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O O O
H B B B
ROC CO O O O P OO O P OO O P O OOCH2
A B A A A
NH3 O O O O
O Adenine
Amino acid ATP
H H
H H
3 end of tRNA
Adenine H Adenine H
2 O 2 O
H OH H B H OH H B
O ROC CO OO PO OO Adenosine O ROC CO OO PO O O Adenosine
A B A A B A
H OH H OH
3 NH3 O O 3
NH3 O O
CH2 H CH2 H
A Aminoacyl-AMP A Aminoacyl-AMP
O O
A A
O OP PO O OP PO
A A
O O
tRNA tRNA
2a AMP 2b AMP
Adenine H Adenine H
2
H 2
H O COCOR H OH
B A
O O
O NH3 H
H 3
OH H 3
O COCOR
B A
CH2
A
H CH2
A
H O NH3
O transesterification
O
A A
O OP PO 3a O OP P O
A A
O O
Aminoacyl-tRNA
8885d_c27_1034-1080 2/12/04 1:19 PM Page 1053 mac76 mac76:385_reb:
CH3 CH2
CH3
Valine Isoleucine
Extra arm
In addition to proofreading after formation of the
aminoacyl-AMP intermediate, most aminoacyl-tRNA
synthetases can also hydrolyze the ester linkage be-
tween amino acids and tRNAs in the aminoacyl-tRNAs.
Anticodon
This hydrolysis is greatly accelerated for incorrectly arm
charged tRNAs, providing yet a third filter to enhance
the fidelity of the overall process. The few aminoacyl-
Anticodon
tRNA synthetases that activate amino acids with no
close structural relatives (Cys-tRNA synthetase, for ex- FIGURE 2716 Nucleotide positions in tRNAs that are recognized
ample) demonstrate little or no proofreading activity; in by aminoacyl-tRNA synthetases. Some positions (blue dots) are the
these cases, the active site for aminoacylation can suf- same in all tRNAs and therefore cannot be used to discriminate one
ficiently discriminate between the proper substrate and from another. Other positions are known recognition points for one
any incorrect amino acid. (orange) or more (green) aminoacyl-tRNA synthetases. Structural fea-
The overall error rate of protein synthesis (~1 mis- tures other than sequence are important for recognition by some of
take per 104 amino acids incorporated) is not nearly as the synthetases.
8885d_c27_1034-1080 2/12/04 1:19 PM Page 1056 mac76 mac76:385_reb:
IF-3 IF-1
tRNA that is distinct from the tRNAMet used at (5)AUG
codons at interior positions in the mRNA. Polypeptides
synthesized by mitochondrial and chloroplast ribo-
somes, however, begin with N-formylmethionine. This
strongly supports the view that mitochondria and P A
30S
chloroplasts originated from bacterial ancestors that Subunit
were symbiotically incorporated into precursor eukary-
otic cells at an early stage of evolution (see Fig. 136). mRNA
How can the single (5)AUG codon distinguish be-
tween the starting N-formylmethionine (or methionine,
in eukaryotes) and interior Met residues? The details of 1 Initiation mRNA
the initiation process provide the answer. codon
5 A U G 3
The Three Steps of Initiation The initiation of polypep- P IF-1
IF-3
tide synthesis in bacteria requires (1) the 30S ribosomal
subunit, (2) the mRNA coding for the polypeptide to be
made, (3) the initiating fMet-tRNAf Met, (4) a set of three
proteins called initiation factors (IF-1, IF-2, and IF-3),
2 IF-2
(5) GTP, (6) the 50S ribosomal subunit, and (7) Mg2. 5
fMet
Formation of the initiation complex takes place in three
GTP
steps (Fig. 2720).
