell andreceive information on all proteins that are assigned to this category

Nucleic Acids Research 2013 3

abundance of the majority of yeast proteins understandard laboratory growth conditions based on the con-struction of the GFP collection (18) Additionally re-analysis of the GFP collection and additional proteinsinvolved in lipid metabolism was performed by high-resolution confocal imaging (25) and is available atthe YPL+DB (httpyplpuni-grazatindexphp) (2627)For a broader range of proteomic data the YeastResource Center (YRC) (httpdeptswashingtoneduyeastrc) is a collaborative web site that gathers severalcore technologies and provides a breadth of informationsuch as quantitative mass-spectrometry sequence-function relationships microscopy structure and compu-tational strategies for structure-function predictionsFinally organelle DB (httporganelledblsiumicheduindexphp) compiles protein localization data that areorganized into gt50 organelles subcellular structures andprotein complexes (2829) The data set includes 138 or-ganisms spanning the eukaryotic kingdom and incorpor-ates ongoing results from large-scale studies of proteinlocalization in yeast S cerevisiaeA similar effort in human cell lines is based on the

library of annotated reporter cell-clones (LARC) inwhich each strain (out of 2180) contains an endogenousprotein fused to yellow fluorescent protein (YFP) (30)This DB (httpwwwweizmannacilmcbUriAlonDynamProt) provides the expression and localization ofeach protein over time in response to various drugs andis regularly updated with newly created clones (30) Amore comprehensive human proteome atlas has recentlybecome available and harbors expression data for thou-sands of human proteins from a large number of healthytissues tumors cell lines and subcellular localizations allbased on antibody staining (httpwwwproteinatlasorg)(31) A principally similar DB called lsquoPeptideAtlasrsquo(httpwwwpeptideatlasorg) harbor growing data onproteins expressed under various conditions for a wide

variety of organisms from yeast to human collected bytandem mass spectrometry (MSMS) (32)

OUTLOOK

The LoQAtE DB in its first version contains easily ac-cessible and user-friendly information about subcellularlocalization and levels for 5330 yeast proteins underthree environmental stress conditions and two genetic per-turbations The richness of post-translational events docu-mented in this DB underlines the significance of studyingthe level of proteins to capture a true picture of a cellrsquosresponse to stress Starting from behavior of individualproteins and up to a birdrsquos eye view on the entire cell asa holistic functional unit this DB enables scientists to gaina new insight on cellular dynamics As more screens areperformed in our laboratory they will be added into thispublicly available atlas This rich resource should serve formultiparametric searches and discovery of new proteinfunctions illuminated only by the combination of suchvast amounts of data

ACKNOWLEDGEMENTS

The authors would like to thank Jonathan Weissman forproviding them with the full GFP library and AmnonHorovitz and Michal Nadler-Holly for their fruitful col-laboration studying the CCT complex They would like tothank Keren Katzav Genia Brodsky and Orit Bechar forthe graphical designs of the database and Rachel Baumanand Anastasia Zarankin from the internet services sectionof the Weizmann Institute for constructing the LoQAtEdatabase

FUNDING

Isreali Ministry of Science the European ResearchCouncil (ERC) Starting Grant [StG 260395] and

Figure 2 Application of LoQAtE to study regulatory networks Two advanced search options were used to put-together a list of all yeast TFs thatchanged abundance (search by abundance) or localization (search by movement) (A) Example of change in localization during nitrogen starvationScale bar represents 5 m (B) The list of 33 TFs was compared with microarray data on expression changes under the same stress condition (14) touncover that 16 of them were not documented to have any change in their transcripts under the exact same experimental conditions (C) Using theDB for regulatory motifs (YEASTRACT) we extracted the targets for these 16 TFs and found that an enormous fraction of all changes in proteinlevels found in LoQAtE during nitrogen starvation (using the Search by Abundance option) (22ndash24) can be explained by 10 of the TFs

4 Nucleic Acids Research 2013

reintegration grant [IRG 239224] EMBO YoungInvestigator Fellow (to MS) Recipient of an MITInternational Science and Technology Initiative (MISTI)(to MS) Funding for open access charge ERC StartingGrant [260395]

