1. Chapter 22
Gene Expression: II. Protein Synthesis and Sorting

2. Gene Expression: II. Protein Synthesis and Sorting
For some genes, the RNA transcript is the final product
But for many other genes, the ultimate product is protein
mRNAs encode instructions for translation, the process of assembling amino acids into a polypeptide 2

3. Translation: The Cast of Characters
Ribosomes carry out the process of polypeptide synthesis
tRNA molecules align the amino acids in the correct order
Aminoacyl-tRNA synthetases attach amino acids to their appropriate tRNA molecules 3

4. The cast of characters
mRNA molecules encode the amino acid sequence information
Protein factors facilitate some of the steps of translation 4

5. The Ribosome Carries Out Polypeptide Synthesis
Ribosomes are particles made of rRNA and protein
In eukaryotes, they are found free in the cytoplasm, and bound to ER and the outer nuclear envelope
In prokaryotes, the ribosomes are smaller 5

6. Ribosome structure
Ribosomes are built from dissociable subunits, the large and small subunits
Bacterial ribosomes are sensitive to different inhibitors of protein synthesis and are composed of fewer proteins and smaller and fewer RNA molecules 6

7. Figure 22-1A

8. Figure 22-1B

9. Table 22-1

10. Ribosomes, machines in polypeptide synthesis
rRNA performs many of the key functions of ribosomes
Ribosomes have four important sites: the mRNA binding site, the A (aminoacyl) site, the P (peptidyl) site, and an E (exit) site

11. Figure 22-2

12. Transfer RNA Molecules Bring Amino Acids to the Ribosome
A tRNA molecule is an adaptor that binds both a specific amino acid and the mRNA sequences that specify the amino acid
Each tRNA is linked to its amino acid by an ester bond
tRNAs are named for the amino acids attached to them, e.g., tRNAAla for alanine

13. Figure 22-3A

14. Figure 22-3B

15. Video: A Stick-and-Ribbon Rendering of a tRNA

16. tRNAs
tRNAs attached to an amino acid are said to be aminoacyl tRNAs—the tRNA is called charged, whereas the amino acid is called activated
Each tRNA recognizes codons in mRNA due to their complementarity to the anticodon in the tRNA
Some tRNA molecules recognize more than one codon

17. The wobble hypothesis
mRNA and tRNA line up on the ribosome in a way that permits flexibility or wobble in the pairing between the third base of the codon and the corresponding base of the anticodon
This is the wobble hypothesis, which allows for some unexpected base pairing

18. Figure 22-4

19. Inosine
The nucleotide inosine is able to pair with U, C, or A and is often found in the wobble position of the anticodon
This allows for several codons to specify one amino acid 19

20. Aminoacyl-tRNA Synthetases Link Amino Acids to the Correct Transfer RNAs
Cells typically have 20 different aminoacyl-tRNA synthetases to attach each amino acid to the appropriate tRNA
There is one aminoacyl-tRNA synthetase for each amino acid
Cells with nontraditional amino acids have special tRNAs and aminoacyl-tRNA synthetases for these amino acids, too

21. Aminoacyl-tRNA synthesis
Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to the tRNAs via an ester bond, using ATP hydrolysis

22. Aminoacyl-tRNA synthesis
Both the anticodon and the 3 end of the tRNA are needed to specify the correct amino acid
After addition of an amino acid the synthetases proofread the final product to ensure the correct amino acid was added
It is the tRNA that then recognizes the appropriate codon in mRNA

23. Figure 22-5, Steps 1, 2

24. Figure 22-5, Steps 3, 4

25. Messenger RNA Brings Polypeptide Coding Information to the Ribosome
The sequence of codons in mRNA directs the order of amino acids in the polypeptide
mRNA is exported to the cytoplasm via binding to proteins that contain nuclear export signals (NES)
An untranslated sequence at the 5 end of the message precedes the start codon, the first to be translated (usually AUG)

26. Coding information
There is an untranslated region at the 3 end of the mRNA that follows the stop codon, which signals the end of translation
The stop codon may be UAG, UAA, or UGA
5 and 3 untranslated regions vary in length and are essential for mRNA function
mRNAs also have a 5 cap and 3 poly(A) tail

27. Figure 22-6

28. Figure 22-6A

29. Figure 22-6B

30. Eukaryotic mRNAs are monocistronic
Most mRNAs in eukaryotes are monocistronic, meaning they encode just one polypeptide
In bacteria and archaea, some are polycistronic, encoding several polypeptides, usually with related functions
These polycistronic transcription units are called operons

