105 hapter 7 rotein Metabolism B B B R B 3 R mino acid 3 class I aminoacyl-tr synthetases B B 1 T i denine denine -minoacyl adenylate (aminoacyl-m) class II aminoacyl-tr synthetases MISM FIGUR 7 14 minoacylation of tr by aminoacyl-tr synthetases. Step 1 is formation of an aminoacyl adenylate, which remains bound to the active site. In the second step the aminoacyl group is transferred to the tr. The mechanism of this step is somewhat different for the two classes of aminoacyl-tr synthetases (see Table 7 7). For class I enzymes, a the aminoacyl group is transferred initially to the -hydroxyl group of the -terminal residue, then 3a to the -hydroxyl group by a transesterification reaction. For class II enzymes, b the aminoacyl group is transferred directly to the -hydroxyl group of the terminal adenylate. end of tr denine R 3 B denosine B minoacyl-m denine R 3 B denosine B minoacyl-m tr tr a M b M denine R B 3 transesterification 3a denine R B 3 minoacyl-tr
7. rotein Synthesis 1053 D arm end of tr denine pg B R incorrect aminoacyl-m products to a separate active site on the enzyme; a substrate that binds in this second active site is hydrolyzed. The R group of valine is slightly smaller than that of isoleucine, so Val-M fits the hydrolytic (proofreading) site of the Ile-tR synthetase but Ile-M does not. Thus Val-M is hydrolyzed to valine and M in the proofreading active site, and tr bound to the synthetase does not become aminoacylated to the wrong amino acid. mino acid arm Tw arm nticodon arm 3 minoacyl group FIGUR 7 15 General structure of aminoacyl-trs. The aminoacyl group is esterified to the position of the terminal residue. The ester linkage that both activates the amino acid and joins it to the tr is shaded pink. low as that of D replication. Because flaws in a protein are eliminated when the protein is degraded and are not passed on to future generations, they have less biological significance. The degree of fidelity in protein synthesis is sufficient to ensure that most proteins contain no mistakes and that the large amount of energy required to synthesize a protein is rarely wasted. ne defective protein molecule is usually unimportant when many correct copies of the same protein are present. Interaction between an minoacyl-tr Synthetase and a tr: Second Genetic ode n individual aminoacyl-tr synthetase must be specific not only for a single amino acid but for certain trs as well. Discriminating among dozens of trs is just as important for the overall fidelity of protein biosynthesis as is distinguishing among amino acids. The interaction between aminoacyl-tr synthetases and trs has been referred to as the second genetic code, reflecting its critical role in maintaining the accuracy of protein synthesis. The coding rules appear to be more complex than those in the first code. Figure 7 16 summarizes what we know about the nucleotides involved in recognition by some aminoacyltr synthetases. Some nucleotides are conserved in all trs and therefore cannot be used for discrimination. mino acid arm 3 3 3 3 D arm Tw arm 3 Valine 3 Isoleucine In addition to proofreading after formation of the aminoacyl-m intermediate, most aminoacyl-tr synthetases can also hydrolyze the ester linkage between amino acids and trs in the aminoacyl-trs. This hydrolysis is greatly accelerated for incorrectly charged trs, providing yet a third filter to enhance the fidelity of the overall process. The few aminoacyltr synthetases that activate amino acids with no close structural relatives (ys-tr synthetase, for example) demonstrate little or no proofreading activity; in these cases, the active site for aminoacylation can sufficiently discriminate between the proper substrate and any incorrect amino acid. The overall error rate of protein synthesis (~1 mistake per 10 4 amino acids incorporated) is not nearly as nticodon xtra arm nticodon arm FIGUR 7 16 ucleotide positions in trs that are recognized by aminoacyl-tr synthetases. Some positions (blue dots) are the same in all trs and therefore cannot be used to discriminate one from another. ther positions are known recognition points for one (orange) or more (green) aminoacyl-tr synthetases. Structural features other than sequence are important for recognition by some of the synthetases.
