Chapter 27: Protein Metabolism

Question 1

Protein Bionsenthesis Stages

Answer 1

Stage 1: Activation of Amino Acids

• carboxyl group of each amino acid must be activated to facilitate formation of a peptide bond
• a link must be established between each new amino acid and the codon
• an amino acid is attached to a tRNA in the cytosol
• Each amino acids is covalently attached to a specific tRNA using ATP
  
  • tRNAs is aminoacylated
  • tRNAs are said to be “charged.”
• aminoacyltRNA synthetases attaches the amino acid to tRNA
  
  • Mg²⁺-dependent

Stage 2: Initiation

• mRNA binds to the smaller of two ribosomal subunits and to the initiating aminoacyltRNA
• large ribosomal subunit then binds to form an initiation complex
• initiating aminoacyl-tRNA base-pairs with the mRNA codon AUG that signals the beginning of the polypeptide
• process requires GTP
• promoted by cytosolic proteins called initiation factors.

Stage 3: Elongation

• polypeptide is lengthened by covalent attachment of successive amino acid units carried to the ribosome by its tRNA
• tRNA base-pairs the amino acid to the codon
• requires cytosolic proteins known as elongation factors
• binding of each incoming aminoacyl-tRNA and the movement of the ribosome are facilitated by the hydrolysis of GTP as each residue is added to the growing polypeptide

Stage 4: Termination and Ribosome Recycling

• Completion is signaled by a termination codon
• new polypeptide is released
• aided by proteins called release factors
• ribosome is recycled for another round of synthesis.

Stage 5: Folding and Posttranslational Processing

• new polypeptide must fold into its proper three-dimensional conformation

Pre/Post Protein Folding Processing

• Before or after folding, the polypeptide may undergo enzymatic processing
• removal of one or more amino acids (usually from the amino terminus)
• addition of acetyl, phosphoryl, methyl, carboxyl, or other groups to certain amino acid residues
• proteolytic cleavage; and/or attachment of oligosaccharides or prosthetic groups

PDF pg. 1146, Table 27-5, Components Required for the Five Major Stages of Protein Synthesis in E. coli

Question 2

Ribosome

Answer 2

• generally promote one of two types of reactions: hydrolytic cleavage of phosphodiester bonds or phosphoryl transfers
• substrates are always RNA molecules
• Ribosomal subunits are identified by their S (Svedberg unit) values
  
  • sedimentation coefficients
  • refer to their rate of sedimentation in a centrifuge
  • not additive when subunits are combined
  • are approximately proportional to the 2/3 power of molecular weight and are also slightly affected by shape

Bacterial

• it’s a ribozyme
• contain about 65% rRNA and 35% protein
• diameter of about 18 nm
• two unequal subunits
• sedimentation coefficients of 30S and 50S; combined sedimentation coefficient of 70S
• contain dozens of ribosomal proteins and at least one large rRNA
  
  • proteins vary in size
• 50S subunit
  
  • the 5S and 23S rRNAs form the structural core
  • proteins are secondary elements decorating the surface
  • no protein within 18 Å of the active site for peptide bond formation
• combined molecular weight of ,2.7 million
• subunits fit together to form a cleft through which the mRNA passes
• Most proteins have globular domains arranged on the ribosome surface
• Some proteins also have snakelike extensions that protrude into the rRNA core of the ribosome, stabilizing its structure
• folding patterns are highly conserved in all organisms, particularly the regions implicated in key functions

Eukaryotic

• larger and more complex than bacterial ribosomes
• diameter of about 23 nm
• sedimentation coefficient of about 80S
• diameter of about 23 nm
• sedimentation coefficient of about 80S
• two subunits, vary in size among species but on average are 60S and 40S
• contain more than 80 different proteins
• ribosomes of mitochondria and chloroplasts are somewhat smaller

PDF pg 1146-1149

Question 3

tRNA

Answer 3

• relatively small
• consist of a single strand of RNA folded into a precise three-dimensional structure
• between 73 and 93 nucleotide residues
• molecular weights of 24,000 to 31,000.
• Mitochondria and chloroplast are smaller
• at least one kind of tRNA for each amino acid, for a total of at least 32 tRNAs
• some tRNA recognize more than one codon
• Eight or more of the nucleotide residues have modified bases and sugars, methylated derivatives
• some have a guanylate (pG) residue at the 5’ end
• all have the trinucleotide sequence CCA(3’) at the 3’ end
• When drawn in two dimensions
  