Conflict of interest statement None declared

REFERENCES

1 Frenkel-MorgensternM CohenAA Geva-ZatorskyNEdenE PriluskyJ IssaevaI SigalA Cohen-SaidonCLironY CohenL et al (2010) Dynamic Proteomics a databasefor dynamics and localizations of endogenous fluorescently-taggedproteins in living human cells Nucleic Acids Res 38D508ndashD512

2 AragonT van AnkenE PincusD SerafimovaIMKorennykhAV RubioCA and WalterP (2009) MessengerRNA targeting to endoplasmic reticulum stress signalling sitesNature 457 736ndash740

3 BeyerA HollunderJ NasheuerHP and WilhelmT (2004)Post-transcriptional expression regulation in the yeastSaccharomyces cerevisiae on a genomic scale Mol CellProteomics 3 1083ndash1092

4 BrekerM GymrekM and SchuldinerM (2013) A novel single-cell screening platform reveals proteome plasticity during yeaststress responses J Cell Biol 200 839ndash850

5 EdenE Geva-ZatorskyN IssaevaI CohenA DekelEDanonT CohenL MayoA and AlonU (2011) Proteomehalf-life dynamics in living human cells Science 331 764ndash768

6 ErjavecN LarssonL GranthamJ and NystromT (2007)Accelerated aging and failure to segregate damaged proteins inSir2 mutants can be suppressed by overproducing the proteinaggregation-remodeling factor Hsp104p Genes Dev 212410ndash2421

7 HedbackerK TownleyR and CarlsonM (2004) Cyclic AMP-dependent protein kinase regulates the subcellular localization ofSnf1-Sip1 protein kinase Mol Cell Biol 24 1836ndash1843

8 LeeMV TopperSE HublerSL HoseJ WengerCDCoonJJ and GaschAP (2011) A dynamic model of proteomechanges reveals new roles for transcript alteration in yeast MolSyst Biol 7 514

9 SigalA MiloR CohenA Geva-ZatorskyN KleinYAlalufI SwerdlinN PerzovN DanonT LironY et al (2006)Dynamic proteomics in individual human cells uncoverswidespread cell-cycle dependence of nuclear proteins NatMethods 3 525ndash531

10 TkachJM YimitA LeeAY RiffleM CostanzoMJaschobD HendryJA OuJ MoffatJ BooneC et al (2012)Dissecting DNA damage response pathways by analysing proteinlocalization and abundance changes during DNA replicationstress Nat Cell Biol 14 966ndash976

11 NewmanJR GhaemmaghamiS IhmelsJ BreslowDKNobleM DeRisiJL and WeissmanJS (2006) Single-cellproteomic analysis of S cerevisiae reveals the architecture ofbiological noise Nature 441 840ndash846

12 Stathopoulos-GerontidesA GuoJJ and CyertMS (1999)Yeast calcineurin regulates nuclear localization of the Crz1ptranscription factor through dephosphorylation Genes Dev 13798ndash803

13 ToulmayA and PrinzWA Direct imaging reveals stablemicrometer-scale lipid domains that segregate proteins in livecells J Cell Biol 202 35ndash44

14 GaschAP SpellmanPT KaoCM Carmel-HarelOEisenMB StorzG BotsteinD and BrownPO (2000) Genomicexpression programs in the response of yeast cells to environmentalchanges Mol Biol Cell 11 4241ndash4257

15 HughesTR MartonMJ JonesAR RobertsCJStoughtonR ArmourCD BennettHA CoffeyE DaiHHeYD et al (2000) Functional discovery via a compendiumof expression profiles Cell 102 109ndash126

16 CaustonHC RenB KohSS HarbisonCT KaninEJenningsEG LeeTI TrueHL LanderES and YoungRA(2001) Remodeling of yeast genome expression in response toenvironmental changes Mol Biol Cell 12 323ndash337

17 RohTY and ZhaoK (2008) High-resolution genome-widemapping of chromatin modifications by GMAT Methods MolBiol 387 95ndash108

18 HuhWK FalvoJV GerkeLC CarrollAS HowsonRWWeissmanJS and OrsquoSheaEK (2003) Global analysis of proteinlocalization in budding yeast Nature 425 686ndash691

