What are the most abundant proteins in a cell?

Transcription

1 What ar th most abundant s in a cll? Evn aftr rading svral txtbooks on s, on may still b lft wondring which of ths critical molcular playrs in th lif of a cll ar th most quantitativly abundant. Though figuring this out by pur thought alon is gnrally not asy, clls in th lavs of plants ar that rar cas in which it is rlativly asy to mak an stimat. Th carbon-fixing nzym Rubisco, th molcular gatkpr btwn th inorganic and th organic worlds is rquird at xtrmly high concntrations. Lt s s why. As schmatically dpictd in Figur 1, th photon flux undr full illumination is about 2000 microeinstin/m 2 -s. About 10-30% of this flux is maximally utilizd and byond that thr is saturation of th photosynthtic apparatus. About vry 10 photons supply nough nrgy to fix on carbon atom. Rubisco works at a sluggish maximal rat of 1-3 pr sc pr catalytic sit. From this alon, w can s that th cll thus nds 0.3-3x10 7 Rubisco molculs pr micron 2 cross sction. A Rubisco monomr has a mass of 60kDalton (BNID ) and so th wight pr micron 2 is 0.3-3x10-12 g. Lt s stimat th total contnt in laf. A charactristic laf has a hight of about 200 micron. 80% of th volum is vacuols (BNID ) and th dry mass will b 30% of this volum with s consisting about half, so w arriv at about 6x10-12 g of pr cll as drivd in Figur 1. W conclud that about 5-50% of th mass is Rubisco. Indd, th xprimntal dtrminations in C3 plants such as what, potato and tobacco find that Rubisco constituts in th rang of 25-60% of all solubl s in such clls (BNID ). Th cnsus for othr organisms, vn modl microorganisms, is mor complicatd. In th lat 1970s, a uniqu catalog of th quantitis of 140 s undr diffrnt growth rats in E. coli was cratd using 2D gl lctrophorsis and 14 C labling (Pdrsn t al, Cll 1978 BNID ). Nwr mthods hav rcntly nabld xtnsiv wid survys of contnt using mass spctromtry (BNID xxx), TAP labling (Ghammaghami 2003, BNID ) and fluorscnt light microscopy (Taniguchi t al., 2010, BNID xxx). A nw databas ( has bn cratd to collct such data on abundancs across organisms. Th pictur mrging from ths kinds of xprimnts shows svral prominnt playrs. First, not surprisingly, ribosomal s and thir ancillary componnts ar highly abundant. Th longation factor EF-TU, rsponsibl for mdiating th ntranc of th trna to th fr sit of th ribosom, was charactrizd as th most abundant in th original 1978 catalog with a copy numbr of ~58,000 s pr bactrial gnom. This absolut molcular count can b rpackagd in concntration units and is roughly quivalnt to 100 μm (BNID ). Rcall that undr diffrnt growth conditions th cll siz and thus total contnt can chang svral fold (s, for xampl, th vigntt on yast siz) and this mdia dpndnc to th cnsus is spcially important for ribosomal s. Anothr contndr for th titl of most abundant is ACP, th Acyl carrir, which plays an important rol in fatty acid biosynthsis. This carris fatty acid chains as th chains ar longatd. It is claimd to b th most abundant in E. coli, with about 60,000 molculs pr cll (BNID ). In a rcnt high throughput mass spctromtry masurmnt on minimal mdium (Lu, 2007 BNID ), a valu of 76,000 was rportd making it th third most abundant rportd. Tabl 1 givs a rank ordring of som of th most ubiquitous s found in E. coli, though it should b notd that thr ar inconsistncis btwn th diffrnt xprimntal approachs that hav not yt bn fully sttld. Th most