  • the hydrogen-bonding pattern of all tRNAs forms a cloverleaf structure with four arms
• In three dimensions, a tRNA has the form of a twisted L
• longer tRNAs have a short fifth arm
• Extra nucleotides occur in the extra arm or in the D arm

Structure

• amino acid arm
  
  • carries a specific amino acid esterified by its carboxyl group to the 2’ or 3’-hydroxyl group of the A residue at the 3’ end of the tRNA
• anticodon arm contains the anticodon
  
  • anticodon loop, contains 7 unpaired nucleotides
• D arm
  
  • contains the nucleotide dihydrouridine (D)
  • contains two or three D (5,6-dihydrouridine) residues
  • contribute with overall folding of tRNA
  • In some tRNAs, the D arm has only three hydrogen-bonded base pairs
• TψC arm contains
  
  • ribothymidine (T), not usually present in RNAs,
  • pseudouridine (ψ), which has an unusual C–C bond between the base and ribose
  • contribute with overall folding of tRNA
  • interacts with the largesubunit rRNA

Question 4

Stage 1: Activation of tRNA

Answer 4

• aminoacyl-tRNA synthetases esterify the 20 amino acids to their corresponding tRNAs
• Each synthetases is specific for one amino acid and one or more corresponding tRNAs
• Most organisms have one synthetase for each amino acid
• two classes based on differences in primary and tertiary structure and in reaction mechanism
• evidence that the two classes share a common ancestor
• PDF pg. 1150, Table 27-7

Aminoacylation of tRNA by aminoacyl-tRNA synthetases

• occurs in two steps
• irreversible
• an enzyme-bound intermediate, aminoacyl adenylate (aminoacyl-AMP), is formed
• aminoacyl group is transferred from enzyme-bound aminoacyl-AMP to its corresponding specific tRNA
  
  • mechanism of this step is different for each class of aminoacyl-tRNA synthetases
  • class I
    
    • the aminoacyl group is transferred initially to the 2’-hydroxyl group of the 3’-terminal A residue
    • then to the 3’-hydroxyl group by a transesterification reaction
  • class II
    
    • aminoacyl group is transferred directly to the 3’-hydroxyl group of the terminal adenylate
• ester linkage between amino acid and tRNA has a highly negative standard free energy of hydrolysis
  
  • ΔG’º = -29 kJ/mol
  • pyrophosphate formed in the activation reaction undergoes hydrolysis to phosphate by inorganic pyrophosphatase
  • two high-energy phosphate bonds are expended for each amino acid molecule activated
• PDF pg. 1151, MECHANISM FIGURE 27–19

Proofreading by Aminoacyl-tRNA Synthetases

• identity of the amino acid attached to a tRNA is not checked on the ribosome
• enzyme specificity is limited by the binding energy available from enzyme-substrate interaction
• if available binding interactions do not provide sufficient discrimination between two substrates, the necessary specificity can be achieved by substrate-specific binding in two successive steps
• first filter
  
  • initial binding of amino acid to the Synthetases and its activation to aminoacyl-AMP
• second filter
  
  • binding of any incorrect aminoacyl-AMP products are placed in a separate proofreading active site
  • there, they are hydrolized to the amino acid and AMP
• third filter
  
  • most synthetases can hydrolyze the ester linkage between amino acids and tRNAs in the aminoacyl-tRNAs
  • This hydrolysis is greatly accelerated for incorrectly charged tRNAs
• synthetases that activate amino acids with no close structural relatives (Cys-tRNA synthetase) have little or no proofreading activity
  
  • the active site for aminoacylation can sufficiently discriminate between the proper & incorrect substrate
• error rate of protein synthesis
  
  • 1 mistake per 10⁴ amino acids incorporated
  • not nearly as low as that of DNA replication
  • flaws in a protein are eliminated when the protein is degraded and are not passed on to future generations
  • have less biological significance
  • degree of fidelity in protein synthesis is sufficient to ensure that most proteins contain no mistakes

Interaction between an Aminoacyl-tRNA Synthetase and a tRNA: A “Second Genetic Code”

• Some nucleotides are conserved in all tRNAs and therefore cannot be used for discrimination
• Other nucleotides are known recognition points for one or more aminoacyl-tRNA synthetases
  