19 Nadler-HollyM BrekerM GruberR AziaA GymrekMEisensteinM WillisonKR SchuldinerM and HorovitzAInteractions of subunit CCT3 in the yeast chaperonin CCTTRiCwith QN-rich proteins revealed by high-throughput microscopyanalysis Proc Natl Acad Sci USA 109 18833ndash18838

20 PicottiP Clement-ZizaM LamH CampbellDS SchmidtADeutschEW RostH SunZ RinnerO ReiterL et alA complete mass-spectrometric map of the yeast proteomeapplied to quantitative trait analysis Nature 494 266ndash270

21 de GodoyLM OlsenJV CoxJ NielsenML HubnerNCFrohlichF WaltherTC and MannM (2008) Comprehensivemass-spectrometry-based proteome quantification of haploidversus diploid yeast Nature 455 1251ndash1254

22 TeixeiraMC MonteiroP JainP TenreiroS FernandesARMiraNP AlenquerM FreitasAT OliveiraAL and Sa-CorreiaI (2006) The YEASTRACT database a tool for theanalysis of transcription regulatory associations in Saccharomycescerevisiae Nucleic Acids Res 34 D446ndashD451

23 MonteiroPT MendesND TeixeiraMC drsquoOreySTenreiroS MiraNP PaisH FranciscoAP CarvalhoAMLourencoAB et al (2008) YEASTRACT-DISCOVERER newtools to improve the analysis of transcriptional regulatoryassociations in Saccharomyces cerevisiae Nucleic Acids Res 36D132ndashD136

24 AbdulrehmanD MonteiroPT TeixeiraMC MiraNPLourencoAB dos SantosSC CabritoTR FranciscoAPMadeiraSC AiresRS et al (2010) YEASTRACT providing aprogrammatic access to curated transcriptional regulatoryassociations in Saccharomyces cerevisiae through a web servicesinterface Nucleic Acids Res 39 D136ndashD140

25 NatterK LeitnerP FaschingerA WolinskiH McCraithSFieldsS and KohlweinSD (2005) The spatial organization oflipid synthesis in the yeast Saccharomyces cerevisiae derived fromlarge scale green fluorescent protein tagging and high resolutionmicroscopy Mol Cell Proteomics 4 662ndash672

26 HabelerG NatterK ThallingerGG CrawfordMEKohlweinSD and TrajanoskiZ (2002) YPLdb the yeastprotein localization database Nucleic Acids Res 30 80ndash83

27 KalsM NatterK ThallingerGG TrajanoskiZ andKohlweinSD (2005) YPLdb2 the yeast protein localizationdatabase version 20 Yeast 22 213ndash218

28 WiwatwattanaN and KumarA (2005) Organelle DB a cross-species database of protein localization and function NucleicAcids Res 33 D598ndash604

29 WiwatwattanaN LandauCM CopeGJ HarpGA andKumarA (2007) Organelle DB an updated resource ofeukaryotic protein localization and function Nucleic Acids Res35 D810ndashD814

30 Frenkel-MorgensternM CohenAA Geva-ZatorskyNEdenE PriluskyJ IssaevaI SigalA Cohen-SaidonCLironY CohenL et al (2010) Dynamic Proteomics a databasefor dynamics and localizations of endogenous fluorescently-taggedproteins in living human cells Nucleic Acids Res 38D508ndashD512

31 UhlenM OksvoldP FagerbergL LundbergE JonassonKForsbergM ZwahlenM KampfC WesterK HoberS et al(2010) Towards a knowledge-based Human Protein Atlas NatBiotechnol 28 1248ndash1250

32 DesiereF DeutschEW NesvizhskiiAI MallickP KingNLEngJK AderemA BoyleR BrunnerE DonohoeS et al(2005) Integration with the human genome of peptidesequences obtained by high-throughput mass spectrometryGenome Biol 6 R9