1056 hapter 7 rotein Metabolism tr that is distinct from the tr Met used at ()UG codons at interior positions in the mr. olypeptides synthesized by mitochondrial and chloroplast ribosomes, however, begin with -formylmethionine. This strongly supports the view that mitochondria and chloroplasts originated from bacterial ancestors that were symbiotically incorporated into precursor eukaryotic cells at an early stage of evolution (see Fig. 1 36). ow can the single ()UG codon distinguish between the starting -formylmethionine (or methionine, in eukaryotes) and interior Met residues? The details of the initiation process provide the answer. The Three Steps of Initiation The initiation of polypeptide synthesis in bacteria requires (1) the 30S ribosomal subunit, () the mr coding for the polypeptide to be made, (3) the initiating fmet-tr fmet, (4) a set of three proteins called initiation factors (IF-1, IF-, and IF-3), (5) GT, (6) the 50S ribosomal subunit, and (7) Mg. Formation of the initiation complex takes place in three steps (Fig. 7 0). In step 1 the 30S ribosomal subunit binds two initiation factors, IF-1 and IF-3. Factor IF-3 prevents the 30S and 50S subunits from combining prematurely. The mr then binds to the 30S subunit. The initiating ()UG is guided to its correct position by the Shine- Dalgarno sequence (named for ustralian researchers John Shine and Lynn Dalgarno, who identified it) in the mr. This consensus sequence is an initiation signal of four to nine purine residues, 8 to 13 bp to the side of the initiation codon (Fig. 7 1a). The sequence base-pairs with a complementary pyrimidine-rich sequence near the end of the 16S rr of the 30S ribosomal subunit (Fig. 7 1b). This mr-rr interaction positions the initiating ()UG sequence of the mr in the precise position on the 30S subunit where it is required for initiation of translation. The particular ()UG where fmet-tr fmet is to be bound is distinguished from other methionine codons by its proximity to the Shine-Dalgarno sequence in the mr. Bacterial ribosomes have three sites that bind aminoacyl-trs, the aminoacyl () site, the peptidyl () site, and the exit () site. Both the 30S and the 50S subunits contribute to the characteristics of the and sites, whereas the site is largely confined to the 50S subunit. The initiating ()UG is positioned at the site, the only site to which fmettr fmet can bind (Fig. 7 0). The fmet-tr fmet is the only aminoacyl-tr that binds first to the site; during the subsequent elongation stage, all other incoming aminoacyl-trs (including the Met-tR Met that binds to interior UG codons) bind first to the site and only subsequently to the and sites. The site is the site from which the uncharged trs leave during elongation. Factor IF-1 binds at the site and prevents tr binding at this site during initiation. 30S Subunit 1 IF-3 fmet () U () nticodon 3 U IF-3 50S Subunit IF-3 Initiation codon IF- GT tr 50S Subunit U G U IF- mr fmet U G U fmet U G IF-1 GT IF-1 GD i IF-1 IF- IF-3 IF-1 mr ext codon FIGUR 7 0 Formation of the initiation complex in bacteria. The complex forms in three steps (described in the text) at the expense of the hydrolysis of GT to GD and i. IF-1, IF-, and IF-3 are initiation factors. designates the peptidyl site, the aminoacyl site, and the exit site. ere the anticodon of the tr is oriented to, left to right, as in Figure 7 8 but opposite to the orientation in Figures 7 16 and 7 18.
7. rotein Synthesis 1057. coli trp. coli arab. coli laci fx174 phage protein l phage cro () G G G G G G U U G U G G G U () U U U G G U G G G U G G U G G G U U G U U G G G U G G U G U G U G G U U U U G G G G U U U U U U U G G U U G U U U U G U U G G G G U U G U U G G G Shine-Dalgarno sequence; pairs with 16S rr Initiation codon; pairs with fmet-tr fmet (a) rokaryotic mr with consensus Shine-Dalgarno sequence nd of 16S rr U U U G U () G U U U G G G G U U U (b) G U U G G G U U U U G U () FIGUR 7 1 Messenger R sequences that serve as signals for initiation of protein synthesis in bacteria. (a) lignment of the initiating UG (shaded in green) at its correct location on the 30S ribosomal subunit depends in part on upstream Shine-Dalgarno sequences (pink). ortions of the mr transcripts of five prokaryotic genes are shown. ote the unusual example of the. coli LacI protein, which initiates with a GUG (Val) codon (see Box 7 ). (b) The Shine- Dalgarno sequence of the mr pairs with a sequence near the end of the 16S rr. In step of the initiation process (Fig. 7 0), the complex consisting of the 30S ribosomal subunit, IF-3, and mr is joined by both GT-bound IF- and the initiating fmet-tr fmet. The anticodon of this tr now pairs correctly with the mr s initiation codon. In step 3 this large complex combines with the 50S ribosomal subunit; simultaneously, the GT bound to IF- is hydrolyzed to GD and i, which are released from the complex. ll three initiation factors depart from the ribosome at this point. ompletion of the steps in Figure 7 0 produces a functional 70S ribosome called the initiation complex, containing the mr and the initiating fmettr fmet. The correct binding of the fmet-tr fmet to the site in the complete 70S initiation complex is assured by at least three points of recognition and attachment: the codon-anticodon interaction involving the initiation UG fixed in the site; interaction between the Shine-Dalgarno sequence in the mr and the 16S rr; and binding interactions between the ribosomal site and the fmet-tr fmet. The initiation complex is now ready for elongation. Initiation in ukaryotic ells Translation is generally similar in eukaryotic and bacterial cells; most of the significant differences are in the mechanism of initiation. ukaryotic mrs are bound to the ribosome as a complex with a number of specific binding proteins. Several of these tie together the and ends of the message. t the end, the mr is bound by the poly() binding protein (B). ukaryotic cells have at least nine initiation factors. complex called eif4f, which includes the proteins eif4, eif4g, and eif4, binds to the cap (see Fig. 6 1) through eif4. The protein eif4g binds to both eif4 and B, effectively tying them together (Fig. 7 ). The protein eif4 has an R helicase activity. It is the eif4f complex that associates UG cap Gene 40S Ribosomal subunit eif4 eif3 ()n B eif4g poly() tail Untranslated region FIGUR 7 rotein complexes in the formation of a eukaryotic initiation complex. The and ends of eukaryotic mrs are linked by a complex of proteins that includes several initiation factors and the poly() binding protein (B). The factors eif4 and eif4g are part of a larger complex called eif4f. This complex binds to the 40S ribosomal subunit.