5
In step 1 the 30S ribosomal subunit binds two ini- fMet
tiation factors, IF-1 and IF-3. Factor IF-3 prevents the
C tRNA
30S and 50S subunits from combining prematurely. The U A
mRNA then binds to the 30S subunit. The initiating (3) UAC (5) IF-2 GTP
(5)AUG is guided to its correct position by the Shine- Anticodon U A C
Dalgarno sequence (named for Australian researchers
5 A U G 3
John Shine and Lynn Dalgarno, who identified it) in the
P IF-1
mRNA. This consensus sequence is an initiation signal IF-3
of four to nine purine residues, 8 to 13 bp to the 5 side
of the initiation codon (Fig. 2721a). The sequence
base-pairs with a complementary pyrimidine-rich se- 50S
quence near the 3 end of the 16S rRNA of the 30S ri- Subunit
GDP Pi
bosomal subunit (Fig. 2721b). This mRNA-rRNA in-
teraction positions the initiating (5)AUG sequence of
the mRNA in the precise position on the 30S subunit IF-1 IF-2 IF-3
where it is required for initiation of translation. The par-
ticular (5)AUG where fMet-tRNAfMet is to be bound is
3
distinguished from other methionine codons by its prox- 5
imity to the Shine-Dalgarno sequence in the mRNA. fMet
Bacterial ribosomes have three sites that bind
aminoacyl-tRNAs, the aminoacyl (A) site, the pep-
50S
tidyl (P) site, and the exit (E) site. Both the 30S Subunit E U A C
and the 50S subunits contribute to the characteristics
of the A and P sites, whereas the E site is largely con- 5 A U G 3
fined to the 50S subunit. The initiating (5)AUG is P A Next
positioned at the P site, the only site to which fMet- codon
tRNAfMet can bind (Fig. 2720). The fMet-tRNAfMet is
the only aminoacyl-tRNA that binds first to the P site;
during the subsequent elongation stage, all other in- FIGURE 2720 Formation of the initiation complex in bacteria. The
coming aminoacyl-tRNAs (including the Met-tRNAMet complex forms in three steps (described in the text) at the expense of
that binds to interior AUG codons) bind first to the A the hydrolysis of GTP to GDP and Pi. IF-1, IF-2, and IF-3 are initia-
site and only subsequently to the P and E sites. The E tion factors. P designates the peptidyl site, A the aminoacyl site, and
site is the site from which the uncharged tRNAs leave E the exit site. Here the anticodon of the tRNA is oriented 3 to 5,
during elongation. Factor IF-1 binds at the A site and left to right, as in Figure 278 but opposite to the orientation in Fig-
prevents tRNA binding at this site during initiation. ures 2716 and 2718.
8885d_c27_1034-1080 2/12/04 1:19 PM Page 1057 mac76 mac76:385_reb:
(a)
3
OH G
3 End of A
Prokaryotic 16S rRNA A U
mRNA U C
with consensus U C C U C C A
Shine-Dalgarno
sequence (5) G A U U C C U A G G A G G U U U G A C C U A U G C G A G C U U U U A G U (3)
(b)
FIGURE 2721 Messenger RNA sequences that serve as signals for shown. Note the unusual example of the E. coli LacI protein, which
initiation of protein synthesis in bacteria. (a) Alignment of the initi- initiates with a GUG (Val) codon (see Box 272). (b) The Shine-
ating AUG (shaded in green) at its correct location on the 30S ribo- Dalgarno sequence of the mRNA pairs with a sequence near the 3
somal subunit depends in part on upstream Shine-Dalgarno sequences end of the 16S rRNA.