31. Protein Factors Are Required for the Initiation, Elongation, and Termination of Polypeptide Chains
Each part of translation requires certain protein factors

32. The Mechanism of Translation
Translation is an ordered, stepwise process that begins at the N-terminus of the polypeptide and adds amino acids to the growing chain until the C-terminus is reached
The mRNA is read in the 5 to 3 direction
Translation is divided into three stages: initiation (1), elongation (2), and termination (3)

33. Figure 22-7

34. The Initiation of Translation Requires Initiation Factors, Ribosomal Subunits, mRNA, and Initiator tRNA
Initiation of translation differs in bacteria and eukaryotes

35. Bacterial Initiation
Initiation of translation in bacteria can be divided into three steps
Three initiation factors (IF1, IF2, and IF3) bind to the small ribosomal subunit, with GTP bound to IF2 (Step 1)
mRNA and the tRNA carrying the first amino acid bind to the small subunit (Step 2)

36. Figure 22-8

37. Bacterial initiation
The mRNA has a ribosome binding site, also called the Shine-Dalgarno sequence
The purines in the mRNA sequence form complementary base pairs with RNA (the mRNA-binding site) in the small subunit
This allows for correct binding of the mRNA to the small ribosome subunit

38. Bacterial initiation
The binding of mRNA to the small subunit places the start codon at the ribosome’s P site
A special methionine, N-formylmethionine (fMet) is used to initiate translation; only its carboxyl group can bind to another amino acid
tRNAfMet is an initiator tRNA

39. Figure 22-9

40. Bacterial initiation
The initiator tRNA binds the P site of the small subunit by action of IF2 (plus GTP)
The small ribosomal subunit, with the initiation factors, mRNA and tRNAfMet, is called the 30S initiation complex
IF3 is released and the 30S initiation complex binds the large subunit, to form the 70S initiation complex (Step 3)

41. Activity: Initiation of Translation

42. Eukaryotic Initiation
The start codon in eukaryotes and archaea specifies methionine rather than N-formylmethionine
The initiation factors are called eIFs; there are about a dozen of these
eIF2 (with GTP attached) binds to the initiator tRNAMet before the tRNA then binds the small ribosomal subunit 42

43. Eukaryotic initiation (continued)
Other initiation factors also bind the small subunit (e.g., eIF1A)
The resulting complex then binds to the 5 end of the mRNA, recognizing the 5 cap with the assistance of eIF4F
In some cases the complex binds an internal ribosome entry sequence instead of the 5 cap 43

44. Eukaryotic initiation (continued)
After binding the mRNA, the small ribosomal subunit (including the initiator tRNA) scans along the transcript and begins translation at the first AUG
Nucleotides to either side of the start codon are involved in the recognition; e.g., a common start sequence is ACCAUGG, called a Kozak sequence
After the initiator tRNA is base-paired with the start codon the large subunit joins the complex, facilitated by GTP hydrolysis 44

45. Chain Elongation Involves Sequential Cycles of Aminoacyl tRNA Binding, Peptide Bond Formation, and Translocation
Once initiation has been completed a polypeptide chain is synthesized
Amino acids are added in sequence to the growing chain (elongation)
Elongation involves a repetitive cycle of three steps 45

46. Binding of Aminoacyl tRNA
As elongation begins, the start codon is located at the P site and the next codon is at the A site
Elongation begins as a tRNA with an anticodon complementary to the second codon binds the A site (1)
This requires two elongation factors, EF-Tu and EF-Ts, and GTP hydrolysis

47. Figure 22-10

48. Binding of aminoacyl tRNA (continued)
Elongation factors don’t recognize particular anticodons, so all types (except initiator tRNAs) are brought to the A site
Only those with an anticodon complementary to the codon stay at the A site long enough for GTP hydrolysis to take place
The final error rate in translation is at most 1/10,000

49. Peptide Bond Formation
Once the aminoacyl tRNA is bound to the A site, a peptide bond forms between the amino group of the amino acid at the A site and the carboxyl group of the amino acid at the P site
The growing peptide chain is transferred to the tRNA at the A site (2)
No ATP or GTP hydrolysis is required for this step

50. rRNA catalyzes peptide bond formation
For many years, it was thought that the protein peptidyl transferase catalyzed peptide bond formation
However, Noller and colleagues showed that rRNA contains the catalytic activity; in the case of bacteria, it is the 23S RNA

