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But large protein assembliesformed from many protein molecules perform the most impressive tasks. Nowthat it is possible to reconstruct most biological processesin cell-free systemsinthe laboratory, it is clear that each of the central processesin a cell-such asDNA replication, protein synthesis,vesicle budding, or transmembrane signaling-is catalyzed by a highly coordinated, linked set of I0 or more proteins. Inmost such protein machines, an energetically favorable reaction such as thehydrolysis of bound nucleoside triphosphates (ATp or GTp) drives an orderedseries of conformational changes in one or more of the individual protein subunits, enabling the ensemble of proteins to move coordinately. In this way, eachenzyme can be moved directly into position, as the machine catalyzessuccessive reactions in a series.This is what occurs, for example, in protein synthesison a ribosome (discussedin chapter 6)-or in DNA replication, where a largemultiprotein complex moves rapidly along the DNA (discussedin chapter 5).cells have evolved protein machines for the same reason that humans haveinvented mechanical and electronic machines.
For accomplishing almost anytask, manipulations that are spatially and temporally coordinated throughlinked processesare much more efficient than the use of individual tools.ProteinMachineswith InterchangeablePartsMakeEfficientuseof GeneticlnformationTo probe more deeply into the nature of protein machines, we shall consider arelatively simple one: the SCF ubiquitin ligase. This protein complex binds different "target proteins" at different times in the cell cycle, and it covalently addsmultiubiquitin polypeptide chains to these proteins. Its c-shaped structure isformed from five protein subunits, the largest of which is a molecule that servesas a scaffold protein on which the rest of the structure is built.
The structureunderlies a remarkable mechanism (Figure 3-zg). At one end of the c is an E2ubiquitin-conjugating enzyme. At the other end is a substrate-binding arm, asubunit knovrn as an F-box protein.These two subunits are separatedby a gap ofabout 5 nm. \Mhen this protein complex is acrivated, the F-box protein binds toa specific site on a target protein, positioning the protein in the gap so that someof its lysine side chains contact the ubiquitin-conjugatingThis enzymecan then catalyze the repeated addition of a ubiquitin "nry-e.polypeptide to theselysines (seeFigure 3-79c), producing a polyubiquitin chain that marks rhe target protein for rapid destruction in a proteasome (seep.
393).In this manner, specific proteins are targeted for rapid destruction inPROTEINFUNCTIONadaptorprotein 21)F-boxprotein( s u b s t r a t e - b i n d i nagr m )E 2u b i q u i t i n conjugatingenzymeubiquitinIItwo oJ manypossiblesubstrate-bind ingarms(B)p o l y u b i q ui t y l a t e dprotein targeted lor destruction,/r(C)u b i q u i t i nl i g a s ein cells, inasmuch as new functions can evolve for the entire complex simply byproducing an alternative version of one of its subunits.OftenInvolvesPositioningTheActivationof ProteinMachinesThemat SpecificSitesAs scientists have learned more of the details of cell biology, they have recognized increasing degreesof sophistication in cell chemistry.
Thus, not only do wenow know that protein machines play a predominant role, but it has recentlybecome clear that most of these machines form at specific sites in the cell, beingactivated only where and when they are needed. Using fluorescent, GFP-taggedfusion proteins in living cells (see p. 593), cell biologists are able to follow therepositioning of individual proteins that occurs in response to specific signals.Thus, when certain extracellular signaling molecules bind to receptor proteinsin the plasma membrane, they often recruit a set of other proteins to the insidesurface of the plasma membrane to form protein machines that pass the signalon. As an example, Figure 3-804 illustrates the rapid movement of a proteinkinase C (PKC)enzyme to a complex in the plasma membrane, where it associates with specific substrate proteins that it phosphorylates.There are more than 10 distinct PKC enzymes in human cells, which differboth in their mode of regulation and in their functions. When activated, theseenz]rynesmove from the cytoplasm to different intracellular locations, formingspecific complexes with other proteins that allow them to phosphorylate different protein substrates (Figure 3-808).
The SCF ubiquitin ligases can also moveto specific sites of function at appropriate times. As will be explained when wediscuss cell signaling in Chapter 15, the mechanisms frequently involve proteinphosphorylation, as well as scaffold proteins that link together a set of activating, inhibiting, adaptol and substrate proteins at a specific location in a cell.This general phenomenon is known as induced proximity, and it explainsthe otherwise puzzling observation that slightly different forms of enzymeswith the same catalltic site will often have very different biological functions.Cells change the locations of their proteins by covalently modifying them in avariety of different ways, as part of a "regulatory code" to be described next.Figure3-79 The structure and mode ofactionof a SCFubiquitinligase.(A)Thestructureof the five-proteincomplexthatTheincludesan E2ubiquitinligase.proteindenoted hereas adapterprotein1 is the Rbxl/Hrt1protein,adaPtorprotein2 is the 5kp1protein,and thecullinis the Cull protein.(B)Comparisonof the samecomplexwith two differentarms,the F-boxsubstrate-bindingproteins Skp2 (top) and p-trCPl (bottom),(C)The binding andrespectively.ubiquitylationof a target protein by theaSCFubiquitinligase.lf,as indicated,chainof ubiquitinmoleculesis addedtothe samelysineof the target protein,thatprotein is markedfor rapid destructionbythe proteasome.(A and B,adaptedfromG.Wu et al.,Mol.Cell11:1445-1456,2003.With oermissionfrom Elsevier.)r86C h a p t e r3 : P r o t e i n s0 min3 min(A),I(B)2 0u m1 0u munstructuredregton, ,,,i(C)1 0m i nrapidcollisions+structuredd o m ar n'fhesernoditicationscreate sites on proteins that bind them to particular scafftlld proteins,therebyclusteringthe proteins required for particular reactionsinspecificregionsof the cell.