1058 hapter 7 rotein Metabolism TBL 7 8 Factor Bacterial IF-1 IF- IF-3 ukaryotic * eif eifb, eif3 eif4 eif4b eif4 eif4g eif5 eif6 rotein Factors Required for Initiation of Translation in Bacterial and ukaryotic ells Function revents premature binding of trs to site Facilitates binding of fmet-tr fmet to 30S ribosomal subunit Binds to 30S subunit; prevents premature association of 50S subunit; enhances specificity of site for fmet-tr fmet Facilitates binding of initiating Met-tR Met to 40S ribosomal subunit First factors to bind 40S subunit; facilitate subsequent steps R helicase activity removes secondary structure in the mr to permit binding to 40S subunit; part of the eif4f complex Binds to mr; facilitates scanning of mr to locate the first UG Binds to the cap of mr; part of the eif4f complex Binds to eif4 and to poly() binding protein (B); part of the eif4f complex romotes dissociation of several other initiation factors from 40S subunit as a prelude to association of 60S subunit to form 80S initiation complex Facilitates dissociation of inactive 80S ribosome into 40S and 60S subunits * The prefix e identifies these as eukaryotic factors. with another factor, eif3, and with the 40S ribosomal subunit. The efficiency of translation is affected by many properties of the mr and proteins in this complex, including the length of the poly() tract (in most cases, longer is better). The end-to-end arrangement of the eukaryotic mr facilitates translational regulation of gene expression, considered in hapter 8. The initiating ()UG is detected within the mr not by its proximity to a Shine-Dalgarno-like sequence but by a scanning process: a scan of the mr from the end until the first UG is encountered, signaling the beginning of the reading frame. The eif4f complex is probably involved in this process, perhaps using the R helicase activity of eif4 to eliminate secondary structure in the untranslated portion of the mr. Scanning is also facilitated by another protein, eif4b. The roles of the various bacterial and eukaryotic initiation factors in the overall process are summarized in Table 7 8. The mechanism by which these proteins act is an important area of investigation. Stage 3: eptide Bonds re Formed in the longation Stage The third stage of protein synthesis is elongation. gain, our initial focus is on bacterial cells. longation requires (1) the initiation complex described above, () aminoacyl-trs, (3) a set of three soluble cytosolic proteins called elongation factors (F-Tu, F-Ts, and F-G in bacteria), and (4) GT. ells use three steps to add each amino acid residue, and the steps are repeated as many times as there are residues to be added. longation Step 1: Binding of an Incoming minoacyl-tr In the first step of the elongation cycle (Fig. 7 3), the appropriate incoming aminoacyl-tr binds to a complex of GT-bound F-Tu. The resulting aminoacyltr F-Tu GT complex binds to the site of the 70S initiation complex. The GT is hydrolyzed and an F-Tu GD complex is released from the 70S ribosome. The F-Tu GT complex is regenerated in a process involving F-Ts and GT. longation Step : eptide Bond Formation peptide bond is now formed between the two amino acids bound by their trs to the and sites on the ribosome. This occurs by the transfer of the initiating -formylmethionyl group from its tr to the amino group of the second amino acid, now in the site (Fig. 7 4). The -amino group of the amino acid in the site acts as a nucleophile, displacing the tr in the site to form the peptide bond. This reaction produces a dipeptidyltr in the site, and the now uncharged (deacylated) tr fmet remains bound to the site. The trs then shift to a hybrid binding state, with elements of each spanning two different sites on the ribosome, as shown in Figure 7 4. The enzymatic activity that catalyzes peptide bond formation has historically been referred to as peptidyl transferase and was widely assumed to be intrinsic to one or more of the proteins in the large ribosomal subunit. We now know that this reaction is catalyzed by the 3S rr (Fig. 7 9), adding to the known catalytic repertoire of ribozymes. This discovery has interesting implications for the evolution of life (Box 7 3).