51. Translocation
After the peptide bond forms, the mRNA advances to bring the next codon into the proper position
During this translocation, the peptidyl tRNA moves from the A to the P site, and the empty tRNA moves to the E site
Hydrolysis of GTP bound to EF-G triggers a conformational change that completes these movements (3)

52. Activity: Elongation of the Polypeptide

53. Translocation (continued)
Once the next mRNA codon reaches the A site, the ribosome is now set to receive the next aminoacyl tRNA
The elongation cycle repeats and the amino terminal of the growing polypeptide passes out of the ribosome through an exit tunnel in the 50S subunit
Here molecular chaperones assist its folding 53

54. Termination of Polypeptide Synthesis Is Triggered by Release Factors That Recognize Stop Codons
Codons are read on the mRNA one after the other, until a stop codon arrives at the A site
Stop codons are recognized by protein release factors, rather than tRNAs
Once release factors bind to the stop codons, translation is terminated through release of the completed polypeptide 54

55. Figure 22-11

56. Activity: Translation Termination

57. Polypeptide Folding Is Facilitated by Molecular Chaperones
Proteins must fold into their correct three-dimensional shapes before they can function
Protein folding is usually facilitated by proteins called molecular chaperones; often several are required, acting in sequence
Chaperones bind polypeptide chains during the early stages of folding 57

58. Molecular chaperones
If folding goes awry, chaperones can sometimes rescue the proteins and fold them properly
Alternatively, improperly folded proteins may be destroyed
Some kinds of incorrectly folded proteins bind to each other and form insoluble aggregates within and between cells

59. Molecular chaperones (continued)
Two of the most widely occurring chaperone families are Hsp70 and Hsp60
Members of each family function differently but both involve ATP-dependent cycles of binding and releasing their protein substrates
Chaperones also perform other functions, such as assembling polypeptides into multisubunit proteins

60. Protein Synthesis Typically Utilizes a Substantial Fraction of a Cell’s Energy Budget
Polypeptide elongation involves hydrolysis of at least four high-energy phosphoanhydride bonds
Assuming each bond has a G°of 7.3 kcal/mol, they represent a free energy input of 29.2 kcal/mol
Additional GTPs are used during formation of the initiation complex, the binding of incorrect aminoacyl tRNAs, and termination

61. A Summary of Translation
Translation converts information in mRNAs into a chain of amino acids linked by peptide bonds
Most messages are read by many ribosomes simultaneously; a cluster of such ribosomes attached to the same mRNA is called a polyribosome
RNA molecules play important roles in translation; mRNA, tRNA, rRNA

62. Mutations and Translation
mRNAs may contain mutant codons that cause errors in the polypeptide chain synthesized
Most codon mutations alter a single amino acid and some (in the third base of a codon) don’t alter the amino acid at all
Mutations that add or remove stop codons or alter the reading frame can severely disrupt translation 62

63. Suppressor tRNA Overcomes the Effects of Some Mutations
Mutations that convert amino acid-coding codons into stop codons, called nonsense mutations, typically lead to incomplete, nonfunctional polypeptides
These mutations are often lethal, but can sometimes be overcome by an independent mutation affecting a tRNA gene
This is called a suppressor tRNA 63

64. Figure 22-12A, B

65. Suppressor tRNAs
Suppressor tRNAs recognize stop codons and insert amino acids, suppressing nonsense mutations
Highly efficient suppressor tRNAs might lead to the production of many abnormal amino acids
However, at the 3 ends of mRNAs, release factors bind stop codons more efficiently than suppressor tRNAs; this limits the effect of the suppressor to internal locations on the mRNA 65

66. Figure 22-12B, C

67. Nonsense-Mediated Decay and Nonstop Decay Promote the Destruction of Defective mRNAs
Without a suppressor tRNA, a nonsense mutation will cause premature termination of translation and an incomplete polypeptide chain
Eukaryotic cells use nonsense-mediated decay to destroy mRNAs containing premature stop codons
In mammals, the exon junction complex (EJC) is used to detect premature stop codons 67

68. The EJC and nonsense mutations
A multiprotein EJC is deposited wherever an intron is removed from pre-mRNA, so each spliced mRNA has at least one complex bound to it
If an mRNA contains a stop codon prior to the final EJC, translation is terminated
EJCs still associated with the tRNA target it for degradation 68

