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In additionto the variation in length of the fatty acyl sidechain (indicated in the figure), some homologslack the isopropyl group at the end and/ora,/3-unsaturation.H<CH2OH-OHOH HHAT-AcetylglucosamineHNHO=CCH3Fatty acyl side chainH OC 3Uracil\I II III N OH Hr/ C H - ( C H 2 ) n - C = C - C - N \ L _ J / CH2CH3{n = 8-11)^^CHOH.O vTunicamineNucleusRough ERTransport vesicleCis side^LysosomeI3^boning ^Trans side\—Secretory vesicleTransport vesiclesCytos<aPlasma membraneFigure 26-39 The pathway taken by proteins destined for lysosomes, the plasma membrane, or secretion. Proteins are moved from the ER to the cisside of the Golgi complex in transport vesicles.Sorting occurs primarily in the trans side of theGolgi complex.due oligosaccharide (Fig. 26-37).
Several antibiotics interfere with oneor more steps in this process. The best-characterized is tunicamycin(Fig. 26-38), which blocks the first step.Proteins are moved from the ER to the Golgi complex in transportvesicles (Fig. 26-39). In the Golgi complex, O-linked oligosaccharidesare added and AMinked oligosaccharides are further modified. Bymechanisms only partially understood, proteins are also sorted hereand sent to their final destinations (Fig. 26-39). Within the Golgi complex, the processes that segregate proteins destined for the cell exteriorfrom those destined for the plasma membrane or lysosomes must distinguish between proteins on the basis of structural features otherthan the signal sequence, which was removed in the lumen of the ER.This sorting process is perhaps best understood in the case of hydrolases destined for transport to lysosomes.
Upon arrival in the Golgicomplex from the ER, some as yet undetermined feature of the threedimensional structure of these hydrolases (sometimes called a "signalpatch") is recognized by a phosphotransferase that catalyzes the phosphorylation of certain mannose residues in the enzymes' oligosaccharides (Fig.
26-40). The presence of one or more mannose-6-phosphateresidues in their AMinked oligosaccharides is the structural signal thattargets these proteins to lysosomes. A receptor protein in the membrane of the Golgi complex recognizes this mannose-6-phosphate signal and binds the hydrolases so marked. Vesicles containing these receptor-hydrolase complexes bud from the trans side of the Golgicomplex and make their way to sorting vesicles. Here, the receptorhydrolase complexes dissociate in a process facilitated by the lower pHwithin the sorting vesicles and by a phosphatase-catalyzed removal ofphosphate groups from the mannose-6-phosphate residues.
The receptor is returned to the Golgi complex, and vesicles containing the hydrolases bud from the sorting vesicles and move to the lysosomes. In cellstreated with tunicamycin (Fig. 26-38), hydrolases normally targetedfor lysosomes do not reach their destination but are secreted instead,confirming that the AMinked oligosaccharide plays a key role in targeting these enzymes to lysosomes.CH 2 OHH/H\Kv OH Hy\o xiYHNHHHv^^HO— Oligosaccharide — N—(Enzyme )— P — O — P - O - Uridine—o—p—o—pO"HHMannoseO"IHydrolasec=oCH 3UDP-iV-Acetylglucosamine(UDP-GlcNAc)V-acet\ lylucosaminepho-photian-fei a^LUMPCH 2 OH0O— Oligosaccharide l— N—( EnzymeHO— Oligosaccharide — N ^ E n z y m e )H 0IIHHMannose-6-phosphateFor proteins destined for the plasma membrane or for secretion,and for those destined to reside permanently in the ER or the Golgicomplex, the signals are less well understood. These targeting pathways are not impeded by tunicamycin, indicating that the signals arenot carbohydrates.Cellular proteins targeted to the mitochondria, chloroplasts, ornucleus use their own distinct signal sequences.
For mitochondria andchloroplasts, the signal sequences are again found at the amino terminus of the proteins and are cleaved once the proteins arrive at theirfinal destinations. The signal sequences that target some proteins tothe nucleus (an example is the sequence -Pro-Lys-Lys-Lys-ArgLys-Val-) are located internally and are not cleaved. These signalspermit proteins such as DNA polymerases and RNA polymerases toenter the nucleus rapidly through nuclear pores.Figure 26-40 The two-step process by which mannose residues on lysosome-targeted enzymes, suchas hydrolases, are phosphorylated.
iV-Acetylglucosamine phosphotransferase recognizes some as yetunidentified structural feature of lysosome-destinedhydrolases.Bacteria Also Use Signal Sequences for Protein TargetingBacteria must also target some proteins to the inner or outer membranes, the periplasmic space between the membranes, or the extracellular medium (secretion). This targeting uses signal sequences at theamino terminus of the proteins much like those found on eukaryoticproteins targeted to the ER (Fig.