Nucleic Acids Research 2013 5

Page 4: LoQAtE—Localization and Quantitation ATlas of the yeast ... · In addition, the results of each search can be downloaded in an excel table or as raw images. ‘Search by movement’

abundance of the majority of yeast proteins understandard laboratory growth conditions based on the con-struction of the GFP collection (18) Additionally re-analysis of the GFP collection and additional proteinsinvolved in lipid metabolism was performed by high-resolution confocal imaging (25) and is available atthe YPL+DB (httpyplpuni-grazatindexphp) (2627)For a broader range of proteomic data the YeastResource Center (YRC) (httpdeptswashingtoneduyeastrc) is a collaborative web site that gathers severalcore technologies and provides a breadth of informationsuch as quantitative mass-spectrometry sequence-function relationships microscopy structure and compu-tational strategies for structure-function predictionsFinally organelle DB (httporganelledblsiumicheduindexphp) compiles protein localization data that areorganized into gt50 organelles subcellular structures andprotein complexes (2829) The data set includes 138 or-ganisms spanning the eukaryotic kingdom and incorpor-ates ongoing results from large-scale studies of proteinlocalization in yeast S cerevisiaeA similar effort in human cell lines is based on the

library of annotated reporter cell-clones (LARC) inwhich each strain (out of 2180) contains an endogenousprotein fused to yellow fluorescent protein (YFP) (30)This DB (httpwwwweizmannacilmcbUriAlonDynamProt) provides the expression and localization ofeach protein over time in response to various drugs andis regularly updated with newly created clones (30) Amore comprehensive human proteome atlas has recentlybecome available and harbors expression data for thou-sands of human proteins from a large number of healthytissues tumors cell lines and subcellular localizations allbased on antibody staining (httpwwwproteinatlasorg)(31) A principally similar DB called lsquoPeptideAtlasrsquo(httpwwwpeptideatlasorg) harbor growing data onproteins expressed under various conditions for a wide

variety of organisms from yeast to human collected bytandem mass spectrometry (MSMS) (32)

OUTLOOK

The LoQAtE DB in its first version contains easily ac-cessible and user-friendly information about subcellularlocalization and levels for 5330 yeast proteins underthree environmental stress conditions and two genetic per-turbations The richness of post-translational events docu-mented in this DB underlines the significance of studyingthe level of proteins to capture a true picture of a cellrsquosresponse to stress Starting from behavior of individualproteins and up to a birdrsquos eye view on the entire cell asa holistic functional unit this DB enables scientists to gaina new insight on cellular dynamics As more screens areperformed in our laboratory they will be added into thispublicly available atlas This rich resource should serve formultiparametric searches and discovery of new proteinfunctions illuminated only by the combination of suchvast amounts of data

ACKNOWLEDGEMENTS

The authors would like to thank Jonathan Weissman forproviding them with the full GFP library and AmnonHorovitz and Michal Nadler-Holly for their fruitful col-laboration studying the CCT complex They would like tothank Keren Katzav Genia Brodsky and Orit Bechar forthe graphical designs of the database and Rachel Baumanand Anastasia Zarankin from the internet services sectionof the Weizmann Institute for constructing the LoQAtEdatabase

FUNDING

Isreali Ministry of Science the European ResearchCouncil (ERC) Starting Grant [StG 260395] and

Figure 2 Application of LoQAtE to study regulatory networks Two advanced search options were used to put-together a list of all yeast TFs thatchanged abundance (search by abundance) or localization (search by movement) (A) Example of change in localization during nitrogen starvationScale bar represents 5 m (B) The list of 33 TFs was compared with microarray data on expression changes under the same stress condition (14) touncover that 16 of them were not documented to have any change in their transcripts under the exact same experimental conditions (C) Using theDB for regulatory motifs (YEASTRACT) we extracted the targets for these 16 TFs and found that an enormous fraction of all changes in proteinlevels found in LoQAtE during nitrogen starvation (using the Search by Abundance option) (22ndash24) can be explained by 10 of the TFs

4 Nucleic Acids Research 2013

reintegration grant [IRG 239224] EMBO YoungInvestigator Fellow (to MS) Recipient of an MITInternational Science and Technology Initiative (MISTI)(to MS) Funding for open access charge ERC StartingGrant [260395]