2 abundant found in this particular survy of E. coli is RplL, a ribosomal (stimatd at 109,000 copis pr cll, and rportd (Subrananlan, 1975) to b in 4 copis pr ribosom in contrast to othr ribosomal s which hav on copy pr ribosom) and TufB (th longation factor also known as EF-TU, stimatd at 87,000 copis pr cll). Th nxt most abundant rportd s ar GroS (MopB, 65,000), a componnt of th chapron systm Gro- EL-Gro-ES ncssary for propr folding of many s and GapA (49,000), a ky nzym in glycolysis. Structural s can also b highly abundant. FimA is th major of th fimbria (pili) of E. coli (BNID ). Evry pilus has about 1000 copis (BNID ) and thus a simpl stimat lads us to xpct hundrds of thousands of this rpating monomr on th outsid of th cll. As notd abov, contnt varis basd on growth conditions and gn induction. For xampl, LacZ, th gn rsponsibl for braking lactos into glucos and galactos is usually rprssd and th has only a small numbr of copis (10 to 20, BNID ), but undr full induction was charactrizd to hav a concntration of 50uM (BNID ), i.. about 100,000 copis pr cll. In summary, though diffrnt masurmnt mthods can vary significantly vn undr similar conditions th ovrall pictur of th most abundant s in E.coli is gnrally consistnt. As usual, it is intrsting to contrast what has bn discovrd in bactria with similar xprimnts in ukaryotic microorganisms. In yast, an ovrall stimat of 50,000,000 s pr cll was rportd (BNID ). Masurmnts basd on a TAP tag (BNID Ghammaghami 2003) rport that out of this hug stor of s, only thr ar found with ovr a million copis pr cll. Ths ar a cll wall (YKL096W-a), th Plasma mmbran H+-ATPas (YGL008C), that pumps protons out of th cll and Fructos 1,6-bisphosphat aldolas (YKL060C), ssntial for glycolysis and gluconognsis. Diffrnt rports on th abundanc of s in glycolysis, an intnsly studid modl systm, ld to an ovrall stimat of 25% of total contnt (BNID ). Lik with E. coli, in yast as wll, nw highthroughput MS data is bcoming availabl (BNID , ). Tabl 1 shows th top 10 most abundant yast s in rich as wll as minimal mdia. In rich mdia, th s with highst abundanc ar mostly glycolytic. In minimal mdia th most abundant s ar still of unclar function, which furthr highlights our limitd knowldg on ths most lmntary qustions to dat. Why ar popl going to all th troubl of carrying out ths incrasingly rfind cnsuss of som of th most favord modl organisms? Many of th biochmical and rgulatory pathways that mak up th lif of a cll hav bn or ar now bing mappd with xquisit dtail and many of th nods hav ssntial rols. But a wiring diagram dos not a cll mak. To rally undrstand th rlativ rats of th various componnts of ths pathways, w nd to know about th abundancs of th various s and thir substrats. Furthr, if on is intrstd in assssing th biosynthtic burdn of ths various molcular playrs, th actual abundanc is critical. Similarly, th many binding ractions that ar th basis for much of th busy biochmical activity of clls, whthr spcific binding of intntional partnrs or spurious nonspcific binding btwn unnatural partnrs is ultimatly dictatd by molcular counts. Finally, thr is a growing apprciation of th constraints that ar inflictd on th cll as a rsult of nois in copy numbrs. For undrstanding and prdicting such ffcts it is vital to know if on is daling with tns of thousands of copis pr cll or only tns of copis pr cll, as turns out to oftn b th cas in unicllular organisms. In ths small-numbrs limits, fluctuations ar a fact of lif and both w and th cll must account for thm.

3 Figur 1: Estimat of th fraction of Rubisco s of total contnt in a laf cll.

4 Tabl 1-2: Most abundant s in prokaryots and ukaryots. Svral mthods using mass spc (APEX, Lu t al., 2007 PMID ), using a yllow fluorscnt fusion library (Taniguchi t al., 2010 PMID ), cration of a yast fusion library whr ach opn rading fram is taggd with a high-affinity pitop and xprssd from its natural chromosomal location (Ghammaghami t al., 2003 PMID ) and mass spctromtry data of mous fibroblast clls (Schwanha ussr t al., 2011 PMID ). Gn annotation: Yast -SGD, E. coli Ecoliwiki, mous-uniprot. Color cod: yllow translation, cyan glycolysis, grn chaprons. Th sum is basd on adding togthr all th absolut valus rportd in ach study. Protin rank E. coli minim al mdia, Nat Biotc hnol, Lu 2007 (total of s/cll, sum of s in rfrn c is 2,500,0 00) 1 RplL, 4.4%, 50S riboso mal 2 TufB, 3.5%, EF-Tu, Elongat ion Factor- Transla tion (***** ***) E. coli M9 minimal mdia, Scinc, Taniguchi 2010 (sum of s in rfrnc is 95,000) CspC, 8.3%, strss TufA, 3.6%, chain longation factor EF-Tu (********) B. subtilis minimal mdium during xponntia l growth, Analytical Chmistry, Maass 2011 (sum of s in rfrnc is 2,300,000) TufA, 4.3%, Elongation factor Tu (********) CspD, 4.0%, Cold shock CspD S. aurus synthtic mdium during xponnti al growth, Anal Chm, Maass 2011 (sum of s in rfrnc is 350,000) Asp23, 7.1%, Alkalin shock 23 SodA, 6.9%, Suproxid dismutas [Mn/F] 1 (***) Lptospira intrrogans EMJH ( Ellinghausn- McCullough-Johnson- Harris) mdium, Malmström 2009 (sum of s in rfrnc is 820,000) LipL32, 4.6%, xtrnal ncapsulating structur Pptidoglycan associatd cytoplasmic mmbran, 3.7%, xtrnal ncapsulating structur