26-41).933934Inner membrane proteinsPhage fd, majorcoat proteincleavagesiteMet Lys Lys Ser Leu Val Leu Lys Ala Ser Val Ala Val Ala Thr Leu Val Pro Met Leu Ser Phe Ala Ala GluPhage fd, minorcoat proteinMet Lys Lys Leu Leu Phe Ala HePro Leu Val Val Pro Phe Tyr Ser His Ser Ala Glu --Periplasmic proteinsMet Lys Gin Ser Thr He Ala Leu Ala Leu Leu Pro Leu Leu Phe Thr Pro Val Thr Lys Ala Arg ThrAlkaline phosphataseLeucine-specificbinding proteinMet Lys Ala Asn Ala Lys Thr liejS-Lactamase ofpBR322Met Ser lie Gin His Phe Arg Val Ala Leu lie Pro Phe Phe Ala Ala Phe Cys Leu Pro Val Phe Ala His Pro --lie Ala Gly Met HeAla Leu Ala He Ser His Thr Ala Met Ala Asp Asp --Outer membrane proteinsLipoproteinLamBOmpAMet Lys Ala Thr Lys Leu Val Leu Gly Ala Val lieLeu Gly Ser Thr Leu Leu Ala Gly Cys Ser -Leu Arg Lys Leu Pro Leu Ala Val Ala Val Ala Ala Gly Val Met Ser Ala Gin Ala Met Ala^Val Asp Met Met He Thr Met Lys Lys Thr Ala lie Ala He Ala Val Ala Leu Ala Gly Phe Ala Thr Val Ala Gin Ala Ala Pro -Figure 26—41 Signal sequences used for targetingto different locations in bacteria.
Basic amino acids(blue) near the amino terminus and hydrophobiccore amino acids (yellow) are highlighted. Thecleavage sites marking the ends of the signal sequences are marked by red arrows. Note that theinner membrane (see Fig. 2-6) is where phage fdcoat proteins and DNA are assembled into phageparticles.Heavy ^chainSome proteins that are translocated through one or more membranes to reach their final destinations must be maintained in a distinct "translocation-competent" conformation until this process is complete. The functional conformation is assumed after translocation, andproteins purified in this final form are often found to be no longercapable of translocation.
There is growing evidence that the translocation conformation is stabilized by a specialized set of proteins in allbacterial cells. These bind to the protein to be translocated while it isbeing synthesized, preventing it from folding into its final threedimensional structure. In E. coli, a protein called trigger factor(Mr 63,000) appears to facilitate the translocation of at least one outermembrane protein through the inner membrane.Cells Import Proteins by Receptor-Mediated EndocytosisSome proteins are imported into certain cells from the surroundingmedium; these include low-density lipoprotein (LDL), the iron-carrying protein transferrin, peptide hormones, and circulating proteinsthat are destined to be degraded.
These proteins bind to receptors onthe outer face of the plasma membrane. The receptors are concentrated in invaginations of the membrane called coated pits, which arecoated on their cytosolic side with a lattice made up of the proteinclathrin (Fig. 26-42). Clathrin forms closed polyhedral structures,and as more of the receptors become occupied with target proteins, theclathrin lattice grows until a complete membrane-bounded endocyticvesicle buds off the plasma membrane and moves into the cytoplasm.-80nm0 1 yumFigure 26-42 Clathrin is a trimer of three light(L) chains (Mr 35,000) and three heavy (H) chains(Mr 180,000).
(a) The (HL)3 clathrin unit is organized as a three-legged structure called a triskelion.(b) Triskelions have a propensity to assemble intopolyhedral lattices, (c) Electron micrograph of acoated pit on the cytosolic face of the plasma membrane of a flbroblast.Apotransferrin9352Fe 3Apotransferrinis released.Diferric-transferrinDiferric-transferrinbinds to receptorin coated pit.ExtracellularfluidApotransferrinand receptorare recycled.Coated pit budsand pinches offfrom membraneClathrin coat isremoved fromvesicle.2 Fe3+ are releasedfrom transferrin.Endocytic vesicle recognizesand fuses with an endosome.The clathrin is quickly removed by uncoating enzymes, and the vesicles fuse with endosomes. The pH of endosomes is lowered by the activity of V-type ATPases in their membranes (see Table 10-5), producingan environment that facilitates dissociation of receptors from their target proteins. Proteins and receptors then go their separate ways, theirfates varying according to the system.
Transferrin and its receptor areeventually recycled (Fig. 26-43). Some hormones, growth factors, andimmune complexes are degraded along with their receptors after theyhave elicited the appropriate response. LDL is degraded after the associated cholesterol has been delivered to its destination, but its receptoris recycled (see Fig. 20-39).Receptor-mediated endocytosis is exploited by some viruses to gainentry to cells. Influenza virus enters cells this way. HIV, the virus thatcauses AIDS, also binds to specific receptors on the cell surface andmay gain entry by endocytosis.
In humans, the receptor that bindsHIV, known as CD4, is a glycoprotein found primarily on the surface ofimmune system cells called helper T cells. CD4 is normally involved inthe complex communication between cells of the immune system thatis required to execute the immune response.Protein Degradation Is Mediated bySpecialized Systems in All CellsProteins are constantly being degraded in all cells to prevent thebuildup of abnormal or unwanted proteins and to facilitate the recycling of amino acids. Degradation is a selective process.
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