7. rotein Synthesis 1059 Initiation complex fmet 50S U U G site site site Initiation codon 30S ext codon Incoming aminoacyltr R 1.. R fmet-tr fmet minoacyltr Tu GT Tu GT mr U UG binding of incoming aminoacyltr Tu Ts Ts GT peptide bond formation i Tu GD fmet Ts GD site site R 1 R site Deacylated tr fmet DipeptidyltR U U G U UG FIGUR 7 3 First elongation step in bacteria: binding of the second aminoacyl-tr. The second aminoacyl-tr enters the site of the ribosome bound to F-Tu (shown here as Tu), which also contains GT. Binding of the second aminoacyl-tr to the site is accompanied by hydrolysis of the GT to GD and i and release of the F-Tu GD complex from the ribosome. The bound GD is released when the F-Tu GD complex binds to F-Ts, and F-Ts is subsequently released when another molecule of GT binds to F-Tu. This recycles F-Tu and makes it available to repeat the cycle. FIGUR 7 4 Second elongation step in bacteria: formation of the first peptide bond. The peptidyl transferase catalyzing this reaction is the 3S rr ribozyme. The -formylmethionyl group is transferred to the amino group of the second aminoacyl-tr in the site, forming a dipeptidyl-tr. t this stage, both trs bound to the ribosome shift position in the 50S subunit to take up a hybrid binding state. The uncharged tr shifts so that its and ends are in the site. Similarly, the and ends of the peptidyl tr shift to the site. The anticodons remain in the and sites.
1060 hapter 7 rotein Metabolism longation Step 3: Translocation In the final step of the elongation cycle, translocation, the ribosome moves one codon toward the end of the mr (Fig. 7 5a). This movement shifts the anticodon of the dipeptidyltr, which is still attached to the second codon of the mr, from the site to the site, and shifts the deacylated tr from the site to the site, from where the tr is released into the cytosol. The third codon of the mr now lies in the site and the second codon in the site. Movement of the ribosome along the mr requires F-G (also known as translocase) and the energy provided by hydrolysis of another molecule of GT. site site R 1 R site Deacylated tr fmet DipeptidyltR change in the three-dimensional conformation of the entire ribosome results in its movement along the mr. Because the structure of F-G mimics the structure of the F-Tu tr complex (Fig. 7 5b), F-G can bind the site and presumably displace the peptidyl-tr. The ribosome, with its attached dipeptidyl-tr and mr, is now ready for the next elongation cycle and attachment of a third amino acid residue. This process occurs in the same way as addition of the second residue (as shown in Figs 7 3, 7 4, and 7 5). For each amino acid residue correctly added to the growing polypeptide, two GTs are hydrolyzed to GD and i as the ribosome moves from codon to codon along the mr toward the end. The polypeptide remains attached to the tr of the most recent amino acid to be inserted. This association maintains the functional connection between the information in the mr and its decoded polypeptide output. t the same time, the ester linkage between this tr and the carboxyl terminus of the growing polypeptide activates the terminal carboxyl group for nucleophilic attack by the incoming amino acid to form a new peptide bond (Fig. 7 4). s the existing ester linkage between the polypeptide and tr is broken during U UG F-G GT translocation F-G GD i FIGUR 7 5 Third elongation step in bacteria: translocation. (a) The ribosome moves one codon toward the end of the mr, using energy provided by hydrolysis of GT bound to F-G (translocase). The dipeptidyl-tr is now entirely in the site, leaving the site open for the incoming (third) aminoacyl-tr. The uncharged tr dissociates from the site, and the elongation cycle begins again. (b) The structure of F-G mimics the structure of F-Tu complexed with tr. Shown here are (left) F-Tu complexed with tr (green) (DB ID 1B3) and (right) F-G complexed with GD (red) (DB ID 1DR). The carboxyl-terminal part of F-G (dark gray) mimics the structure of the anticodon loop of tr in both shape and charge distribution. site site site R 1 Incoming aminoacyl-tr 3 R U UG Direction of ribosome movement (a) (b)