Most biologicalreactionsare catalyzedby setsof 5 ormore proteins, and such a clustering of proteins is often required for the reaction to occur. Scaffoldsthereby allow cells to compartmentalizereactionsevenirr the absence of membranes. Although onty recently recognized as awidespread phenomenon, this tvpe of clustering is particularly obvious in thecell nucleus (seeFigure4-69).Many scaffolds appear to be quite different from the cullin illustrated previously in Figure 3-79: rather than holding their bound proteins in precisepositions lelative to each other, the interacting proteins are linked by unstructuredregionsof polvpeptide chain.
This tethersthe proteins together,causingthem tocollicle frequently with each other in random orientations-some of which willlead to a productive reaction (Figure3-B0c). In essence,this mechanism greatlyspeeds reactions by creating a very high local concentration of the reactingspecies.For this reason,the use ofscaffold proteins representsan especiallyversatileway of controlling cell chemistry (seealso Figure l5-61).Figure3-80 The assemblyof proteinmachinesat specificsitesin a cell.(A)In responseto a signal(herea phorbolester),the gammasubspeciesof proteinkinaseC movesrapidlyfrom the cytosolto the plasmamembrane.The proteinkinaseis fluorescentin theselivingcellsbecausean engineeredgeneinsidethecellencodesa fusionproteinthat linksthe kinaseto greenfluorescentprotein(GFP).(B)Thespecificassociationof adifferentsubspeciesof proteinkinaseC(aPKC)with the apicaltip of adifferentiatingneuroblastin an earlyDrosophilaembryo.The kinaseis stainedred,andthe cellnucleusgreen.(C)Diagramillustratinghow a simpleproximitycreatedby scaffoldproteinscangreatlyspeedreactionsin a cell.Inthis example,long unstructuredregionsof polypeptidechainin a largescaffoldproteinconnecta seriesof structureddomainsthat bind a setof reactingproteins.The unstructuredregionsserveasflexible"tethers"thatgreatlyspeedreactionratesby causinga rapid,randomcollisionof all of the proteinsthat are(Fora simpleboundto the scaffold.exampleof tethering,seeFigure16-38.)(A,from N.
Sakaiet al,J. CellBiol.139:1465-1476, 1997.With permissionfrom The RockefellerUniversityPress.B,courtesyof AndreasWodarz,Instituteof Genetics,Universityof Dr.isseldorf,Germany.)Many ProteinsAre controlled by Multisitecovalent Modificationwe have thus far described only one type of posttranslational modification ofproteins-that in which a phosphate is covalentlv attached to an amino acidside chain (seeFigure3-64). But a largenumber of other such modifications alsooccLlr,rnore than 200 distinct types being known. To give a senseof the variety,lable 3-3 presents a subset of modifying groups with known regulatory roles.AsTable3-3 SomeMoleculesCovalentlyAttachedto ProteinsRegulateProteinFunctionMODIFYINGGROUPSOMEPROMINENTFUNCTIONSPhosphateon Ser,Thr,or TyrMethylon LysDrivesthe assemblyof a proteininto largercomplexes(seeFigure15-,|9).Helpsto createshistonecodein chromatinthroughformingeithermono-,di-,or tri-methyllysine(seeFigure4-38).Helpsto createshistonecodein chromatin(seeFigure4-38).Thisfattyacidadditiondrivesproteinassociation(seewith membranesFigurel0-20).Controlsenzymeactivityandgeneexpressionin glucosehomeostasis.Monoubiquitinadditionregulatesthe transportof membraneproteinsin vesicles(seeFigure13-58).A polyubiquitinchaintargetsa proteinfor degradation(seeFigure3-79).Acetylon LysPalmitylgroupon CysN-acetylglucosamineon Seror ThrUbiquitinon LysU b i q u i tsi na / 6 a m i n o a c i d p o y p e pt ht iedree;a r e eaat stl0otherubiquitin-relatedproteins,suchasSUMo,thatmodifyproteinsins187PROTEINFUNCTIONPpliltlPPPPPAc ActtlPPIubiquitinw:"PT E T R A M E R I Z A T I OCN- T E R M I N A LREGULATORYDOMAINDOMAIND N A - B I N D I NDGO M A I NP R O T E IpNs 3.e.
phosphateSUMOr,lPTRANSACTIVATIONDOMAIN(A){ti'$P,' methylS I G N A L I N IGN P U T Sts+J/t+\/,iiVAe\/VV( B ) H I S T O NHE3T H EC O D EI SR E A DB I N DT OM O V ET OM O V ET O^ r n \ / ET ^pRoTEASoMEpRoTErNsoror PLASMAill;.];,',: orF O RD E G R A D A T I O N M E M B R A N EY ANDZ(c)in phosphate addition, these groups are added and then removed from proteinsaccording to the needs of the cell.A large number of proteins are now knor,vnto be modified on more than oneamino acid side chain, with different regulatory events producing a different pattern of such modifications. A striking example is the protein p53, which plays acentral part in controlling a cell'sresponseto adversecircumstances (seep. I 105).Through one of four different tlpes of molecular additions, this protein can bemodified at 20 different sites (Figure 3-SfA).
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