5 3 AcpP 3.0%, acyl carrir (ACP) RpsV, 3.3%, 30S ribosomal IlvC, 3.3%, Ktol-acid rductoiso mras CspA, 4.3%, Cold shock 60 kda chapronin (Protin Cpn60) (groel ) (Hat shock 58 kda ), 2.2%, nuclotid binding 4 GroS, 2.6%, 10 kda chapr onin 5 GapA, 2.0%, glycra ldhyd 3- phosph at dhydr ognas -A 6 MtE, 1.6%, Mthio nin synthas 7 CspC, 1.6%, strss 8 RplW, 1.5%, 50S riboso mal CspE, 3.2%, DNA-binding transcriptional rprssor DnaK, 2.5%, chapron Hsp70 GapA, 2.5%, glycraldhyd- 3-phosphat dhydrognas A TufB, 2.3%, chain longation factor EF-Tu (********) Rho, 2.3%, transcription trmination factor AhpC, 3.0%, Alkyl hydroprox id rductas C (***) YfmK, 2.5%, Uncharact rizd N- actyltrans fras YhA, 2.0%, UPF0342 Icd, 1.8%, Isocitrat dhydrog nas [NADP] participats in mapk signaling pathway GroS, 1.8%, 10 kda chapronin Tuf, 3.7%, Elongatio n factor Tu (******** ) RplL, 2.9%, 50S ribosomal L7/L12 GapA1, 2.8%, Glycrald hyd-3- phosphat dhydrog nas 1 Eno, 2.1%, Enolas (***) (no nam, locus SACOL2595), 1.8%, Putativ uncharact rizd Elongation factor Tu (EF- Tu), 1.7%, hydrolas activity (********) LipL36, 1.7%, xtrnal ncapsulating structur Flagllin, 1.7%, flagllum Elctron transfr flavo alpha, 1.5%, nuclotid binding transcriptional rgulator (ArsR family), 1.5%, transcription factor & rgulators

6 (***) 9 RpsP, 1.2%, 30S riboso mal 10 Mdh, 1.2%, Compo nnt of malat dhydr ognas GroS, 2.2%, 10 kda chapronin GlyA, 1.7%, srin hydroxymthyltr ansfras SodA, 1.6%, Suproxid dismutas [Mn] (***) TrxA, 1.5%, Thiordoxi n (no nam, locus SACOL0427), 1.7%, Putativ uncharact rizd AhpC, 1.4%, Alkyl hydropro xid rductas C (***) LipL41, 1.3%, xtrnal ncapsulating structur LipL21, 1.1%, xtrnal ncapsulating structur Protin rank S. crvisia - rich mdia, Nat Biotchnol, Lu 2007 (total of s/cll according to primary sourc, sum of s in rfrnc is also 50,000,000) 1 ENO2, 6.2%, Enolas II S. crvisia minimal mdia, Nat Biotchnol, Lu 2007 (total of s/c ll according to primary sourc, sum of s in rfrnc is also 50,000,000 ) ABM1, 4.6%, unknown function, rquird for normal microtubul organizatio n S. crvisia rich mdia, Natur, Ghammagha mi 2003 (sum of s in rfrnc is 47,000,000) CWP2, 3.4%, Cll Wall Protin M. musculus (NIH3T3 clls)- light (L) SILAC mdium, Natur, Schwanha ussr t al., 2011 (sum of s in rfrnc is 570,000,000) ACTB, 2.8%, Actin, cytoplasmic 1

7 2 FBA1, 4.0%, Fructos 1,6- bisphosphat aldolas (***) 3 TDH3, 4.0%, Glycraldhyd -3-phosphat dhydrognas 4 PGK1, 3.8%, 3- phosphoglycr at kinas 5 ENO1, 3.6%, Enolas I (***) 6 PDC1, 2.6%, Major of thr pyruvat dcarboxylas isozyms 7 ADH1, 2.6%, Alcohol dhydrognas 8 TEF2, 2.4%, Translational longation factor EF-1 alpha YMR181C, 4.2%, unknown function YLR407W, 4.2%, unknown function ORT1, 3.0%, Ornithin transportr of th mitochond rial innr mmbran YMR115W (SGD nam: MGR3), 2.6%, Subunit of th mitochond rial (mt) i- AAA protas suprcomp lx YIL077C, 2.2%, unknown function YDR193W, 2.0%, Dubious opn rading fram DOA1, 1.8%, WD rpat PMA1, 2.8%, Plasma Mmbran ATPas FBA1, 2.1%, Fructos 1,6- bisphosphat aldolas (***) ILV5, 1.9%, IsoLucinplus-Valin rquiring YEF3, 1.9%, Yast Elongation Factor (translation) HHF2, 1.4%, Histon H Four RPP2B, 1.4%, Ribosomal Protin P2 Bta HHF1, 1.1%, Histon H Four HIST1H4A, 2.6%, Histon H4 HIST1H2AF, 2.6%, Histon H2A typ 1-F HIST2H2BB, 1.9%, Histon H2B typ 2-B HIST1H3B, 1.5%, Histon H3.2 EEF1A1, 0.93%, Elongation factor 1-alpha 1 (translation) RPS27A, 0.9%, Ubiquitin-40S ribosomal S27a S100A4, 0.75%, Protin S100-A4 (similar to Glycraldhyd- 3-phosphat dhydrognas (GAPDH) isoform 1)

8 9 TDH2, 1.9%, Glycraldhyd -3-phosphat dhydrognas 10 CDC19, 1.8%, Pyruvat kinas CCZ1, 1.6%, Protin involvd in vacuolar assmbly RPS26A, 1.5%, small (40S) ribosomal SOD1, 1.1%, SuprOxid Dismutas RPS26B, 1.1%, Ribosomal Protin of th Small TUBB5, 0.75%, Tubulin bta-5 chain ANXA2, 0.67%, Annxin A2

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