106 hapter 7 rotein Metabolism RF U G polypeptidyl-tr link hydrolyzed Release factor binds is a single molecule of mr that is being translated simultaneously by many closely spaced ribosomes, allowing the highly efficient use of the mr. In bacteria, transcription and translation are tightly coupled. Messenger Rs are synthesized and translated in the same n direction. Ribosomes begin translating the end of the mr before transcription is complete (Fig. 7 8). The situation is quite different in eukaryotic cells, where newly transcribed mrs must leave the nucleus before they can be translated. Bacterial mrs generally exist for just a few minutes (p. 100) before they are degraded by nucleases. In order to maintain high rates of protein synthesis, the mr for a given protein or set of proteins must be made continuously and translated with maximum efficiency. The short lifetime of mrs in bacteria allows a rapid cessation of synthesis when the protein is no longer needed. U G Rapid Translation of a Single Message by olysomes Large clusters of 10 to 100 ribosomes that are very active in protein synthesis can be isolated from both eukaryotic and bacterial cells. lectron micrographs show a fiber between adjacent ribosomes in the cluster, which is called a polysome (Fig. 7 7). The connecting strand RF components dissociate U G FIGUR 7 6 Termination of protein synthesis in bacteria. Termination occurs in response to a termination codon in the site. First, a release factor, RF (RF-1 or RF-, depending on which termination codon is present), binds to the site. This leads to hydrolysis of the ester linkage between the nascent polypeptide and the tr in the site and release of the completed polypeptide. Finally, the mr, deacylated tr, and release factor leave the ribosome, and the ribosome dissociates into its 30S and 50S subunits. RF Stage 5: ewly Synthesized olypeptide hains Undergo Folding and rocessing In the final stage of protein synthesis, the nascent polypeptide chain is folded and processed into its biologically active form. During or after its synthesis, the polypeptide progressively assumes its native conformation, with the formation of appropriate hydrogen bonds and van der Waals, ionic, and hydrophobic interactions. In this way the linear, or one-dimensional, genetic message in the mr is converted into the threedimensional structure of the protein. Some newly made proteins, both prokaryotic and eukaryotic, do not attain their final biologically active conformation until they have been altered by one or more processing reactions called posttranslational modifications. mino-terminal and arboxyl-terminal Modifications The first residue inserted in all polypeptides is -formylmethionine (in bacteria) or methionine (in eukaryotes). owever, the formyl group, the amino-terminal Met residue, and often additional amino-terminal (and, in some cases, carboxyl-terminal) residues may be removed enzymatically in formation of the final functional protein. In as many as 50% of eukaryotic proteins, the amino group of the amino-terminal residue is -acetylated after translation. arboxyl-terminal residues are also sometimes modified. Loss of Signal Sequences s we shall see in Section 7.3, the 15 to 30 residues at the amino-terminal end of some proteins play a role in directing the protein to its ultimate destination in the cell. Such signal sequences are ultimately removed by specific peptidases. Modification of Individual mino cids The hydroxyl groups of certain Ser, Thr, and Tyr residues of some proteins are enzymatically phosphorylated by T (Fig.
7. rotein Synthesis 1063 0.5 mm (b) FIGUR 7 7 olysome. (a) Four ribosomes translating a eukaryotic mr molecule simultaneously, moving from the end to the end and synthesizing a polypeptide from the amino terminus to the carboxyl terminus. (b) lectron micrograph and explanatory diagram of a polysome from the silk gland of a silkworm larva. The mr is being translated by many ribosomes simultaneously. The nascent polypeptides become longer as the ribosomes move toward the end of the mr. The final product of this process is silk fibroin. 7 9a); the phosphate groups add negative charges to these polypeptides. The functional significance of this modification varies from one protein to the next. For example, the milk protein casein has many phosphoserine groups that bind a. alcium, phosphate, and D duplex R polymerase amino acids are all valuable to suckling young, so casein efficiently provides three essential nutrients. nd as we have seen in numerous instances, phosphorylationdephosphorylation cycles regulate the activity of many enzymes and regulatory proteins. xtra carboxyl groups may be added to Glu residues of some proteins. For example, the blood-clotting protein prothrombin contains a number of -carboxyglutamate residues (Fig. 7 9b) in its amino-terminal region, introduced by an enzyme that requires vitamin K. These carboxyl groups bind a, which is required to initiate the clotting mechanism. Ribosome mr 3 3 Direction of translation Direction of transcription FIGUR 7 8 oupling of transcription and translation in bacteria. The mr is translated by ribosomes while it is still being transcribed from D by R polymerase. This is possible because the mr in bacteria does not have to be transported from a nucleus to the cytoplasm before encountering ribosomes. In this schematic diagram the ribosomes are depicted as smaller than the R polymerase. In reality the ribosomes (M r.7 10 6 ) are an order of magnitude larger than the R polymerase (M r 3.9 10 5 ).