  • concentrated in the amino acid arm and the anticodon arm, including the nucleotides of the anticodon itself
  • also located in other parts
• Structural features other than sequence are important for recognition by some of the synthetases

Question 5

Stage 2: Protein Synthesis - Initiation

Answer 5

APE sites

• aminoacyl (A) site
  
  • bind to aminoacyl-tRNAs
• peptidyl (P) site
  
  • bind to aminoacyl-tRNAs
• exit (E) site
  
  • binds to uncharged tRNAs
• In bacteria
  
  • 30S and the 50S subunits contribute to the characteristics of the A and P sites
  • E site is largely confined to the 50S
• In Eukaryotic
  
  • ribosomes do not have an E site
  • uncharged tRNAs are expelled directly from the P site

Initiation Codon

• Protein synthesis begins at the amino-terminal end and proceeds by the stepwise addition of amino acids to the carboxyl-terminal end
• AUG initiation codon specifies an amino-terminal methionine residue
• there are 2 tRNAs for methionine
  
  • one used for the initiation codon and the other for internal positions in the polypeptide
• In bacteria
  
  • tRNA^Met
  • tRNA^fMet
    
    • amin acid used is Nformylmethionine (fMet)
    • methionine is attached to tRNA^fMet by the Met-tRNA synthetase
    • transformylase transfers a formyl group from N¹⁰-formyltetrahydrofolate to the amino group of the Met residue
      
      • specific for Met residues attached to tRNA^fMet
      • does not act on tRNA^Met
    • Addition of the N-formyl group prevents fMet from entering interior positions in a polypeptide
• in eukaryotes
  
  • polypeptides begin with a Met residue instead of fMet
  • cell uses a specialized initiating tRNA
  • Polypeptides synthesized by mitochondrial and chloroplast begin with fMet
• Dintzis experiment: PDF pg 1158, Figure 27-24

Bacterial Initiation

• ​30S ribosomal subunit binds two initiation factors, IF-1 and IF-3
  
  • IF-1 binds at A site and prevents tRNA binding at this site during initiation
  • IF-3 prevents the 30S and 50S subunits from combining
• mRNA then binds to the 30S subunit
• Shine-Dalgarno sequence base-pairs with a complementary pyrimidine-rich sequence near the 3’ end of the 16S rRNA of the 30S
  
  • consensus sequence
  • four to nine purine residues
  • 8 to 13 bp to the 5’ (upstream) side of the initiation codon
• Shine-Dalgarno binding positions the initiating (5’)AUG sequence at the P site
  
  • initiating (5’)AUG is distinguished from other methionine codons by its proximity to the Shine-Dalgarno sequence
• GTP-bound IF-2
• initiating fMet-tRNA^fMet binds to the P site
  
  • can only bind to this site
  • only tRNA that can do this
• anticodon pairs with initiation codon in P site
• 50S attaches and GTP bound to IF-2 is hydrolyzed to GDP and Pi,
• GDP and Pi are released
• all 3 initiation factors depart
• 70s ribosome, initiation complex, is complete
• correct binding of fMet-tRNA^fMet ​to intiation codon depends on
  
  • Shine-Dalgarno sequence in the mRNA binding to 16S rRNA
  • ribosomal P site and fMet-tRNAfMet binding
  • codon-anticodon interaction
• alternate initiation codons
  
  • E. coli, AUG is the start codon in 91% of the genes, GUG (7%) and UUG (2%)

Eukaryotic Initiation

• ​similar to bacterial cells
• have at least 12 initiation factors
• eIF1A and eIF3 bind the 40S subunit in step
  
  • eIF1A and eIF3 are homologs of the bacterial IF-1 and IF-3
  • eIF1A blocks A site
  • eIF3 stops premature joining of large/small ribosomal subunits
• factor eIF1 binds to the E site
• GTP bound to eIF2 binds to charged initiator tRNA
• GTP-IF2-tRNA complex, eIF5 and eIF5B binds to the 40S ribosomal subunit creating the 43S preinitiation complex
• mRNA binds to eIF4F complex
  
  • mediates binding with the 43S preinitiation complex
  • eIF4E: binds to the 5’ cap of mRNA
  • eIF4A: ATPase and RNA helicase
  • eIF4G: a linker protein
• eIF4G bind to eIF3 (43S preinitiation complex) and eIF4E (in eIF4F complex) creating the first link between mRNA and 43S preinitiation complex
• eIF4G bind poly(A) binding protein (PABP) at the 3’ end of the mRNA
  