Conflict of interest statement None declared

REFERENCES

1 Frenkel-MorgensternM CohenAA Geva-ZatorskyNEdenE PriluskyJ IssaevaI SigalA Cohen-SaidonCLironY CohenL et al (2010) Dynamic Proteomics a databasefor dynamics and localizations of endogenous fluorescently-taggedproteins in living human cells Nucleic Acids Res 38D508ndashD512

2 AragonT van AnkenE PincusD SerafimovaIMKorennykhAV RubioCA and WalterP (2009) MessengerRNA targeting to endoplasmic reticulum stress signalling sitesNature 457 736ndash740

3 BeyerA HollunderJ NasheuerHP and WilhelmT (2004)Post-transcriptional expression regulation in the yeastSaccharomyces cerevisiae on a genomic scale Mol CellProteomics 3 1083ndash1092

4 BrekerM GymrekM and SchuldinerM (2013) A novel single-cell screening platform reveals proteome plasticity during yeaststress responses J Cell Biol 200 839ndash850

5 EdenE Geva-ZatorskyN IssaevaI CohenA DekelEDanonT CohenL MayoA and AlonU (2011) Proteomehalf-life dynamics in living human cells Science 331 764ndash768

6 ErjavecN LarssonL GranthamJ and NystromT (2007)Accelerated aging and failure to segregate damaged proteins inSir2 mutants can be suppressed by overproducing the proteinaggregation-remodeling factor Hsp104p Genes Dev 212410ndash2421

7 HedbackerK TownleyR and CarlsonM (2004) Cyclic AMP-dependent protein kinase regulates the subcellular localization ofSnf1-Sip1 protein kinase Mol Cell Biol 24 1836ndash1843

8 LeeMV TopperSE HublerSL HoseJ WengerCDCoonJJ and GaschAP (2011) A dynamic model of proteomechanges reveals new roles for transcript alteration in yeast MolSyst Biol 7 514

9 SigalA MiloR CohenA Geva-ZatorskyN KleinYAlalufI SwerdlinN PerzovN DanonT LironY et al (2006)Dynamic proteomics in individual human cells uncoverswidespread cell-cycle dependence of nuclear proteins NatMethods 3 525ndash531

10 TkachJM YimitA LeeAY RiffleM CostanzoMJaschobD HendryJA OuJ MoffatJ BooneC et al (2012)Dissecting DNA damage response pathways by analysing proteinlocalization and abundance changes during DNA replicationstress Nat Cell Biol 14 966ndash976

11 NewmanJR GhaemmaghamiS IhmelsJ BreslowDKNobleM DeRisiJL and WeissmanJS (2006) Single-cellproteomic analysis of S cerevisiae reveals the architecture ofbiological noise Nature 441 840ndash846

12 Stathopoulos-GerontidesA GuoJJ and CyertMS (1999)Yeast calcineurin regulates nuclear localization of the Crz1ptranscription factor through dephosphorylation Genes Dev 13798ndash803

13 ToulmayA and PrinzWA Direct imaging reveals stablemicrometer-scale lipid domains that segregate proteins in livecells J Cell Biol 202 35ndash44

14 GaschAP SpellmanPT KaoCM Carmel-HarelOEisenMB StorzG BotsteinD and BrownPO (2000) Genomicexpression programs in the response of yeast cells to environmentalchanges Mol Biol Cell 11 4241ndash4257

15 HughesTR MartonMJ JonesAR RobertsCJStoughtonR ArmourCD BennettHA CoffeyE DaiHHeYD et al (2000) Functional discovery via a compendiumof expression profiles Cell 102 109ndash126

16 CaustonHC RenB KohSS HarbisonCT KaninEJenningsEG LeeTI TrueHL LanderES and YoungRA(2001) Remodeling of yeast genome expression in response toenvironmental changes Mol Biol Cell 12 323ndash337

17 RohTY and ZhaoK (2008) High-resolution genome-widemapping of chromatin modifications by GMAT Methods MolBiol 387 95ndash108

18 HuhWK FalvoJV GerkeLC CarrollAS HowsonRWWeissmanJS and OrsquoSheaEK (2003) Global analysis of proteinlocalization in budding yeast Nature 425 686ndash691