1066 hapter 7 rotein Metabolism site peptidyl-tr site puromycin R.. 3 3 3 mr R 3 peptidyl transferase 3 (b) 3 (a) FIGUR 7 31 Disruption of peptide bond formation by puromycin. (a) The antibiotic puromycin resembles the aminoacyl end of a charged tr, and it can bind to the ribosomal site and participate in peptide bond formation. The product of this reaction, instead of being translocated to the site, dissociates from the ribosome, causing premature chain termination. (b) eptidyl puromycin. uromycin, made by the mold Streptomyces alboniger, is one of the best-understood inhibitory antibiotics. Its structure is very similar to the end of an aminoacyl-tr, enabling it to bind to the ribosomal site and participate in peptide bond formation, producing peptidyl-puromycin (Fig. 7 31). owever, because puromycin resembles only the end of the tr, it does not engage in translocation and dissociates from the ribosome shortly after it is linked to the carboxyl terminus of the peptide. This prematurely terminates polypeptide synthesis. Tetracyclines inhibit protein synthesis in bacteria by blocking the site on the ribosome, preventing the binding of aminoacyl-trs. hloramphenicol inhibits protein synthesis by bacterial (and mitochondrial 3 3 Tetracycline 3 hloramphenicol l
7.3 rotein Targeting and Degradation 1069 Signal sequence 1 GU cap mr 8 ytosol SR 3 SR cycle 5 GD i Ribosome cycle ()n Ribosome receptor GT 4 6 ndoplasmic reticulum eptide translocation complex SR receptor Signal peptidase 7 R lumen FIGUR 7 33 Directing eukaryotic proteins with the appropriate signals to the endoplasmic reticulum. This process involves the SR cycle and translocation and cleavage of the nascent polypeptide. The steps are described in the text. SR is a rod-shaped complex containing a 300 nucleotide R (7SL-R) and six different proteins (combined M r 35,000). ne protein subunit of SR binds directly to the signal sequence, inhibiting elongation by sterically blocking the entry of aminoacyl-trs and inhibiting peptidyl transferase. nother protein subunit binds and hydrolyzes GT. The SR receptor is a heterodimer of (M r 69,000) and (M r 30,000) subunits, both of which bind and hydrolyze multiple GT molecules during this process. steps 1 through 8 in Figure 7 33. 1 The targeting pathway begins with initiation of protein synthesis on free ribosomes. The signal sequence appears early in the synthetic process, because it is at the amino terminus, which as we have seen is synthesized first. 3 s it emerges from the ribosome, the signal sequence and the ribosome itself are bound by the large signal recognition particle (SR); SR then binds GT and halts elongation of the polypeptide when it is about 70 amino acids long and the signal sequence has completely emerged from the ribosome. 4 The GT-bound SR now directs the ribosome (still bound to the mr) and the incomplete polypeptide to GT-bound SR receptors in the cytosolic face of the R; the nascent polypeptide is delivered to a peptide translocation complex in the R, which may interact directly with the ribosome. 5 SR dissociates from the ribosome, accompanied by hydrolysis of GT in both SR and the SR receptor. 6 longation of the polypeptide now resumes, with the T-driven translocation complex feeding the growing polypeptide into the R lumen until the complete protein has been synthesized. 7 The signal sequence is removed by a signal peptidase within the R lumen; 8 the ribosome dissociates and is recycled. Glycosylation lays a Key Role in rotein Targeting In the R lumen, newly synthesized proteins are further modified in several ways. Following the removal of signal sequences, polypeptides are folded, disulfide bonds formed, and many proteins glycosylated to form glycoproteins. In many glycoproteins the linkage to their oligosaccharides is through sn residues. These - linked oligosaccharides are diverse (hapter 7), but the pathways by which they form have a common first step. 14 residue core oligosaccharide is built up in a stepwise fashion, then transferred from a dolichol phosphate donor molecule to certain sn residues in the protein (Fig. 7 34). The transferase is on the lumenal face of the R and thus cannot catalyze glycosylation of cytosolic proteins. fter transfer, the core oligosaccharide is trimmed and elaborated in different ways on different 3 n 3 Dolichol phosphate (n 9 ) 3 3