  • circulerizes the mRNA
  • facilitates translational regulation of gene expression
• addition of mRNA and its factors creates a 48S complex
• 48S complex scans the bound mRNA, starting at the 5’ cap, until an AUG codon is found
  
  • scanning process maybe facilitated by e1F4A (RNA helicase) and eIF4B
• 60S ribosomal subunit associates when initiating AUG site is encountered
• eIF5 promotes the GTPase activity of eIF2, producing an eIF2-GDP complex with reduced affinity for the initiator tRNA
• eIF5B protein is homologous to the bacterial IF-2. It hydrolyzes its bound GTP and triggers dissociation of eIF2-GDP and other initiation factors
• 60s subunit binds
• This completes formation of the initiation complex
• PDF pg. 1161, Figure 27-28
• PDF pg. 1162, Table 27-8 Protein factors

Question 6

Stage 3: Protein Synthesis - Elongation

Answer 6

Cells use three steps to add each amino acid residue, and the steps are repeated as many times as there are residues to be added.

In Bacteria

Elongation Step 1: Binding of an Incoming Aminoacyl-tRNA

• incoming aminoacyl-tRNA binds to a complex of GTP-bound EF-Tu forming an aminoacyltRNA–EF-Tu–GTP complex
• aminoacyltRNA–EF-Tu–GTP complex binds to the A site of the 70S initiation complex
• GTP is hydrolyzed and an EF-Tu–GDP complex is released
• EF-Tu–GTP complex is regenerated

Elongation Step 2: Peptide Bond Formation

• N-formylmethionyl group is transferred to the amino group of the second aminoacyl-tRNA in the A site, forming a dipeptidyltRNA
  
  • α-amino group of the amino acid in the A site acts as a nucleophile, displacing the tRNA in the P site to form the peptide bond
  • the enzymatic activity, peptidyl transferase, catalyzed by the 23S rRNA
• both tRNAs bound to the ribosome shift position in the 50S subunit to take up a hybrid binding state
• uncharged tRNA shifts so that its 3’ and 5’ ends are in the E site
• the 39 and 59 ends of the peptidyl tRNA shift to the P site
• anticodons remain in the P and A sites
• PDF pg. 1163, figure 27-30

Elongation Step 3: Translocation

• ribosome moves one codon toward the 3’ end of the mRNA
• movement shifts the anticodon of the dipeptidyl-tRNA (in A site) attached to the second codon of mRNA, from the A site to the P site
• the deacylated tRNA moves from P to E site and is released into the cytosol
• The dipeptidyltRNA is now entirely in the P site, leaving the A site open for the incoming (third) aminoacyl-tRNA
• The third codon of the mRNA now lies in the A site and the second codon in the P site
• Movement requires EF-G (translocase) and the energy provided by hydrolysis of another GTP
  
  • it’s structure mimics EF-Tu–tRNA complex
  • can bind the A site and presumably displace the peptidyl-tRNA
• For each amino acid residue added to the growing polypeptide, two GTPs are hydrolyzed to GDP and Pi
• change in the three-dimensional conformation of the entire ribosome results in its movement
• after translocation, the ribosome is ready for the next elongation cycle
• PDF pg. 1163

In Eukaryotes

• ​similar to bacteria
• eEF1α, eEF1βγ, and eEF2 have functions analogous to those of the bacterial elongation factors EF-Tu, EF-Ts, and EF-G, respectively
• Eukaryotic ribosomes do not have an E site; uncharged tRNAs are expelled directly from the P site

Proofreading

• ​The GTPase activity of EF-Tu contributes to the rate and fidelity
• Both EF-Tu–GTP and EF-Tu–GDP complexes exist for a few milliseconds before they dissociate
• These two intervals provide opportunities for the codon-anticodon interactions to be proofread
• Incorrect aminoacyl-tRNAs dissociate from the A site during one of these periods
• If the GTP analog guanosine 59-O-(3-thiotriphosphate) (GTPγS) is used in place of GTP, hydrolysis is slowed, improving the fidelity, but reducing the rate of protein synthesis
• proofreading mechanism on the ribosome establishes that the proper codon-anticodon pairing has taken place, not that the correct amino acid is attached to the tRNA