19 Nadler-HollyM BrekerM GruberR AziaA GymrekMEisensteinM WillisonKR SchuldinerM and HorovitzAInteractions of subunit CCT3 in the yeast chaperonin CCTTRiCwith QN-rich proteins revealed by high-throughput microscopyanalysis Proc Natl Acad Sci USA 109 18833ndash18838

20 PicottiP Clement-ZizaM LamH CampbellDS SchmidtADeutschEW RostH SunZ RinnerO ReiterL et alA complete mass-spectrometric map of the yeast proteomeapplied to quantitative trait analysis Nature 494 266ndash270

21 de GodoyLM OlsenJV CoxJ NielsenML HubnerNCFrohlichF WaltherTC and MannM (2008) Comprehensivemass-spectrometry-based proteome quantification of haploidversus diploid yeast Nature 455 1251ndash1254

22 TeixeiraMC MonteiroP JainP TenreiroS FernandesARMiraNP AlenquerM FreitasAT OliveiraAL and Sa-CorreiaI (2006) The YEASTRACT database a tool for theanalysis of transcription regulatory associations in Saccharomycescerevisiae Nucleic Acids Res 34 D446ndashD451

23 MonteiroPT MendesND TeixeiraMC drsquoOreySTenreiroS MiraNP PaisH FranciscoAP CarvalhoAMLourencoAB et al (2008) YEASTRACT-DISCOVERER newtools to improve the analysis of transcriptional regulatoryassociations in Saccharomyces cerevisiae Nucleic Acids Res 36D132ndashD136

24 AbdulrehmanD MonteiroPT TeixeiraMC MiraNPLourencoAB dos SantosSC CabritoTR FranciscoAPMadeiraSC AiresRS et al (2010) YEASTRACT providing aprogrammatic access to curated transcriptional regulatoryassociations in Saccharomyces cerevisiae through a web servicesinterface Nucleic Acids Res 39 D136ndashD140

25 NatterK LeitnerP FaschingerA WolinskiH McCraithSFieldsS and KohlweinSD (2005) The spatial organization oflipid synthesis in the yeast Saccharomyces cerevisiae derived fromlarge scale green fluorescent protein tagging and high resolutionmicroscopy Mol Cell Proteomics 4 662ndash672

26 HabelerG NatterK ThallingerGG CrawfordMEKohlweinSD and TrajanoskiZ (2002) YPLdb the yeastprotein localization database Nucleic Acids Res 30 80ndash83

27 KalsM NatterK ThallingerGG TrajanoskiZ andKohlweinSD (2005) YPLdb2 the yeast protein localizationdatabase version 20 Yeast 22 213ndash218

28 WiwatwattanaN and KumarA (2005) Organelle DB a cross-species database of protein localization and function NucleicAcids Res 33 D598ndash604

29 WiwatwattanaN LandauCM CopeGJ HarpGA andKumarA (2007) Organelle DB an updated resource ofeukaryotic protein localization and function Nucleic Acids Res35 D810ndashD814

30 Frenkel-MorgensternM CohenAA Geva-ZatorskyNEdenE PriluskyJ IssaevaI SigalA Cohen-SaidonCLironY CohenL et al (2010) Dynamic Proteomics a databasefor dynamics and localizations of endogenous fluorescently-taggedproteins in living human cells Nucleic Acids Res 38D508ndashD512

31 UhlenM OksvoldP FagerbergL LundbergE JonassonKForsbergM ZwahlenM KampfC WesterK HoberS et al(2010) Towards a knowledge-based Human Protein Atlas NatBiotechnol 28 1248ndash1250

32 DesiereF DeutschEW NesvizhskiiAI MallickP KingNLEngJK AderemA BoyleR BrunnerE DonohoeS et al(2005) Integration with the human genome of peptidesequences obtained by high-throughput mass spectrometryGenome Biol 6 R9

Nucleic Acids Research 2013 5

Page 5: LoQAtE—Localization and Quantitation ATlas of the yeast ... · In addition, the results of each search can be downloaded in an excel table or as raw images. ‘Search by movement’

reintegration grant [IRG 239224] EMBO YoungInvestigator Fellow (to MS) Recipient of an MITInternational Science and Technology Initiative (MISTI)(to MS) Funding for open access charge ERC StartingGrant [260395]