1070 hapter 7 rotein Metabolism tunicamycin -cetylglucosamine (Glcc) Mannose (Man) Glucose (Glc) 5 GD 5 GD-Man UM UD UD-Glcc translocation 3 1 dolichol phosphate recycled 4 Dolichol Man 4 Dolichol 3 Dolichol Glc 3 Dolichol 4 ndoplasmic reticulum 3 Dolichol 3 5 sn 6 7 3 8 9 i ytosol mr FIGUR 7 34 Synthesis of the core oligosaccharide of glycoproteins. The core oligosaccharide is built up by the successive addition of monosaccharide units. 1, The first steps occur on the cytosolic face of the R. 3 Translocation moves the incomplete oligosaccharide across the membrane (mechanism not shown), and 4 completion of the core oligosaccharide occurs within the lumen of the R. The precursors that contribute additional mannose and glucose residues to the growing oligosaccharide in the lumen are dolichol phosphate derivatives. In the first step in the construction of the - linked oligosaccharide moiety of a glycoprotein, 5, 6 the core oligosaccharide is transferred from dolichol phosphate to an sn residue of the protein within the R lumen. The core oligosaccharide is then further modified in the R and the Golgi complex in pathways that differ for different proteins. The five sugar residues shown surrounded by a beige screen (after step 7 ) are retained in the final structure of all -linked oligosaccharides. 8 The released dolichol pyrophosphate is again translocated so that the pyrophosphate is on the cytosolic face of the R, then 9 a phosphate is hydrolytically removed to regenerate dolichol phosphate. proteins, but all -linked oligosaccharides retain a pentasaccharide core derived from the original 14 residue oligosaccharide. Several antibiotics act by interfering with one or more steps in this process and have aided in elucidating the steps of protein glycosylation. The best-characterized is tunicamycin, which mimics the structure of UD--acetylglucosamine and blocks the first step of the process (Fig. 7 34, step 1 ). few proteins are -glycosylated in the R, but most -glycosylation occurs in the Golgi complex or in the cytosol (for proteins that do not enter the R). Suitably modified proteins can now be moved to a variety of intracellular destinations. roteins travel from the R to the Golgi complex in transport vesicles (Fig. 7 35). In the Golgi complex, oligosaccharides are - linked to some proteins, and -linked oligosaccharides are further modified. By mechanisms not yet fully understood, the Golgi complex also sorts proteins and sends them to their final destinations. The processes that segregate proteins targeted for secretion from those targeted for the plasma membrane or lysosomes must distinguish among these proteins on the basis of structural features other than signal sequences, which were removed in the R lumen. Fatty acyl side chain 3 a b ( ) n 3 3 (n 8 11) -cetylglucosamine Tunicamycin Tunicamine Uracil
107 hapter 7 rotein Metabolism B B Uridine 3 UD -cetylglucosamine (UD-Glcc) -acetylglucosamine phosphotransferase Mannose residue UM ligosaccharide nzyme ydrolase B 3 ligosaccharide nzyme phosphodiesterase Glcc FIGUR 7 36 hosphorylation of mannose residues on lysosome-targeted enzymes. -cetylglucosamine phosphotransferase recognizes some as yet unidentified structural feature of hydrolases destined for lysosomes. B ligosaccharide nzyme Mannose 6-phosphate residue proteins in the cytosol. The complex of the LSbearing protein and the importin docks at a nuclear pore and is translocated through the pore by an energydependent mechanism that requires the Ran GTase. The two importin subunits separate during the translocation, and the LS-bearing protein dissociates from importin inside the nucleus. Importin and are then exported from the nucleus to repeat the process. ow importin remains dissociated from the many LSbearing proteins inside the nucleus is not yet clear. Bacteria lso Use Signal Sequences for rotein Targeting Bacteria can target proteins to their inner or outer membranes, to the periplasmic space between these membranes, or to the extracellular medium. They use signal sequences at the amino terminus of the proteins (Fig. 7 38), much like those on eukaryotic proteins targeted to the R, mitochondria, and chloroplasts. Most proteins exported from. coli make use of the pathway shown in Figure 7 39. Following translation, a protein to be exported may fold only slowly, the amino-terminal signal sequence impeding the folding. The soluble chaperone protein SecB binds to the protein s signal sequence or other features of its incompletely folded structure. The bound protein is then delivered to Sec, a protein associated with the inner surface of the plasma membrane. Sec acts as both a receptor and a translocating Tase. Released from SecB and bound to Sec, the protein is delivered to a translocation complex in the membrane, made up of SecY,, and G, and is translocated stepwise through the membrane at the SecYG complex in lengths of about 0 amino acid residues. ach step is facilitated by the hydrolysis of T, catalyzed by Sec.