Question 7

Stage 4: Protein Synthesis - Temination

Answer 7

• signaled by the presence of one of three termination codons in the mRNA (UAA, UAG, UGA)
• Mutations in a tRNA anticodon that allow an amino acid to be inserted at a termination codon are generally deleterious to the cell

In bacteria

• once a termination codon occupies the A site
• RF-1 recognizes the termination codons UAG and UAA
• RF-2 recognizes UGA and UAA
• Depending on which codon is present, either RF-1 or RF-2 binds the termination codon
  
  • have domains thought to mimic the structure of tRNA so can bind to A site
• peptidyl transferase transfer the growing polypeptide to a water molecule rather than to another amino acid
• This leads to hydrolysis of the ester linkage between the nascent polypeptide and the tRNA in the P site
• polypeptide is released
• mRNA, deacylated tRNA, and release factor leave the ribosome
• RF-3 is thought to release the ribosomal subunit

In Eukaryotes

• a single release factor, eRF, recognizes all three termination codons
• Ribosome recycling leads to dissociation of the translation components
• release factors dissociate from the posttermination complex (with an uncharged tRNA in the P site) and are replaced by EF-G and ribosome recycling factor (RRF; Mr 20,300)
• Hydrolysis of GTP by EF-G leads to dissociation of the 50S subunit from the 30S–tRNA–mRNA complex
• EF-G and RRF are replaced by IF-3, which promotes the dissociation of the tRNA
• mRNA is then released
• The complex of IF-3 and the 30S subunit is then ready to initiate another round of protein synthesis

Question 8

Energy Cost of Fidelity in Protein Synthesis

Answer 8

• Formation of each aminoacyl-tRNA uses two high-energy phosphate groups.
• An additional ATP is consumed each time an incorrectly activated amino acid is hydrolyzed
• A GTP is cleaved to GDP and Pi during the first elongation step, and another during the translocation ste
• hydrolysis of more than four NTPs to NDPs is required for the formation of each peptide bond of a polypeptide
• least 4 X 30.5 kJ/mol = 122 kJ/mol of phosphodiester bond energy to generate a peptide bond
• has a standard free energy of hydrolysis of only about -21 kJ/mol
• The net free-energy change during peptide bond synthesis is thus -101 kJ/mol.

Question 9

Polysomes

Answer 9

• in both eukaryotic and bacterial cells
• Large clusters of 10 to 100 ribosomes that are very active in protein synthesis
• a mRNA between adjacent ribosomes in the cluster is being translated simultaneously by many closely spaced ribosomes
• In bacteria Ribosomes begin translating the 5’ end of the mRNA before transcription is complete

Question 10

Stage 5: Protein Synthesis - Posttranslational Modifications

Answer 10

Amino-Terminal and Carboxyl-Terminal Modifications

• The first residue inserted in all polypeptides is N-formylmethionine (in bacteria) or methionine (in eukaryotes)
• the formyl group, the amino-terminal Met residue, and often additional amino-terminal (in some cases, carboxylterminal) residues may be removed

Loss of Signal Sequences

• signal sequences are 15 to 30 residues at the amino-terminal end of some proteins
• they play a role directing the protein to its ultimate destination
• these are eventually removed by specific peptidases

Modification of Individual Amino Acids

• hydroxyl groups of certain Ser, Thr, and Tyr residues of some proteins are enzymatically phosphorylated by ATP
• the phosphate groups add negative charges
• phosphorylation-dephosphorylation cycles regulate the activity of many enzymes and regulatory proteins
• Extra carboxyl groups may be added to Glu of some proteins
• In other proteins, the carboxyl groups of some Glu residues undergo methylation, removing their negative charge

Attachment of Carbohydrate Side Chains

• carbohydrate side chains of glycoproteins are attached covalently during or after synthesis of the polypeptide
• some glycoproteins, the carbohydrate side chain is attached enzymatically to Asn residues (N-linked oligosaccharides), in others to Ser or Thr residues (O-linked oligosaccharides)
• Many proteins that function extracellularly, as well as lubricating proteoglycans, contain oligosaccharide side chains

Addition of Isoprenyl Groups A

• some eukaryotic proteins are added groups derived from isoprene (isoprenyl groups)
• A thioether bond is formed between the isoprenyl group and a Cys residue of the protein
  
  • helps to anchor the protein in a membrane
• The isoprenyl groups are derived from pyrophosphorylated intermediates of the cholesterol biosynthetic pathway (farnesyl pyrophosphate)