Conflict of interest statement None declared

REFERENCES

1 Frenkel-MorgensternM CohenAA Geva-ZatorskyNEdenE PriluskyJ IssaevaI SigalA Cohen-SaidonCLironY CohenL et al (2010) Dynamic Proteomics a databasefor dynamics and localizations of endogenous fluorescently-taggedproteins in living human cells Nucleic Acids Res 38D508ndashD512

2 AragonT van AnkenE PincusD SerafimovaIMKorennykhAV RubioCA and WalterP (2009) MessengerRNA targeting to endoplasmic reticulum stress signalling sitesNature 457 736ndash740

3 BeyerA HollunderJ NasheuerHP and WilhelmT (2004)Post-transcriptional expression regulation in the yeastSaccharomyces cerevisiae on a genomic scale Mol CellProteomics 3 1083ndash1092

4 BrekerM GymrekM and SchuldinerM (2013) A novel single-cell screening platform reveals proteome plasticity during yeaststress responses J Cell Biol 200 839ndash850

5 EdenE Geva-ZatorskyN IssaevaI CohenA DekelEDanonT CohenL MayoA and AlonU (2011) Proteomehalf-life dynamics in living human cells Science 331 764ndash768

6 ErjavecN LarssonL GranthamJ and NystromT (2007)Accelerated aging and failure to segregate damaged proteins inSir2 mutants can be suppressed by overproducing the proteinaggregation-remodeling factor Hsp104p Genes Dev 212410ndash2421

7 HedbackerK TownleyR and CarlsonM (2004) Cyclic AMP-dependent protein kinase regulates the subcellular localization ofSnf1-Sip1 protein kinase Mol Cell Biol 24 1836ndash1843

8 LeeMV TopperSE HublerSL HoseJ WengerCDCoonJJ and GaschAP (2011) A dynamic model of proteomechanges reveals new roles for transcript alteration in yeast MolSyst Biol 7 514

9 SigalA MiloR CohenA Geva-ZatorskyN KleinYAlalufI SwerdlinN PerzovN DanonT LironY et al (2006)Dynamic proteomics in individual human cells uncoverswidespread cell-cycle dependence of nuclear proteins NatMethods 3 525ndash531

10 TkachJM YimitA LeeAY RiffleM CostanzoMJaschobD HendryJA OuJ MoffatJ BooneC et al (2012)Dissecting DNA damage response pathways by analysing proteinlocalization and abundance changes during DNA replicationstress Nat Cell Biol 14 966ndash976

11 NewmanJR GhaemmaghamiS IhmelsJ BreslowDKNobleM DeRisiJL and WeissmanJS (2006) Single-cellproteomic analysis of S cerevisiae reveals the architecture ofbiological noise Nature 441 840ndash846

12 Stathopoulos-GerontidesA GuoJJ and CyertMS (1999)Yeast calcineurin regulates nuclear localization of the Crz1ptranscription factor through dephosphorylation Genes Dev 13798ndash803

13 ToulmayA and PrinzWA Direct imaging reveals stablemicrometer-scale lipid domains that segregate proteins in livecells J Cell Biol 202 35ndash44

14 GaschAP SpellmanPT KaoCM Carmel-HarelOEisenMB StorzG BotsteinD and BrownPO (2000) Genomicexpression programs in the response of yeast cells to environmentalchanges Mol Biol Cell 11 4241ndash4257

15 HughesTR MartonMJ JonesAR RobertsCJStoughtonR ArmourCD BennettHA CoffeyE DaiHHeYD et al (2000) Functional discovery via a compendiumof expression profiles Cell 102 109ndash126

16 CaustonHC RenB KohSS HarbisonCT KaninEJenningsEG LeeTI TrueHL LanderES and YoungRA(2001) Remodeling of yeast genome expression in response toenvironmental changes Mol Biol Cell 12 323ndash337

17 RohTY and ZhaoK (2008) High-resolution genome-widemapping of chromatin modifications by GMAT Methods MolBiol 387 95ndash108