69. The fate of mRNAs with no stop codon
In eukaryotes, translation is stalled when a ribosome reaches the end of a transcript that lacks a stop codon
An RNA degrading enzyme binds the empty A site of the ribosome and degrades the defective mRNA via nonstop decay
In bacteria, a transfer messenger RNA (tmRNA) binds the A site and directs addition of amino acids that target the protein for destruction 69

70. Posttranslational Processing
After polypeptide chains are synthesized, they often must undergo posttranslational modification before they can perform their functions
In bacteria, the N-formyl methionine at the N-terminus is removed
In eukaryotes, the methionine at the N-terminus is released, too 70

71. Posttranslational processing (continued)
Sometimes whole blocks of amino acids are removed from the polypeptide, for instance certain enzymes synthesized as inactive precursors
These are activated by removal of sequences from one end of the protein
Transport of proteins may require removal of a signal sequence and some have internal amino acids that must be removed 71

72. Posttranslational processing (continued)
Other common processing events include chemical modification of amino acids—methylation, phosphorylation, acetylation
Some proteins undergo a rare process called protein splicing
Similar to RNA splicing, protein sequences called inteins are removed and the remaining sequences called exeins are spliced together 72

73. Protein Targeting and Sorting
As proteins are synthesized, they must be sorted and directed to their final locations
The compartments of eukaryotic cells can be divided into three categories: the endomembrane system; the cytosol; and the mitochondria, chloroplasts, and peroxisomes and interior of the nucleus
Polypeptides are routed to compartments via several mechanisms 73

74. Translation is mainly cytosolic
Up to 10% of ribosomes may reside in the nucleus, translating new mRNAs in a mainly quality control capacity
Most polypeptide synthesis takes place in the cytosol with transcripts leaving the nucleus and associating with free ribosomes
Shortly after translation begins, two pathways for routing the protein products diverge 74

75. Cotranslational import
The first pathway is utilized by ribosomes synthesizing polypeptides destined for export from the cell
These ribosomes become attached to ER membranes early in translation, and polypeptide chains are transferred across the ER membrane as synthesis takes place
This is called cotranslational import 75

76. Figure 22-13A

77. Posttranslational import
An alternative mechanism is employed for polypeptides destined for the cytosol or mitochondria, chloroplasts, peroxisomes, or nuclear interior
After translation is complete, the polypeptides are released from ribosomes and remain in the cytosol, or are taken up by the appropriate organelle
Special targeting signals are required for this posttranslational import 77

78. Figure 22-13B

79. Cotranslational Import Allows Some Polypeptides to Enter the ER as They Are Being Synthesized
Proteins are synthesized on ribosomes that become attached to the ER shortly after the beginning of translation
Labeling experiments show that polypeptides synthesized on rough ER are localized inside the ER lumen
They enter the ER as they are being synthesized 79

80. Figure 22-14

81. The signal hypothesis
Blobel and Sabatini first suggested the signal hypothesis, which proposed that proteins that move into the ER during synthesis possess an intrinsic signal, directing them to the ER
The ER signal sequence directs the mRNA polypeptide complex to the rough ER surface
Milstein and associates found evidence that a 20 amino acid sequence at the N-terminus of the light chain of immunoglobulin G is a signal sequence 81

82. Figure 22-15

83. ER targeting of proteins
Other polypeptides destined for the ER also contain N-terminal sequences to target them to the ER
Proteins containing these signal sequences are referred to as preproteins 83

84. Features of signal sequences
The signal sequences are usually 15–30 amino acids long
They have a positively charged N-terminal region, a central hydrophobic region, and a polar region near where the cleavage from the mature protein will take place 84

85. The Signal Recognition Particle (SRP) Binds the Ribosome-mRNA-Polypeptide Complex to the ER Membrane
Contact with the ER is mediated by a signal recognition particle (SRP), which binds the ER signal sequence of the newly forming polypeptide
Then it binds the ER membrane
The SRP contains both proteins and an RNA 85

86. Cotranslational import
Polypeptide synthesis proceeds until the ER signal sequence has been formed
The SRP binds to the signal sequence and blocks further translation (1)
The SRP binds the ribosome to a structure in the ER membrane called the translocon

87. Figure 22-16, Step 1

88. Cotranslational import (continued)
The translocon is a protein complex that includes an SRP receptor and a ribosome receptor
It also has a pore protein that forms a channel to allow the polypeptide to enter the ER, and a signal peptidase to remove the ER signal sequence

