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The pathways bywhich proteins are sorted and transported to their proper cellular location are often referred to as protein targeting pathways.The most important element in all of these targeting systems (withthe exception of cytosolic and nuclear proteins) is a short amino acidsequence at the amino terminus of a newly synthesized polypeptidecalled the signal sequence. This signal sequence, whose function wasfirst postulated by David Sabatini and Gunter Blobel in 1970, directs aprotein to its appropriate location in the cell and is removed duringtransport or when the protein reaches its final destination. In manycases, the targeting capacity of particular signal sequences has beenconfirmed by fusing the signal sequence from one protein, say proteinA, to a different protein B, and showing that the signal directs proteinB to the location where protein A is normally found.The selective degradation of proteins no longer needed in the cellalso relies largely on a set of molecular signals embedded in each protein's structure; most of these signals are not yet understood.
The finalpart of this chapter is devoted to the processes of targeting and degradation, with emphasis on the underlying signals and molecular regulation that are so crucial to cellular metabolism. Except where noted, thefocus is on eukaryotic cells.Gunter BlobelPosttranslational Modification of Many EukaryoticProteins Begins in the Endoplasmic ReticulumPerhaps the best-characterized targeting system begins in the endoplasmic reticulum (ER).
Most lysosomal, membrane, or secreted proteins have an amino-terminal signal sequence that marks them fortranslocation into the lumen of the ER. More than 100 signal sequencesfor proteins in this group have been determined (Fig. 26-35). The se-Human influenzavirus AHumanFigure 26—35 Amino-terminal signal sequences ofsome eukaryotic proteins, directing translocationinto the endoplasmic reticulum. The hydrophobiccore (yellow) is preceded by one or more basic residues (blue).
Note the presence of polar and shortside-chain residues immediately preceding thecleavage sites (indicated by red arrows).cleavagesiteMet Lys Ala Lys Leu Leu Val Leu Leu Tyr Ala Phe Val Ala Gly Asp Gin+preproinsulinMet Ala Leu Trp Met Arg Leu Leu Pro Leu Leu Ala Leu Leu Ala Leu Trp Gly Pro Asp Pro Ala Ala Ala Phe Val —Bovinegrowth|hormoneMet Met Ala Ala Gly Pro Arg Thr Ser Leu Leu Leu Ala Phe Ala Leu Leu Cys Leu Pro Trp Thr Gin Val Val Gly Ala Phe -BeepromellitinDrosophila glueprotein+Met Lys Phe Leu Val Asn Val Ala Leu Val Phe Met Val Val Tyr He Ser Tyr He Tyr Ala Ala Pro IMet Lys Leu Leu Val Val Ala Val He Ala Cys Met Leu lie Gly Phe Ala Asp Pro Ala Ser Gly Cys Lys930Part IV Information PathwaysGeorge PaladeFigure 26—36 Directing eukaryotic proteins withthe appropriate signals to the endoplasmic reticulum: the SRP cycle and nascent polypeptide translocation and cleavage.
(T) The ribosomal subunitsassemble in an initiation complex at the initiationcodon and begin protein synthesis. (2) If an appropriate signal sequence appears at the amino terminus of the nascent polypeptide, (3) the SRP bindsto the ribosome and halts elongation. (4) The ribosome-SRP complex is bound by receptors on theER, and (§) the SRP dissociates and is recycled.(6) Protein synthesis resumes, coupled to translocation of the polypeptide chain into the lumen of theER. (7) The signal sequence is cleaved by a signalpeptidase within the lumen of the ER. (8) The ribosome is recycled.The SRP is a rod-shaped complex containing a300 nucleotide RNA (called 7SL-RNA) and six different proteins, with a combined molecular weightof 325,000. One protein subunit of the SRP bindsdirectly to the signal sequence, inhibiting elongation by sterically blocking entry of aminoacyltRNAs and inhibiting peptidyl transferase.
TheSRP receptor is a heterodimer of a (Mr 69,000)and p (Mr 30,000) subunits.CytosolEndoplasmicreticulumquences vary in length (13 to 36 amino acid residues), but all have (1) asequence of hydrophobic amino acids, typically 10 to 15 residues long,(2) one or more positively charged amino acid residues, usually nearthe amino terminus preceding the hydrophobic sequence, and (3) ashort sequence at the carboxyl terminus (near the cleavage site) that isrelatively polar, with amino acid residues having short side chains(especially Ala) predominating in the positions closest to the cleavagesite.As originally demonstrated by George Palade, proteins with thesesignal sequences are synthesized on ribosomes attached to the ER.