18 HuhWK FalvoJV GerkeLC CarrollAS HowsonRWWeissmanJS and OrsquoSheaEK (2003) Global analysis of proteinlocalization in budding yeast Nature 425 686ndash691

19 Nadler-HollyM BrekerM GruberR AziaA GymrekMEisensteinM WillisonKR SchuldinerM and HorovitzAInteractions of subunit CCT3 in the yeast chaperonin CCTTRiCwith QN-rich proteins revealed by high-throughput microscopyanalysis Proc Natl Acad Sci USA 109 18833ndash18838

20 PicottiP Clement-ZizaM LamH CampbellDS SchmidtADeutschEW RostH SunZ RinnerO ReiterL et alA complete mass-spectrometric map of the yeast proteomeapplied to quantitative trait analysis Nature 494 266ndash270

21 de GodoyLM OlsenJV CoxJ NielsenML HubnerNCFrohlichF WaltherTC and MannM (2008) Comprehensivemass-spectrometry-based proteome quantification of haploidversus diploid yeast Nature 455 1251ndash1254

22 TeixeiraMC MonteiroP JainP TenreiroS FernandesARMiraNP AlenquerM FreitasAT OliveiraAL and Sa-CorreiaI (2006) The YEASTRACT database a tool for theanalysis of transcription regulatory associations in Saccharomycescerevisiae Nucleic Acids Res 34 D446ndashD451

23 MonteiroPT MendesND TeixeiraMC drsquoOreySTenreiroS MiraNP PaisH FranciscoAP CarvalhoAMLourencoAB et al (2008) YEASTRACT-DISCOVERER newtools to improve the analysis of transcriptional regulatoryassociations in Saccharomyces cerevisiae Nucleic Acids Res 36D132ndashD136

24 AbdulrehmanD MonteiroPT TeixeiraMC MiraNPLourencoAB dos SantosSC CabritoTR FranciscoAPMadeiraSC AiresRS et al (2010) YEASTRACT providing aprogrammatic access to curated transcriptional regulatoryassociations in Saccharomyces cerevisiae through a web servicesinterface Nucleic Acids Res 39 D136ndashD140

25 NatterK LeitnerP FaschingerA WolinskiH McCraithSFieldsS and KohlweinSD (2005) The spatial organization oflipid synthesis in the yeast Saccharomyces cerevisiae derived fromlarge scale green fluorescent protein tagging and high resolutionmicroscopy Mol Cell Proteomics 4 662ndash672

26 HabelerG NatterK ThallingerGG CrawfordMEKohlweinSD and TrajanoskiZ (2002) YPLdb the yeastprotein localization database Nucleic Acids Res 30 80ndash83

27 KalsM NatterK ThallingerGG TrajanoskiZ andKohlweinSD (2005) YPLdb2 the yeast protein localizationdatabase version 20 Yeast 22 213ndash218

28 WiwatwattanaN and KumarA (2005) Organelle DB a cross-species database of protein localization and function NucleicAcids Res 33 D598ndash604

29 WiwatwattanaN LandauCM CopeGJ HarpGA andKumarA (2007) Organelle DB an updated resource ofeukaryotic protein localization and function Nucleic Acids Res35 D810ndashD814

30 Frenkel-MorgensternM CohenAA Geva-ZatorskyNEdenE PriluskyJ IssaevaI SigalA Cohen-SaidonCLironY CohenL et al (2010) Dynamic Proteomics a databasefor dynamics and localizations of endogenous fluorescently-taggedproteins in living human cells Nucleic Acids Res 38D508ndashD512

31 UhlenM OksvoldP FagerbergL LundbergE JonassonKForsbergM ZwahlenM KampfC WesterK HoberS et al(2010) Towards a knowledge-based Human Protein Atlas NatBiotechnol 28 1248ndash1250

32 DesiereF DeutschEW NesvizhskiiAI MallickP KingNLEngJK AderemA BoyleR BrunnerE DonohoeS et al(2005) Integration with the human genome of peptidesequences obtained by high-throughput mass spectrometryGenome Biol 6 R9

Nucleic Acids Research 2013 5