(pink). Portions of the mRNA transcripts of five prokaryotic genes are
In step 2 of the initiation process (Fig. 2720), the protein (PAB). Eukaryotic cells have at least nine initi-
complex consisting of the 30S ribosomal subunit, IF-3, ation factors. A complex called eIF4F, which includes
and mRNA is joined by both GTP-bound IF-2 and the the proteins eIF4E, eIF4G, and eIF4A, binds to the 5
initiating fMet-tRNAfMet. The anticodon of this tRNA cap (see Fig. 2612) through eIF4E. The protein eIF4G
now pairs correctly with the mRNAs initiation codon. binds to both eIF4E and PAB, effectively tying them to-
In step 3 this large complex combines with the 50S gether (Fig. 2722). The protein eIF4A has an RNA he-
ribosomal subunit; simultaneously, the GTP bound to licase activity. It is the eIF4F complex that associates
IF-2 is hydrolyzed to GDP and Pi, which are released
from the complex. All three initiation factors depart
from the ribosome at this point. 40S Ribosomal subunit
Completion of the steps in Figure 2720 produces
a functional 70S ribosome called the initiation com-
eIF3
plex, containing the mRNA and the initiating fMet- 5 cap 3 poly(A) tail
tRNAfMet. The correct binding of the fMet-tRNAfMet to A
the P site in the complete 70S initiation complex is as- AA A
(A) n
sured by at least three points of recognition and at-
eIF4E PAB
tachment: the codon-anticodon interaction involving the eIF4G
initiation AUG fixed in the P site; interaction between AUG
the Shine-Dalgarno sequence in the mRNA and the 16S
rRNA; and binding interactions between the ribosomal
P site and the fMet-tRNAfMet. The initiation complex is
now ready for elongation.
Gene 3 Untranslated
region
Initiation in Eukaryotic Cells Translation is generally sim-
ilar in eukaryotic and bacterial cells; most of the signif- FIGURE 2722 Protein complexes in the formation of a eukaryotic
icant differences are in the mechanism of initiation. Eu- initiation complex. The 3 and 5 ends of eukaryotic mRNAs are linked
karyotic mRNAs are bound to the ribosome as a complex by a complex of proteins that includes several initiation factors and
with a number of specific binding proteins. Several of the poly(A) binding protein (PAB). The factors eIF4E and eIF4G are
these tie together the 5 and 3 ends of the message. At part of a larger complex called eIF4F. This complex binds to the 40S
the 3 end, the mRNA is bound by the poly(A) binding ribosomal subunit.
8885d_c27_1034-1080 2/12/04 1:19 PM Page 1058 mac76 mac76:385_reb:
TABLE 278 Protein Factors Required for Initiation of Translation in Bacterial and Eukaryotic Cells
Factor Function
Bacterial
IF-1 Prevents premature binding of tRNAs to A site
IF-2 Facilitates binding of fMet-tRNAfMet to 30S ribosomal subunit
IF-3 Binds to 30S subunit; prevents premature association of 50S
subunit; enhances specificity of P site for fMet-tRNAfMet
Eukaryotic*
eIF2 Facilitates binding of initiating Met-tRNAMet to 40S ribosomal subunit
eIF2B, eIF3 First factors to bind 40S subunit; facilitate subsequent steps
eIF4A RNA helicase activity removes secondary structure in the mRNA to permit binding
to 40S subunit; part of the eIF4F complex
eIF4B Binds to mRNA; facilitates scanning of mRNA to locate the first AUG
eIF4E Binds to the 5 cap of mRNA; part of the eIF4F complex
eIF4G Binds to eIF4E and to poly(A) binding protein (PAB); part of the eIF4F complex
eIF5 Promotes dissociation of several other initiation factors from 40S subunit as a
prelude to association of 60S subunit to form 80S initiation complex
eIF6 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 Elongation Step 1: Binding of an Incoming Aminoacyl-tRNA In
subunit. The efficiency of translation is affected by many the first step of the elongation cycle (Fig. 2723), the
properties of the mRNA and proteins in this complex, appropriate incoming aminoacyl-tRNA binds to a com-
including the length of the 3 poly(A) tract (in most plex of GTP-bound EF-Tu. The resulting aminoacyl-
cases, longer is better). The end-to-end arrangement of tRNAEF-TuGTP complex binds to the A site of the
the eukaryotic mRNA facilitates translational regulation 70S initiation complex. The GTP is hydrolyzed and an
of gene expression, considered in Chapter 28. EF-TuGDP complex is released from the 70S ribosome.