Thesignal sequence itself is instrumental in directing the ribosome to theER. The overall pathway summarized in Figure 26-36 begins with theinitiation of protein synthesis on free ribosomes. The signal sequenceappears early in the synthetic process because it is at the amino terminus. As it leaves the ribosome, this sequence and the ribosome itselfare rapidly bound by a large complex called the signal recognitionparticle (SRP). This binding event halts elongation when the peptideis about 70 amino acids long and the signal sequence has emergedcompletely from the ribosome.
The bound SRP directs the ribosomewith the incomplete polypeptide to a specific set of SRP receptors in thecytosolic face of the ER. The nascent polypeptide is delivered to a peptide translocation complex in the ER, the SRP dissociates from theribosome, and synthesis of the protein resumes. The translocation complex feeds the growing polypeptide into the lumen of the ER in a reaction that is driven by the energy of ATP. The signal sequence is removed by a signal peptidase within the lumen of the ER. Once thecomplete protein has been synthesized, the ribosome dissociates fromthe ER.In the lumen of the ER, newly synthesized proteins are modified inseveral ways.
In addition to the removal of signal sequences, polypeptide chains fold and disulfide bonds form. Many proteins are alsoglycosylated.Signal/ON sequenceCOmRNAChapter 26 Protein Metabolism931Glycosylation Plays a Key Role in Protein TargetingGlycosylated proteins, or glycoproteins, often are linked to their oligosaccharides through Asn residues. These AMinked oligosaccharidesare very diverse (Chapter 11), but the many pathways by which theyform all have a common first step. A 14 residue core oligosaccharide(containing two iV-acetylglucosamine, nine mannose, and three glucoseresidues) is transferred from a dolichol phosphate donor molecule tocertain Asn residues on the proteins.OCHSCH3O—P—O—CH2 —CH2 —C—CH20"\CH3g— CH=C—CH2 / —CH2—CH=C—CH3HDolichol phosphate(n = 9-22)The core oligosaccharide is built up on the phosphate group ofdolichol phosphate (an isoprenoid derivative) by the successive addition of monosaccharide units.
Once this core oligosaccharide is complete, it is enzymatically transferred from dolichol phosphate to theprotein (Fig. 26-37). The transferase is located on the lumenal face ofthe ER and thus does not catalyze glycosylation of cytosolic proteins.After the transfer, the core oligosaccharide is trimmed and elaboratedin different ways on different proteins, but all AMinked oligosaccharides retain a pentasaccharide core derived from the original 14 resi-W iV-Acetylglucosamine (GlcNAc)S Mannose (Man)0 Glucose (Glc)5 GDPtunicamycin5 GDP-ManUMP+UDP i2 UDP-GlcNAcFigure 26-37 Synthesis of the core oligosaccharideof glycoproteins.
The core oligosaccharide is builtup in a series of steps as shown. The first few stepsoccur on the cytosolic face of the ER. Completionoccurs within the lumen of the ER after a translocation step (upper left) in which the incomplete oligosaccharide is moved across the membrane. Themechanism of this translocation is not shown. Thesynthetic precursors that contribute additionalmannose and glucose residues to the growing oligosaccharide in the lumen are themselves dolicholphosphate derivatives.
The dolichol—(P)—Man anddolichol—(P)—Glc are synthesized from dolicholphosphate and GDP-mannose or UDP-glucose, respectively. After it is transferred to the protein, thecore oligosaccharide is further modified in the ERand the Golgi complex in pathways that differ fordifferent proteins. The five sugar residues enclosedin a beige screen (lower right) are retained in thefinal structure of all iV-linked oligosaccharides. Inthe first step in the construction of the AMinkedoligosaccharide moiety of a glycoprotein, the coreoligosaccharide is transferred from dolichol phosphate to an Asn residue of the protein within thelumen of the ER. The released dolichol pyrophosphate is recycled.PTranslocationDolicholrecycledEndoplasmicreticulumL— 4 Dolichol-®-Manr ^ * 4 Dolichol<P)V ^ — 3 DolichoKg^ Glc\ ^ > 3 Dolichol<P)CytosolmRNADolichol<P)932Part IV Information PathwaysFigure 26-38 The structure of tunicamycin, anantibiotic produced by Streptomyces that mimicsUDP-iV-acetylglucosamine and blocks the first stepin the synthesis of the core oligosaccharide of glycoproteins on dolichol phosphate (see Fig.
26—37).Tunicamycin is actually a family of antibiotics produced by (and isolated as a mixture from) Streptomyces lysosuperficens. They all contain uracil, Nacetylglucosamine, an 11 carbon aminodialdosecalled tunicamine, and a fatty acyl side chain. Thestructure of the fatty acyl side chain varies in thedifferent compounds within the family.
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