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The lifetime ofany particular protein is regulated by proteolytic systems specializedfor this task, as opposed to proteolytic events that might occur duringposttranslational processing. The half-lives of different proteins canvary from half a minute to many hours or even days in eukaryotes.Figure 26-43 The transferrin cycle transports ironinto cells. Diferric-transferrin (transferrin containing two bound Fe3^ ions) is bound by receptors incoated pits (top right), which form endocytic vesicles coated with clathrin. Uncoating is catalyzed byATP-dependent enzymes. This is followed by receptor-mediated fusion of the vesicles with endosomes (bottom). The low pH within the endosomecauses dissociation of the Fe 3+ . At low pH, the receptor retains a high affinity for apotransferrin,which is returned to the cell surface still bound tothe receptors.
Here the neutral pH lowers the affinity of the receptor for apotransferrin, permitting itsdissociation. At neutral pH, the receptor has a highaffinity for diferric-transferrin, allowing more molecules of diferric-transferrin to bind, thereby continuing the cycle.Ubiquitin —CFigure 26-44 The three-step process by whichubiquitin is attached to a protein targeted for destruction in eukaryotes.
Two different enzymeubiquitin intermediates are involved. The free carboxyl of ubiquitin's carboxyl-terminal Gly residueis ultimately linked through an amide (isopeptide)bond to an e-amino group of a Lys residue of thetarget protein.ATPAMP + PPiOUbiquitin — C —S—(Enzx;—SH*EnzxV-SHOUbiquitin — C — S —(Enz2i\Target protein^—Lys —NH2Enz3!OUbiquitin — C — NH—Lys -\Target protein)Table 26—9 Relationship between thehalf-life of a protein and its amino-terminalamino acidAmino-terminal residueStabilizingMet, Gly, Ala, Ser, Thr, ValDestabilizingHe, GinTyr, GluProLeu, Phe, Asp, LysArgHalf-life*>20h- 3 0 min—10 min—7 min—3 min—2 minSource Modified from Bachmair, A , Finley, D , & Varshavsky,A (1986) In vivo half-life of a protein is a function of itsamino-terminal residue Science 234, 179-186* Half-lives were measured in yeast for a single protein thatwas modified so that in each experiment it had a differentamino-terminal amino acid residue (See Chapter 28 for a discussion of techniques used to engineer proteins with alteredamino acid sequences ) Half-lives may vary for different proteins and in different organisms, but this general pattern appears to hold for all organisms amino acids listed here as stabilizing when present at the amino terminus have a stabilizingeffect on proteins in all cells936Most proteins are turned over rapidly in relation to the lifetime of acell, although a few stable proteins (such as hemoglobin) can last forthe life span of a cell (about 110 days for an erythrocyte).
Proteins thatare degraded rapidly include those that are defective because of one ormore incorrect amino acids inserted during synthesis or because ofdamage that occurs during normal functioning. Also targeted for rapidturnover are many enzymes that act at key regulatory points in metabolic pathways.Defective proteins and those with characteristically short halflives are generally degraded in both bacteria and eukaryotes by ATPdependent cytosolic systems.
A second system in vertebrates operatesin lysosomes and serves to recycle membrane proteins, extracellularproteins, and proteins with characteristically long half-lives.In E. coli, many proteins are degraded by an ATP-dependent protease called La. The ATPase is activated only in the presence of defectiveproteins or those slated for rapid turnover; two ATP molecules arehydrolyzed for every peptide bond cleaved. The precise molecular function of ATP hydrolysis during peptide-bond cleavage is unclear. Once aprotein is reduced to small inactive peptides, other ATP-independentproteases complete the degradation process.In eukaryotes, the ATP-dependent pathway is quite different.
Akey component in this system is the 76 amino acid protein ubiquitin,so named because of its presence throughout the eukaryotic kingdoms.One of the most highly conserved proteins known, ubiquitin is essentially identical in organisms as different as yeasts and humans. Ubiquitin is covalently linked to proteins slated for destruction via anATP-dependent pathway involving three separate enzymes (Fig.26-44). How attachment of one or more molecules of ubiquitin to aprotein targets that protein for proteolysis is not yet understood.
TheATP-dependent proteolytic system in eukaryotes is a large complex(Mr > l x 106). The mode of action of the protease component of thesystem and the role of ATP are unknown.The signals that trigger ubiquitination are also not all understood,but one simple one has been found. The amino-terminal residue (i.e.,the residue remaining after removal of methionine and any other proteolytic processing of the amino-terminal end) has a profound influenceon the half-lives of many proteins (Table 26-9).