Addition of Prosthetic Groups

• acetyl-CoA carboxylase
• heme group of hemoglobin or cytochrome c

Proteolytic Processing

• Many proteins are initially synthesized as large, inactive precursor polypeptides that are proteolytically trimmed to form their smaller, active forms

Formation of Disulfide Cross-Links

• After folding some proteins form intrachain or interchain disulfide bridges between Cys residues
• common in proteins to be exported from cells
• cross-links help to protect the native conformation of the protein molecule from denaturation in the extracellular environment

Question 11

Protein Synthesis Inhibition by Antibiotics and Toxins

Answer 11

• Protein synthesis is the primary target of many naturally occurring antibiotics and toxins
• Puromycin
  
  • in bacteria
  • made by the mold Streptomyces alboniger
  • structure is very similar to the 3’ end of an aminoacyltRNA
  • binds to A site
  • participates in peptide bond formation, producing peptidylpuromycin
  • because puromycin resembles only the 3’ end of the tRNA, it does not engage in translocation and dissociates from the ribosome
  • This prematurely terminates polypeptide synthesis
• Tetracyclines
  
  • in bacteria
  • blocks A site
• Chloramphenicol
  
  • in bacteria
    
    • mitochondrial and chloroplast too
    • does not affect cytosolic protein synthesis in eukaryotes
• Cycloxheximide
  
  • in eukaryotes
  • blocks the peptidyl transferase of 80S
  • but not that of 70S bacterial (and mitochondrial and chloroplast) ribosomes
• Streptomycin
  
  • a basic trisaccharide
  • causes misreading of the genetic code (in bacteria) at relatively low concentrations
  • inhibits initiation at higher concentrations
• Diphtheria toxin
  
  • toxic to mammals
  • catalyzes the ADP-ribosylation of a diphthamide (a modified histidine) residue of eukaryotic elongation factor eEF2, thereby inactivating it
• Ricin
  
  • an extremely toxic protein of the castor bean
  • inactivates the 60S subunit of eukaryotic ribosomes by depurinating a specific adenosine in 23S rRNA

Question 12

Protein Targeting and Degradation

Answer 12

• ​Signal sequece
  
  • directs a protein to its appropriate location
  • is removed during transport or after the protein has reached its final destination
  • In proteins slated for transport into mitochondria, chloroplasts, or the ER, the signal sequence is at the amino terminus
  • vary in length from 13 to 36 amino acid residues
  • structure
    
    • about 10 to 15 hydrophobic amino acid residues
    • one or more positively charged residues, usually near the amino terminus, preceding the hydrophobic sequence
    • a short sequence at the carboxyl terminus
      
      • relatively polar
      • typically having amino acid residues with short side chains (especially Ala) at the positions closest to the cleavage site
    • carboxyl terminus of the signal sequence has a cleavage site
• Degradation of proteins no longer needed by the cell also relies largely on a set of molecular signals embedded in each protein’s structure
• focus is now on eukaryotic cells

Proteins destined for secretion, integration in plasma membrane, or inclusion in lysosomes

• signal sequence appears early in the synthetic process
  
  • because it is at the amino terminus, and it’ synthesized first
• the signal sequence and ribosome are bound by the signal recognition particle (SRP)
  
  • a rod-shaped complex containing a 300 nucleotide RNA and 6 different proteins
  • One protein binds to the signal sequence
  • This inhibits elongation by sterically blocking the entry of aminoacyltRNAs and inhibiting peptidyl transferase
  • Another protein subunit binds and hydrolyzes GTP
  • The SRP receptor is a heterodimer of α and β
• SRP then binds GTP
• elongation of polypeptide halts when it is about 70 amino acids long
• now the signal sequence has completely emerged from the ribosome
• GTP-bound SRP directs ribosome & mRNA to GTP-bound SRP receptors in the cytosolic face of the ER
• the polypeptide is delivered to a peptide translocation complex in the ER
  
  • which may interact directly with the ribosome
• hydrolysis of GTP in both SRP and the SRP receptor disassociates SRP
• Elongation of the polypeptide resumes
• ATP-driven translocation complex feeding the growing polypeptide into the ER lumen
• when protein synthesis is complete, signal sequence is removed by a signal peptidase within the ER lumen
• ribosome dissociates and is recycled
• Glycosylation
  