The initiating (5)AUG is detected within the mRNA The EF-TuGTP complex is regenerated in a process in-
not by its proximity to a Shine-Dalgarno-like sequence volving EF-Ts and GTP.
but by a scanning process: a scan of the mRNA from the
5 end until the first AUG is encountered, signaling the Elongation Step 2: Peptide Bond Formation A peptide bond
beginning of the reading frame. The eIF4F complex is is now formed between the two amino acids bound by
probably involved in this process, perhaps using the their tRNAs to the A and P sites on the ribosome. This
RNA helicase activity of eIF4A to eliminate secondary occurs by the transfer of the initiating N-formylme-
structure in the 5 untranslated portion of the mRNA. thionyl group from its tRNA to the amino group of the
Scanning is also facilitated by another protein, eIF4B. second amino acid, now in the A site (Fig. 2724). The
The roles of the various bacterial and eukaryotic ini- -amino group of the amino acid in the A site acts as a
tiation factors in the overall process are summarized in nucleophile, displacing the tRNA in the P site to form
Table 278. The mechanism by which these proteins act the peptide bond. This reaction produces a dipeptidyl-
is an important area of investigation. tRNA in the A site, and the now uncharged (deacy-
lated) tRNAfMet remains bound to the P site. The tRNAs
Stage 3: Peptide Bonds Are Formed in the then shift to a hybrid binding state, with elements of
each spanning two different sites on the ribosome, as
Elongation Stage
shown in Figure 2724.
The third stage of protein synthesis is elongation. The enzymatic activity that catalyzes peptide bond
Again, our initial focus is on bacterial cells. Elongation formation has historically been referred to as peptidyl
requires (1) the initiation complex described above, (2) transferase and was widely assumed to be intrinsic to
aminoacyl-tRNAs, (3) a set of three soluble cytosolic one or more of the proteins in the large ribosomal sub-
proteins called elongation factors (EF-Tu, EF-Ts, and unit. We now know that this reaction is catalyzed by the
EF-G in bacteria), and (4) GTP. Cells use three steps to 23S rRNA (Fig. 279), adding to the known catalytic
add each amino acid residue, and the steps are repeated repertoire of ribozymes. This discovery has interesting
as many times as there are residues to be added. implications for the evolution of life (Box 273).
8885d_c27_1034-1080 2/12/04 1:19 PM Page 1059 mac76 mac76:385_reb:
Initiation 5
fMet
complex
50S U A C
E E site P site A site
5 A U G 3
Initiation P A Next
codon codon
H fMet-tRNAfMet
30S 5
AA2 C O
.. Aminoacyl-
Incoming NH NH2 tRNA2
aminoacyl- R1 C H R2 C H
tRNA
O C O C
O O
5 5
5 AA
2
Tu GTP
E U A C
Tu GTP mRNA 5 A U G 3
P A
Ts
binding of
incoming
aminoacyl-
tRNA GTP
peptide bond
formation
Tu Ts
E U A C
E U A C
5 A U G 3 5 3
A U G
P A P A
FIGURE 2723 First elongation step in bacteria: binding of the sec- FIGURE 2724 Second elongation step in bacteria: formation of the
ond aminoacyl-tRNA. The second aminoacyl-tRNA enters the A site first peptide bond. The peptidyl transferase catalyzing this reaction is
of the ribosome bound to EF-Tu (shown here as Tu), which also con- the 23S rRNA ribozyme. The N-formylmethionyl group is transferred
tains GTP. Binding of the second aminoacyl-tRNA to the A site is ac- to the amino group of the second aminoacyl-tRNA in the A site, form-
companied by hydrolysis of the GTP to GDP and Pi and release of the ing a dipeptidyl-tRNA. At this stage, both tRNAs bound to the ribo-
EF-TuGDP complex from the ribosome. The bound GDP is released some shift position in the 50S subunit to take up a hybrid binding
when the EF-TuGDP complex binds to EF-Ts, and EF-Ts is subse- state. The uncharged tRNA shifts so that its 3 and 5 ends are in the
quently released when another molecule of GTP binds to EF-Tu. This E site. Similarly, the 3 and 5 ends of the peptidyl tRNA shift to the
recycles EF-Tu and makes it available to repeat the cycle. P site. The anticodons remain in the A and P sites.