These amino-terminalsignals have evidently been conserved during billions of years of evolution; the signals are the same in bacterial protein degradation systemsand in the human ubiquitination pathway. The degradation of proteinsis as important to a cell's survival in a changing environment as is theprotein synthetic process, and much remains to be learned about theseinteresting pathways.Chapter 26 Protein MetabolismProteins are synthesized with a particular aminoacid sequence through the translation of information encoded in messenger RNA by an RNAprotein complex called a ribosome. Amino acids arespecified by informational units in the mRNAcalled codons. Translation requires adapter molecules, the transfer RNAs, which recognize codonsand insert amino acids into their appropriate sequential positions in the polypeptide.The codons for the amino acids consist of specific nucleotide triplets.
The base sequences of thecodons were deduced from experiments using synthetic mRNAs of known composition and sequence.The genetic code is degenerate: it has multiple codewords for nearly all the amino acids. The third position in each codon is much less specific than thefirst and second and is said to wobble.
The standard genetic code words are probably universal inall species, although some minor deviations existin mitochondria and a few single-celled organisms.The initiating amino acid, iV-formylmethionine inbacteria, is coded by AUG. Recognition of a particular AUG as the initiation codon requires a purine-rich initiating signal (the Shine-Dalgarno sequence) on the 5' side of the AUG. The tripletsUAA, UAG, and UGA do not code for amino acidsbut are signals for chain termination. In some viruses two different proteins may be coded by thesame nucleotide sequence but translated with different reading frames.Protein synthesis occurs on the ribosomes.
Bacteria have 70S ribosomes, with a large (50S) subunit and a small (30S) subunit. Ribosomes of eukaryotes are significantly larger and contain moreproteins than do bacterial ribosomes.In stage 1 of protein synthesis, amino acids areactivated by specific aminoacyl-tRNA synthetasesin the cytosol. These enzymes catalyze the formation of aminoacyl-tRNAs, with simultaneous cleavage of ATP to AMP and PPi. The fidelity of proteinsynthesis depends to a large extent on the accuracyof this reaction, and some of these enzymes carryout proofreading steps at separate active sites.Transfer RNAs have 73 to 93 nucleotide units, several of which have modified bases.
They have anamino acid arm with the terminal sequenceCCA(3') to which an amino acid is esterified, ananticodon arm, a Ti//C arm, and a DHU arm; sometRNAs have a fifth or extra arm. The anticodonnucleotide triplet of tRNA is responsible for thespecificity of interaction between the aminoacyltRNA and the complementary codon on the mRNA.The growth of polypeptide chains on ribosomes937begins with the amino-terminal amino acid andproceeds by successive additions of new residues tothe carboxyl-terminal end.In bacteria, the initiating aminoacyl-tRNA inall proteins is AT-formylmethionyl-tRNA^^. Initiation of protein synthesis (stage 2) involves formation of a complex between the 30S ribosomal subunit, mRNA, GTP, fMet-tRNA™*, two initiationfactors, and the 50S subunit; GTP is hydrolyzed toGDP and Pj. In the subsequent elongation steps(stage 3), GTP and three elongation factors are required for binding the incoming aminoacyl-tRNAto the aminoacyl site on the ribosome.
In the firstpeptidyl transfer reaction, the fMet residue istransferred to the amino group of the incomingaminoacyl-tRNA. Movement of the ribosome alongthe mRNA then translocates the dipeptidyl-tRNAfrom the aminoacyl site to the peptidyl site, a process requiring hydrolysis of GTP. After many suchelongation cycles, synthesis of the polypeptidechain is terminated (stage 4) with the aid of releasefactors. A poly some consists of an mRNA moleculeto which are attached several or many ribosomes,each independently reading the mRNA and forming a polypeptide. At least four high-energy phosphate bonds are required to generate each peptidebond, an energy investment required to guaranteefidelity of translation.
In stage 5 of protein synthesis, polypeptides undergo folding into their active,three-dimensional forms. Many proteins also arefurther processed by posttranslational modification reactions.After synthesis, many proteins are directed toparticular locations in the cell. One targetingmechanism involves peptide signal sequences generally found at the amino terminus of newly synthesized proteins. In eukaryotes, one class of thesesignal sequences is recognized and bound by alarge protein-RNA complex called the signal recognition particle (SRP). The SRP binds the signalsequence as soon as it appears on the ribosome andtransfers the entire ribosome and incomplete polypeptide to the endoplasmic reticulum.
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