  • In the ER lumen, newly synthesized proteins are further modified
  • are folded, disulfide bonds formed, and many proteins glycosylated to form glycoproteins
  • in many glycoproteins the linkage to their oligosaccharides is through Asn residues
  • A 14 residue core oligosaccharide is built up in a stepwise fashion
  • its transferred from a dolichol phosphate donor molecule to certain Asn residues in the protein
  • The transferase is on the lumenal face
  • After transfer, the core oligosaccharide is trimmed and elaborated in different ways on different proteins, but all N-linked oligosaccharides retain a pentasaccharide core derived from the original 14 residue oligosaccharide
  • tunicamycin, mimics the structure of UDP-N-acetylglucosamine and blocks the first step of the process
  • A few proteins are O-glycosylated in the ER, but most O-glycosylation occurs in the Golgi complex or in the cytosol (for proteins that do not enter the ER)
• modified proteins are moved to a variety of intracellular destinations
• Proteins travel from the ER to the Golgi complex in transport vesicles
• In the Golgi complex, oligosaccharides are O-linked to some proteins, and N-linked oligosaccharides are further modified
• Golgi complex also sorts proteins and sends them to their final destinations.
• The processes that segregate proteins must distinguish among these proteins on the basis of structural features
  
  • PDF pg. 1173, pink highlight, for an example of hydrolase

Proteins destined for mitochondria, chloroplasts

• pathways that target proteins to mitochondria and chloroplasts also rely on amino-terminal signal sequences
• mitochondrial and chloroplast pathways begin only after a precursor protein has been completely synthesized and released from the ribosome
• proteins are bound by cytosolic chaperone proteins and delivered to receptors on the exterior surface of the target organelle
• Specialized translocation mechanisms then transport the protein to its final destination in the organelle
• signal sequence is removed

Proteins destined for nucleus

• A variety of nuclear proteins (rRNA, RNA and DNA polymerases, histones, topoisomerases, proteins that regulate gene expression, and so forth) are synthesized in the cytosol and imported into the nucleus for assemply
• completed subunits are then exported back to the cytosol
• modulated by a complex system of molecular signals and transport proteins
• nuclear localization sequence (NLS)
  
  • signal sequence that targets a protein to the nucleus
  • not removed after the protein arrives at its destination
  • may be located almost anywhere along the primary sequence of the protein
  • can vary considerably
  • consist of four to eight amino acid residues and include several consecutive basic (Arg or Lys) residues
• Nuclear importation is mediated by a proteins that cycle between the cytosol and the nucleus
  
  • importin α
  • importin β
  • GTPase known as Ran
• A heterodimer of importin α and β functions as a soluble receptor for proteins targeted to the nucleus
  
  • α subunit binds NLS-bearing proteins in the cytosol
• NLS-bearing protein and the importin docks at a nuclear pore and is translocated through the pore into the nucleus by an energy-dependent mechanism
• Importin β is bound by Ran and by CAS (cellular apoptosis susceptibility protein) and separated from the NLS-bearing protein.
• Importin α-Importin β-Ran-CAS complex are exported from nucleus
• Ran hydrolyzes GTP in the cytosol to release the importins
• Ran-GDP binds to nuclear transport factor 2 (NTF2) and is brought back to the nucleus
• Inside the nucleus, the GDP bound to Ran is replaced with GTP by Ran guanosine nucleotide–exchange factor (RanGEF)

Proteins destined for the cytosol

• simply remain where they are synthesized.

Bacteria

• can target proteins to their inner or outer membranes, periplasmic space, or to the extracellular medium
• use signal sequences at the amino terminus of the proteins like on eukaryotic proteins
• Following translation, a protein to be exported folds slowly
  
  • aminoterminal signal sequence impedes folding
• chaperone protein SecB binds to the protein’s signal sequence or other features of its incompletely folded structure
• protein is then delivered to SecA
  
  • protein associated with the inner surface of the plasma membrane.
  • acts as both a receptor and a translocating ATPase
• SecB is released, and SecA inserts itself into the membrane
• forcing about 20 amino acid residues of the protein to be exported through the translocation complex
  