8885d_c27_1034-1080 2/12/04 1:19 PM Page 1060 mac76 mac76:385_reb:
Elongation Step 3: Translocation In the final step of the A change in the three-dimensional conformation of the
elongation cycle, translocation, the ribosome moves entire ribosome results in its movement along the mRNA.
one codon toward the 3 end of the mRNA (Fig. 2725a). Because the structure of EF-G mimics the structure of
This movement shifts the anticodon of the dipeptidyl- the EF-TutRNA complex (Fig. 2725b), EF-G can bind
tRNA, which is still attached to the second codon of the the A site and presumably displace the peptidyl-tRNA.
mRNA, from the A site to the P site, and shifts the de-
acylated tRNA from the P site to the E site, from where The ribosome, with its attached dipeptidyl-tRNA and
the tRNA is released into the cytosol. The third codon mRNA, is now ready for the next elongation cycle and
of the mRNA now lies in the A site and the second codon attachment of a third amino acid residue. This process
in the P site. Movement of the ribosome along the mRNA occurs in the same way as addition of the second residue
requires EF-G (also known as translocase) and the en- (as shown in Figs 2723, 2724, and 2725). For each
ergy provided by hydrolysis of another molecule of GTP. amino acid residue correctly added to the growing
polypeptide, two GTPs are hydrolyzed to GDP and Pi as
the ribosome moves from codon to codon along the
E site P site A site
mRNA toward the 3 end.
H
The polypeptide remains attached to the tRNA of
O the most recent amino acid to be inserted. This associ-
C
NH ation maintains the functional connection between the
H Deacylated
1 C
tRNAfMet
information in the mRNA and its decoded polypeptide
R
C
O output. At the same time, the ester linkage between this
NH tRNA and the carboxyl terminus of the growing polypep-
H Dipeptidyl-
2 C
OH R C tRNA2 tide activates the terminal carboxyl group for nucleo-
O
O philic attack by the incoming amino acid to form a new
5 5 5 5 peptide bond (Fig. 2724). As the existing ester linkage
between the polypeptide and tRNA is broken during
E U A C
FIGURE 2725 Third elongation step in bacteria: translocation.
5 A U G 3
(a) The ribosome moves one codon toward the 3 end of the mRNA,
P A
using energy provided by hydrolysis of GTP bound to EF-G (translo-
case). The dipeptidyl-tRNA is now entirely in the P site, leaving the A
site open for the incoming (third) aminoacyl-tRNA. The uncharged
tRNA dissociates from the E site, and the elongation cycle begins again.
(b) The structure of EF-G mimics the structure of EF-Tu complexed with
EF-G GTP
tRNA. Shown here are (left) EF-Tu complexed with tRNA (green) (PDB
translocation ID 1B23) and (right) EF-G complexed with GDP (red) (PDB ID 1DAR).
EF-G GDP Pi The carboxyl-terminal part of EF-G (dark gray) mimics the structure of
the anticodon loop of tRNA in both shape and charge distribution.
U A C
5 A U G 3
P A
Direction of
ribosome movement
(a) (b)
8885d_c27_1034-1080 2/12/04 1:19 PM Page 1062 mac76 mac76:385_reb:
0.25 mm
(b)
2729a); the phosphate groups add negative charges to amino acids are all valuable to suckling young, so casein
these polypeptides. The functional significance of this efficiently provides three essential nutrients. And as we
modification varies from one protein to the next. For have seen in numerous instances, phosphorylation-
example, the milk protein casein has many phospho- dephosphorylation cycles regulate the activity of many
serine groups that bind Ca2. Calcium, phosphate, and enzymes and regulatory proteins.