89. Cotranslational import (continued)
The SRP, with its attached ribosome, binds the SRP receptor, allowing the ribosome to bind the ribosome receptor
Next, GTP binds both SRP and the SRP receptor, unblocking translation and causing transfer of the signal sequence to the pore protein (2)

90. Figure 22-16, Step 2

91. Cotranslational import (continued)
The central channels open as the signal sequence is inserted (3)
GTP is hydrolyzed and the SRP is released (4)
As the polypeptide elongates, it passes into the ER lumen where signal peptidase cleaves the signal peptide (6)

92. Figure 22-16, Steps 3–5

93. Cotranslational import (continued)
The signal peptide is quickly degraded in the ER lumen
When polypeptide synthesis is complete, the polypeptide is released into the lumen and the ribosome detaches from the ER membrane
The subunits dissociate and release the mRNA (6)

94. Figure 22-16, Step 6

95. Protein Folding and Quality Control Take Place Within the ER
After polypeptides are released in the ER lumen, they fold into their final shape
An abundant chaperone in the ER lumen is a member of the Hsp70 chaperones, called BiP (binding protein)
BiP binds to hydrophobic regions of polypeptide chains and prevents aggregation of polypeptides with similar regions 95

96. Protein folding and quality control (continued)
BiP prevents interaction between hydrophobic regions of different proteins
Then it releases the polypeptide chain, accompanied by ATP hydrolysis, giving the polypeptide a short opportunity to fold
If it folds correctly, the hydrophobic region is buried in the interior of the molecule; if it folds incorrectly, it interacts with BiP again 96

97. Protein folding and quality control (continued)
Protein folding is often accompanied by formation of disulfide bonds
This is facilitated by protein disulfide isomerase, which acts before the synthesis of the polypeptide is complete
Various combinations of disulfide bonds are tested until the most stable arrangement is reached 97

98. The unfolded protein response
Proteins that repeatedly fail to fold properly activate various quality control mechanisms
One mechanism is the unfolded protein response (UPR), in which sensor molecules in the ER lumen detect the misfolded proteins
The sensors activate signaling pathways that enhance production of proteins needed for folding and degradation 98

99. The ERAD
The ER-associated degradation (ERAD) mechanism recognizes misfolded or unassembled proteins and exports them to the cytosol
Here they are degraded by proteasomes 99

100. Proteins Released into the ER Lumen Are Routed to the Golgi Complex, Secretory Vesicles, Lysosomes, or Back to the ER
Most proteins synthesized on rough ER are glycoproteins
The initial glycosylation takes place in the ER as the polypeptide is being synthesized
In the Golgi complex, further glycosylation and processing of carbohydrate side chains occurs, and the proteins are sorted and distributed to other locations
100

101. Soluble proteins
Soluble proteins move from the Golgi complex to secretory vesicles for secretion from the cell
Those that are not destined for secretion have specific side chains or signal sequences that target them to destinations within the endomembrane system
E.g., many lysosomal enzymes have side chains with mannose-6-phosphate

102. The KDEL sequence
Proteins whose final destination is the ER have a KDEL sequence (Lys-Asp-Glu-Leu) or related sequence
The Golgi complex has a receptor protein that binds the KDEL sequence and delivers the protein back to the ER

103. Stop-Transfer Sequences Mediate the Insertion of Integral Membrane Proteins
The other major group of polypeptides synthesized on rough ER is molecules destined to become integral membrane proteins
The completed polypeptide chain remains embedded in the ER membrane, anchored by one or more -helical transmembrane segments
There are two main mechanisms postulated for this

104. The stop-transfer sequence
The first postulated mechanism involves polypeptides with an ER signal sequence at their N-terminus
This allows an SRP to bind the ribosome-mRNA complex to the ER membrane
Elongation of the polypeptide continues until the hydrophobic transmembrane domain is synthesized

105. The stop-transfer sequence (continued)
The hydrophobic amino acids function as a stop-transfer sequence
The stop-transfer sequence halts translocation of the polypeptide through the ER membrane; as translation continues, the rest of the polypeptide stays on the cytosolic side of the ER membrane
The stop-transfer sequence moves laterally, forming a permanent transmembrane segment

106. Figure 22-17A

107. The start-transfer sequence
The second mechanism involves membrane proteins without a typical signal sequence at the N-terminus
Instead, these proteins have an internal start-transfer sequence that SRP binds and targets to the ER membrane
Then its hydrophobic region functions as a membrane anchor