7.3 rotein Targeting and Degradation 1073 (a) uclear protein ytosol a Importin b LS 1 uclear envelope (b) 6 uclear pore complex 3 Ran GT GD i 4 b a 5 a ucleoplasm FIGUR 7 37 Targeting of nuclear proteins. (a) 1 protein with an appropriate nuclear localization signal (LS) is bound by a complex of importin and. The resulting complex binds to a nuclear pore, and 3 translocation is mediated by the Ran GTase. 4 Inside the nucleus, importin dissociates from importin, and 5 importin then releases the nuclear protein. 6 Importin and are transported out of the nucleus and recycled. (b) Scanning electron micrograph of the surface of the nuclear envelope, showing numerous nuclear pores. 0. m Inner membrane proteins hage fd, major coat protein hage fd, minor coat protein cleavage site Met Lys Lys Ser Leu Val Leu Lys la Ser Val la Val la Thr Leu Val ro Met Leu Ser he la la Glu Met Lys Lys Leu Leu he la Ile ro Leu Val Val ro he Tyr Ser is Ser la Glu eriplasmic proteins lkaline phosphatase Met Lys Gln Ser Thr Ile la Leu la Leu Leu ro Leu Leu he Thr ro Val Thr Lys la rg Thr Leucine-specific binding protein Met Lys la sn la Lys Thr Ile Ile la Gly Met Ile la Leu la Ile Ser is Thr la Met la sp sp -Lactamase of pbr3 Met Ser Ile Gln is he rg Val la Leu Ile ro he he la uter membrane proteins Lipoprotein Met Lys la Thr Lys Leu Val Leu Gly la Val Ile LamB Leu rg Lys Leu ro Leu la Val la Val la la Gly mp Met Met Ile Thr Met Lys Lys Thr la Ile la Ile la Val la Leu la la he ys Leu ro Val he la is ro Leu Gly Ser Thr Leu Leu la Gly ys Ser Val Met Ser la Gln la Met la Val sp Gly he la Thr Val la Gln la la ro FIGUR 7 38 Signal sequences that target proteins to different locations in bacteria. Basic amino acids (blue) near the amino terminus and hydrophobic core amino acids (yellow) are highlighted. The cleavage sites marking the ends of the signal sequences are indicated by red arrows. ote that the inner bacterial cell membrane (see Fig. 1 6) is where phage fd coat proteins and D are assembled into phage particles. mp is outer membrane protein ; LamB is a cell surface receptor protein for bacteriophage lambda.
1074 hapter 7 rotein Metabolism 1 Sec SecYG SecB SecB T D i T 3 4 5 ytosol 6 eriplasmic space FIGUR 7 39 Model for protein export in bacteria. 1 newly translated polypeptide binds to the cytosolic chaperone protein SecB, which delivers it to Sec, a protein associated with the translocation complex (SecYG) in the bacterial cell membrane. 3 SecB is released, and Sec inserts itself into the membrane, forcing about 0 amino acid residues of the protein to be exported through the translocation complex. 4 ydrolysis of an T by Sec provides the energy for a conformational change that causes Sec to withdraw from the membrane, releasing the polypeptide. 5 Sec binds another T, and the next stretch of 0 amino acid residues is pushed across the membrane through the translocation complex. Steps 4 and 5 are repeated until 6 the entire protein has passed through and is released to the periplasm. The electrochemical potential across the membrane (denoted by and ) also provides some of the driving force required for protein translocation. n exported protein is thus pushed through the membrane by a Sec protein located on the cytoplasmic surface, rather than being pulled through the membrane by a protein on the periplasmic surface. This difference may simply reflect the need for the translocating Tase to be where the T is. The transmembrane electrochemical potential can also provide energy for translocation of the protein, by an as yet unknown mechanism. lthough most exported bacterial proteins use this pathway, some follow an alternative pathway that uses signal recognition and receptor proteins homologous to components of the eukaryotic SR and SR receptor (Fig. 7 33). ells Import roteins by Receptor-Mediated ndocytosis Some proteins are imported into cells from the surrounding medium; examples in eukaryotes include lowdensity lipoprotein (LDL), the iron-carrying protein transferrin, peptide hormones, and circulating proteins destined for degradation. The proteins bind to receptors in invaginations of the membrane called coated pits, which concentrate endocytic receptors in preference to other cell-surface proteins. The pits are coated on their cytosolic side with a lattice of the protein clathrin, which forms closed polyhedral structures (Fig. 7 40). The clathrin lattice grows as more recep- eavy chain Light chain ~80 nm (a) (b) (c) 0.1 mm FIGUR 7 40 lathrin. (a) Three light (L) chains (M r 35,000) and three heavy () chains (M r 180,000) of the (L) 3 clathrin unit, organized as a three-legged structure called a triskelion. (b) Triskelions tend to assemble into polyhedral lattices. (c) lectron micrograph of a coated pit on the cytosolic face of the plasma membrane of a fibroblast.