Extra carboxyl groups may be added to Glu residues
of some proteins. For example, the blood-clotting pro-
DNA RNA
tein prothrombin contains a number of -carboxygluta-
3 mate residues (Fig. 2729b) in its amino-terminal re-
5 duplex polymerase 5
3 gion, introduced by an enzyme that requires vitamin K.
These carboxyl groups bind Ca2, which is required to
initiate the clotting mechanism.
Ribosome
FIGURE 2728 Coupling of transcription and translation in bacte-
5 ria. The mRNA is translated by ribosomes while it is still being tran-
mRNA scribed from DNA by RNA polymerase. This is possible because the
Direction of transcription mRNA in bacteria does not have to be transported from a nucleus to
NH3 the cytoplasm before encountering ribosomes. In this schematic dia-
NH3
gram the ribosomes are depicted as smaller than the RNA polymerase.
Direction of translation
In reality the ribosomes (Mr 2.7 106) are an order of magnitude
larger than the RNA polymerase (Mr 3.9 105).
8885d_c27_1034-1080 2/12/04 1:19 PM Page 1066 mac76 mac76:385_reb:
P site A site
peptidyl-tRNA puromycin
CH2 OCH3
..
H2N C H
NH
C O
R C H
NH OH
C O H H
H H
O OH CH O2
H H
H H HO N N
CH O2 N N
O N N N
CH3 CH3
P N N
5 NH2
AA
5
NH
R C H CH2 OCH3
O C N C H
E H
5 C O
3
mRNA P A NH OH
H H
H H
CH O2
peptidyl N
HO N
transferase
N N
N
CH3 CH3
5
(b)
5 cap
Signal
sequence
2 1
mRNA
GUA
8
Ribosome
cycle
SRP SRP
Cytosol
cycle )n
A
3 A(
AA
5 GDP Pi
Ribosome
receptor GTP 4 6
ER lumen
FIGURE 2733 Directing eukaryotic proteins with the appropriate the signal sequence, inhibiting elongation by sterically blocking the
signals to the endoplasmic reticulum. This process involves the SRP entry of aminoacyl-tRNAs and inhibiting peptidyl transferase. Another
cycle and translocation and cleavage of the nascent polypeptide. The protein subunit binds and hydrolyzes GTP. The SRP receptor is a het-
steps are described in the text. SRP is a rod-shaped complex con- erodimer of (Mr 69,000) and (Mr 30,000) subunits, both of which
taining a 300 nucleotide RNA (7SL-RNA) and six different proteins bind and hydrolyze multiple GTP molecules during this process.
(combined Mr 325,000). One protein subunit of SRP binds directly to
steps 1 through 8 in Figure 2733. 1 The targeting Glycosylation Plays a Key Role in Protein Targeting
pathway begins with initiation of protein synthesis on
In the ER lumen, newly synthesized proteins are further
free ribosomes. 2 The signal sequence appears early in
modified in several ways. Following the removal of sig-
the synthetic process, because it is at the amino termi-
nal sequences, polypeptides are folded, disulfide bonds
nus, which as we have seen is synthesized first. 3 As it
formed, and many proteins glycosylated to form glyco-
emerges from the ribosome, the signal sequenceand
proteins. In many glycoproteins the linkage to their
the ribosome itselfare bound by the large signal
oligosaccharides is through Asn residues. These N-
recognition particle (SRP); SRP then binds GTP and
linked oligosaccharides are diverse (Chapter 7), but the
halts elongation of the polypeptide when it is about 70
pathways by which they form have a common first step.