108. Figure 22-17B

109. The fate of transmembrane proteins
Once the polypeptide has been incorporated into the ER membrane it can remain in place to function as an ER protein
Alternatively it can be transported to other components of the endomembrane system
Transport to the appropriate compartment is carried out by a series of membrane budding and fusion events

110. Posttranslational Import Allows Some Polypeptides to Enter Organelles After They Have Been Synthesized
Proteins destined for the nuclear interior, mitochondrion, chloroplast, or peroxisome are imported into these organelles after completion of translation
These are synthesized on free ribosomes and released into the cytosol
110

111. Nuclear import
Each protein released to the cytosol has localization signals specific to the destination
E.g. import into the nucleus requires nuclear localization signals that target proteins for transport through nuclear pores

112. Importing Polypeptides into Mitochondria and Chloroplasts
Nearly all polypeptides encoded by mitochondrial or chloroplast genes are subunits of multimeric proteins with one or more subunits encoded by nuclear genes
Most mitochondrial and chloroplast polypeptides are synthesized on cytoplasmic ribosome, released into the cytosol, and taken up by the organelle within a few minutes
112

113. The transit sequence
The targeting signal for mitochondrial and chloroplast polypeptides is a transit sequence
It is located at the N-terminus of the polypeptide
Once inside the mitochondrion or chloroplast, the transit sequence is removed by transit peptidase in the organelle, often before transport is complete
113

114. Transport complexes
Polypeptide uptake is mediated by transport complexes in the outer and inner mitochondrial and chloroplast membranes
Mitochondrial transport complexes are called TOM (translocase of the outer mitochondrial membrane) and TIM (translocase of the inner mitochondrial membrane)
In chloroplasts they are called TOC and TIC
114

115. Transport complexes and polypeptide uptake
Uptake is initiated by components of the transport complexes called transit sequence receptors
Once the transit sequence binds its receptor, the polypeptide is translocated across the outer membrane through a pore in the TOM or TOC
If the polypeptide is destined for the interior of the organelle, passage through the TIM or TIC quickly follows
115

116. Figure 22-18

117. Evidence for contact sites
Transport to the interior of the organelle presumably occurs at a contact site where outer and inner membranes lie close together
Evidence for the model comes from EM, which shows sites of close contact, and from cell-free mitochondrial import systems incubated on ice
Polypeptides midway through transport can be seen

118. Figure 22-19

119. Polypeptides are transported in an unfolded state
Polypeptides entering mitochondria and chloroplasts must be unfolded as they cross the membranes
Polypeptides experimentally maintained in a folded state are unable to move across the membrane
To maintain an unfolded state, the polypeptides are bound to chaperone proteins

120. The role of chaperones
Hsp70 class chaperones bind a newly forming polypeptide, keeping it loosely folded (1)
The transit sequence on the polypeptide binds the receptor component of TOM on the outer mitochondrial membrane (2)
Then the chaperones are released, accompanied by ATP hydrolysis as the polypeptide moves into the mitochondrial matrix (3)

121. Figure 22-20

122. The role of chaperones (continued)
When the transit sequence emerges into the matrix, it is removed by transit peptidase (4)
As the remainder of the polypeptide enters the mitochondria, mitochondrial Hsp70 molecules bind to it temporarily; subsequent release of Hsp70 requires ATP hydrolysis (5)
In many cases, Hsp60 chaperones bind to the polypeptide, then, and help it fold fully (6)
122

123. Targeting of Polypeptides to the Proper Compartments Within Mitochondria and Chloroplasts
Proteins to be imported from the cytosol must be targeted to the correct compartment within the organelle
Mitochondria have four compartments: the outer membrane, intermembrane space, inner membrane, and matrix
Chloroplasts have four compartments, too
123

124. Targeting polypeptides to mitochondria and chloroplasts
Given the complexity of mitochondria and chloroplasts, many polypeptides require multiple signals for correct targeting
E.g., targeting to the inner or outer mitochondrial membranes requires signals directing the polypeptide to the mitochondrion, plus a hydrophobic sorting signal to send it to the final destination
124

125. Targeting polypeptides to mitochondria and chloroplasts
Multiple signals are also involved in directing chloroplast polypeptides to their final destinations
E.g. In the stroma the transit sequence required for chloroplast targeting is cleaved, unmasking a hydrophobic thylakoid signal sequence
It targets the polypeptide for the thylakoid membrane or lumen
125