  • made up of SecY, E, and G
• Hydrolysis of an ATP by SecA provides the energy for a conformational change that causes SecA to withdraw from the membrane, releasing the polypeptide
• SecA binds another ATP, and the next stretch of 20 amino acid residues is pushed across the membrane through the translocation complex
• process continues until the entire protein has passed through and is released to the periplasm
• electrochemical potential across the membrane also provides some of the driving force required for protein translocation
• An exported protein is pushed through the membrane by a SecA protein located on the cytoplasmic surface
• some proteins follow an alternative pathway that uses signal recognition and receptor proteins homologous to components of the eukaryotic SRP and SRP receptor

Question 13

Endocytosis

Answer 13

• Some proteins are imported into eukaryotic cells from the surrounding medium
  
  • LDL, transferrin, peptide hormones
• clathrin pathway
  
  • proteins bind to receptors in invaginations of the membrane called coated pits
  • pits are coated on their cytosolic side with a lattice of the protein clathrin, which forms closed polyhedral structures
  • The clathrin lattice grows as more receptors are occupied by target proteins
  • Eventually, a complete membrane bounded endocytic vesicle is pinched off the plasma membrane with the aid of the large GTPase dynamin, and enters the cytoplasm
  • clathrin is quickly removed by uncoating enzymes
  • the vesicle fuses with an endosome
  • ATPase activity in the endosomal membranes reduces the pH therein, facilitating dissociation of receptors from their target proteins
• caveolin
  
  • caveolin causes invagination of patches of membrane containing lipid rafts associated with certain types of receptors
  • makes use of the GTPase dynamin to pinch vesicles from the plasma membrane
  • These endocytic vesicles then fuse with caveolin-containing internal structures, caveosomes
  • there the imported molecules are sorted and redirected to other parts of the cell
  • caveolins recycled to the membrane surface
• Some pathways do not use clathrin or caveolin; some of these make use of dynamin and some do not
• PDF pg 1177 Figure 27-45

Question 14

Protein Degredation

Answer 14

• Protein degradation prevents the buildup of abnormal or unwanted proteins and permits the recycling of amino acids
• two systems
  
  • selective ATP-dependent cytosolic systems
  • in vertebrates, operating in lysosomes, recycles the amino acids of membrane proteins, extracellular proteins, and proteins with characteristically long half-lives
• In E. coli
  
  • many proteins are degraded by an ATPdependent protease called Lon
  • protease is activated in the presence of defective proteins or those slated for rapid turnover
  • two ATP molecules are hydrolyzed for every peptide bond cleaved
  • Once a protein has been reduced to small inactive peptides, other ATP-independent proteases complete the degradation process
• ubiquitin
  
  • protein occurs throughout the eukaryotic kingdoms
  • 76 amino acid residues
  • covalently linked to proteins slated for destruction via an ATP-dependent pathway
  • involves three separate types of enzymes
    
    • E1 activating enzymes
    • E2 conjugating enzymes
    • E3 ligases
  • Ubiquitinated proteins are degraded by a large complex known as the 26S proteasome
    
    • consists of two copies each of at least 32 different subunits
    • contains two main types of subcomplexes, a barrel-like core particle and regulatory particles on either end of the barrel
    • The two subassemblies are the 20S core particle and the 19S regulatory particle, or cap
    • 20S core particle
      
      • consists of four stacked rings arranged to form a barrel-like structure
      • outer rings each have seven different α subunits
      • inner rings from seven β subunits
        
        • 3 rings have protease activities with different substrate specificities.
      • in the core the proteins are are degraded to peptides of 3 to 25 amino acid residues.
    • 19S regulatory particle
      
      • forms a cap on each end of the core particle
      • contains approximately 18 subunits,
      • some recognize and bind ubiquitinated proteins, unfolds them, and translocates them into the core particle
      • Six are AAA1 ATPases
        
        • unfold the ubiquitinated proteins and translocates them into the core particle for degradation
      • regulatory particle deubiquitinates the proteins as they are degraded in the proteasome
    • can be effectively “accessorized,” with regulatory complexes changing with changing cellular conditions
  • Most cells have additional regulatory complexes that can replace the 19S particle
    
    • do not hydrolyze ATP and do not bind to ubiquitin
  • we don’t understand all the signals that trigger ubiquitination
    
    • one is the identity of the first residue that remains after removal of the amino-terminal Met residue, and any other posttranslational proteolytic processing of the amino-terminal end
  • The E1, E2, and E3 components of the ubiquitination pathway are large families of proteins
  • Different E1, E2, and E3 enzymes exhibit different specificities for target proteins and thus regulate different cellular processes