amino acids long and the signal sequence has completely
A 14 residue core oligosaccharide is built up in a step-
emerged from the ribosome. 4 The GTP-bound SRP
wise fashion, then transferred from a dolichol phosphate
now directs the ribosome (still bound to the mRNA) and
donor molecule to certain Asn residues in the protein
the incomplete polypeptide to GTP-bound SRP recep-
(Fig. 2734). The transferase is on the lumenal face of
tors in the cytosolic face of the ER; the nascent polypep-
the ER and thus cannot catalyze glycosylation of cyto-
tide is delivered to a peptide translocation complex
solic proteins. After transfer, the core oligosaccharide is
in the ER, which may interact directly with the ribo-
trimmed and elaborated in different ways on different
some. 5 SRP dissociates from the ribosome, accompa-
nied by hydrolysis of GTP in both SRP and the SRP re-
ceptor. 6 Elongation of the polypeptide now resumes,
CH3 CH3
with the ATP-driven translocation complex feeding the
growing polypeptide into the ER lumen until the com- P CH3
plete protein has been synthesized. 7 The signal se- CH3 n
P P
translocation
P 2 P 1 P dolichol
3 phosphate
recycled
Dolichol P Pi
Endoplasmic
P
P
reticulum 9
H 3N
4 Dolichol P Man
4 Dolichol P P P
H3N
3 Dolichol P Glc NH3
3 Dolichol P
4 P P
8
P 5 Asn 6 7 P
Cytosol
5
mRNA
3
FIGURE 2734 Synthesis of the core oligosaccharide of glycopro- oligosaccharide is transferred from dolichol phosphate to an Asn
teins. The core oligosaccharide is built up by the successive addition residue of the protein within the ER lumen. The core oligosaccharide
of monosaccharide units. 1 , 2 The first steps occur on the cytoso- is then further modified in the ER and the Golgi complex in pathways
lic face of the ER. 3 Translocation moves the incomplete oligosac- that differ for different proteins. The five sugar residues shown sur-
charide across the membrane (mechanism not shown), and 4 com- rounded by a beige screen (after step 7 ) are retained in the final
pletion of the core oligosaccharide occurs within the lumen of the ER. structure of all N-linked oligosaccharides. 8 The released dolichol
The precursors that contribute additional mannose and glucose pyrophosphate is again translocated so that the pyrophosphate is on
residues to the growing oligosaccharide in the lumen are dolichol the cytosolic face of the ER, then 9 a phosphate is hydrolytically re-
phosphate derivatives. In the first step in the construction of the N- moved to regenerate dolichol phosphate.
linked oligosaccharide moiety of a glycoprotein, 5 , 6 the core
CH2OH CH2OH
O O
H H H H
H H
O O
OH H B B OH HO H
HO OO PO OO PO O O Uridine HO OO Oligosaccharide O NO Enzyme
A A
H NH O O H H Hydrolase
A
CPO Mannose residue
A
CH3
UDP N-Acetylglucosamine
(UDP-GlcNAc) N-acetylglucosamine
phosphotransferase UMP
CH2OH
O
H H H
O
OH H B
HO O OP OO OCH2
A
H NH O O
A
H H H
CPO
A
CH3 OH HO H
HO OO Oligosaccharide O NO Enzyme
H H
phosphodiesterase GlcNAc
O
B
O OP OO OCH2
A
O
O
H H
H
OH HO H
FIGURE 2736 Phosphorylation of mannose residues HO OO Oligosaccharide O NO Enzyme
on lysosome-targeted enzymes. N-Acetylglucosamine
phosphotransferase recognizes some as yet unidentified H H
structural feature of hydrolases destined for lysosomes. Mannose 6-phosphate residue
proteins in the cytosol. The complex of the NLS- 2738), much like those on eukaryotic proteins targeted
bearing protein and the importin docks at a nuclear pore to the ER, mitochondria, and chloroplasts.
and is translocated through the pore by an energy- Most proteins exported from E. coli make use of
dependent mechanism that requires the Ran GTPase. the pathway shown in Figure 2739. Following transla-
The two importin subunits separate during the translo- tion, a protein to be exported may fold only slowly, the
cation, and the NLS-bearing protein dissociates from im- amino-terminal signal sequence impeding the folding.
portin inside the nucleus. Importin and are then The soluble chaperone protein SecB binds to the pro-
exported from the nucleus to repeat the process. How teins signal sequence or other features of its incom-
importin remains dissociated from the many NLS- pletely folded structure. The bound protein is then de-
bearing proteins inside the nucleus is not yet clear. livered to SecA, a protein associated with the inner
surface of the plasma membrane. SecA acts as both a
Bacteria Also Use Signal Sequences receptor and a translocating ATPase. Released from
SecB and bound to SecA, the protein is delivered to a
for Protein Targeting
translocation complex in the membrane, made up of
Bacteria can target proteins to their inner or outer mem- SecY, E, and G, and is translocated stepwise through the
branes, to the periplasmic space between these mem- membrane at the SecYEG complex in lengths of about
branes, or to the extracellular medium. They use signal 20 amino acid residues. Each step is facilitated by the
sequences at the amino terminus of the proteins (Fig. hydrolysis of ATP, catalyzed by SecA.
8885d_c27_1034-1080 2/12/04 1:19 PM Page 1073 mac76 mac76:385_reb:
(a)
Nuclear
protein Cytosol
Nuclear
NLS envelope
2
1 (b)
a
Importin b
Ran
6 Nuclear 3 GTP GDP Pi
pore
complex
4
5 a
Nucleoplasm
FIGURE 2737 Targeting of nuclear proteins. (a) 1 A protein with an appropriate nuclear 0.2 m
localization signal (NLS) is bound by a complex of importin and . 2 The resulting complex
binds to a nuclear pore, and 3 translocation is mediated by the Ran GTPase. 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.
FIGURE 2738 Signal sequences that target proteins to different lo- by red arrows. Note that the inner bacterial cell membrane (see Fig.
cations in bacteria. Basic amino acids (blue) near the amino termi- 16) is where phage fd coat proteins and DNA are assembled into
nus and hydrophobic core amino acids (yellow) are highlighted. The phage particles. OmpA is outer membrane protein A; LamB is a cell
cleavage sites marking the ends of the signal sequences are indicated surface receptor protein for bacteriophage lambda.
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An exported protein is thus pushed through the Cells Import Proteins by Receptor-Mediated
membrane by a SecA protein located on the cytoplas-
Endocytosis
mic surface, rather than being pulled through the mem-
brane by a protein on the periplasmic surface. This dif- Some proteins are imported into cells from the sur-
ference may simply reflect the need for the translocating rounding medium; examples in eukaryotes include low-
ATPase to be where the ATP is. The transmembrane density lipoprotein (LDL), the iron-carrying protein
electrochemical potential can also provide energy for transferrin, peptide hormones, and circulating proteins
translocation of the protein, by an as yet unknown destined for degradation. The proteins bind to recep-
mechanism. tors in invaginations of the membrane called coated
Although most exported bacterial proteins use this pits, which concentrate endocytic receptors in prefer-
pathway, some follow an alternative pathway that uses ence to other cell-surface proteins. The pits are coated
signal recognition and receptor proteins homologous to on their cytosolic side with a lattice of the protein
components of the eukaryotic SRP and SRP receptor clathrin, which forms closed polyhedral structures
(Fig. 2733). (Fig. 2740). The clathrin lattice grows as more recep-
Heavy
chain
Light ~80
chain nm
FIGURE 2740 Clathrin. (a) Three light (L) chains (Mr 35,000) and tend to assemble into polyhedral lattices. (c) Electron micrograph
three heavy (H) chains (Mr 180,000) of the (HL)3 clathrin unit, or- of a coated pit on the cytosolic face of the plasma membrane of a
ganized as a three-legged structure called a triskelion. (b) Triskelions fibroblast.