1. Chapter 12 Intracellular Compartments and Protein Sorting
I. The compartmentalization of cells (pages ; figures 12-1 to 12-7; tables to 12-3)
II. The transport of molecules between the nucleus and the cytosol (pages ; figures 12-8 to 12-20)
III. The transport of proteins into mitochondria and chloroplasts (pages ; figures to 12-28)
IV. Peroxisomes (pages ; figures to 12-33)
V. The endoplasmic reticulum (pages ; figures to 12-58)

2. All eucaryotic cells have the same basic set of membrane-enclosed organelles
The major intracellular compartments of an animal cell

5. An electron micrograph of part of a liver cell seen in cross section

6. The topological relationships of membrane-enclosed organelles can be
interpreted in terms of their evolutionary origins
Development of plastids

7. A possible pathway for the evolution of the cell nucleus and the ER

8. A possible pathway for the evolution of mitochondria (and plastids)

9. Topological relationships between compartments
Topologically equivalent compartments are shown in red
The intracellular compartments in eucaryotic cells can be grouped into four distinct families:
the nucleus and the cytosol
ER, Golgi apparatus, endosomes, lysosomes, transport vesicles, and possibly
peroxisomes
mitochondria
(4) plastids (in plants only)

10. Proteins can move between compartments in different ways

11. A simplified “roadmap” of protein traffic

12. Vesicle budding and fusion during
vesicular transport

13. Signal sequences and signal patches direct proteins to the correct
cellular address

15. General properties of signal sequences that direct proteins from the
cytosol to organelles

17. Transport of molecules between the nucleus and the cytosol

18. Nuclear pore complexes perforate the nuclear envelope
Bidirectional traffic occurs continuously between the cytosol and the nucleus

19. The arrangement of nuclear pore complexes in the nuclear envelope

20. Possible paths for free diffusion through the nuclear pore complex

21. Nuclear localization signals direct nuclear proteins to the nucleus

22. with peptides containing nuclear localization signals
Visualizing active import through nuclear pores using gold particles coated
with peptides containing nuclear localization signals

23. Nuclear export works like nuclear import, but in reverse
Nuclear import receptors bind nuclear localization signals and nucleoporins
The majority of nucleocytoplasmic transport cargoes are recognized by a family of soluble transport factors called importins, exportins or transportins, and collectively referred to as karyopherins. The interactions between karyopherins and the NPC occur largely through several nucleoporins containing so-called 'FG domains'.
Nuclear export works like nuclear import, but in reverse

24. The Ran GTPase drives directional transport through nuclear pore complexes
Passage through the pore in either direction seems to be independent of metabolic energy. However, once on the target side, transport is terminated by the asymmetric distribution of the GTP- and GDP-bound forms of the GTPase Ran. The Ran-GTP form is maintained predominantly in the nucleus, where it breaks apart import complexes while stabilizing export complexes. Conversely, hydrolysis of GTP in the cytoplasm produces Ran-GDP and triggers the release of export cargo. It is generally considered that the directionality of transport is conferred, in whole or at least in part, by this differential localization of the two Ran species.

31. Transport of proteins into mitochondria and chloroplasts

32. Protein transport in mitochondria and chloroplasts
- Posttranslational
- Separate translocation complexes in each membrane
- Contact sites
- Require energy
- Amphipathic N-terminal signal sequences that are removed after use

33. Translocation into the mitochondrial matrix depends on a signal sequence ____

34. ____ and protein translocators
The TOM complex is required for the import of all nucleus-encoded mitochondrial proteins.
The TIM23 complex transports some of these proteins into the matrix space; helps insert
transmembrane proteins into the inner membrane.
The TIM22 complex mediates insertion of a subclass of inner membrane proteins.
The OXA complex mediates insertion of inner membrane proteins synthesized in the
mitochondria.

35. Translocator complexes in mitochondria
Translocator complexes in mitochondria. The TOM complex (pink) in the outer membrane, the TIM23 complex (yellow) and the TIM22 complex (light green) in the inner membrane are shown. Ssc1p and Yge1p (Mge1p), peripheral components assisting the function of TIM23 complex (blue) in the matrix, are also included.
Endo, T. et al. J Cell Sci (2003) 116:

36. Translocator complexes for mitochondrial protein import

37. Mitochondrial precursor proteins are imported as unfolded
polypeptide chains

38. Mitochondrial precursor proteins are imported into the matrix
at contact sites that join the inner and outer membranes

39. Protein import by Mitochondria

40. unfolded prior to adding to purified mitochondria.
ATP hydrolysis and a H+ gradient are required for protein import into mitochondria
hsp70 and ATP in the cytosol is not required if the precursor protein is artificially
unfolded prior to adding to purified mitochondria.

41. Functional cooperation of the TOM and TIM complexes
(A) A presequence-containing precursor protein is transferred from the TOM complex to the TIM23 complex. (1) The N-terminal domain of Tim23 tethers the TIM23 complex to the outer membrane. (2) The presequence of the precursor protein reaches the presequence-binding site on the IMS side of the TOM complex (trans site) and is close to Tim50 of the TIM23 complex. (3) ΔΨ facilitates transfer of the presequence from the TOM complex to the TIM23 complex via Tim50 and translocation of the presequence across the inner membrane. (4) Translocation of the entire precursor protein through the TIM23 complex is facilitated by mHsp70 (Ssc1p), in most cases, at the expense of matrix ATP.
Endo, T. et al. J Cell Sci (2003) 116:

42. Functional cooperation of the TOM and TIM complexes
(B) Polytopic inner membrane proteins including AAC are transferred from the TOM complex to the TIM22 complex. (1) AAC enters the import pathway via Tom70. (2) ATP drives translocation of AAC through the TOM channel probably in a loop conformation to bind to the Tim9-Tim10 complex. (3) AAC is transferred to the TIM22 complex via the Tim9/10/12 complex with the aid of ΔΨ. (4) AAC is inserted into the inner membrane by the TIM22 complex and ΔΨ, and forms a dimer.
Endo, T. et al. J Cell Sci (2003) 116:

43. Bacteria and mitochondria use similar mechanisms to insert porins
into their outer membranes
Integration of porins into the outer mitochondrial membrane

44. Transport into the inner mitochondrial membrane and
intermembrane space occurs via several routes

45. Arrangement of targeting sequences
in imported mitochondrial proteins

46. Two signal sequences are required to direct proteins to the thylakoid membrane in
chloroplasts

48. Two pathways for transporting proteins from the cytosol to the thylakoid space

50. “peroxidative” reaction detoxifying various toxic molecules
Peroxisomes are found in all eucaryotic cells and are major sites of oxygen utilization.
They contain one or more enzymes that use molecular oxygen to remove hydrogen atoms from organic compounds and produce H2O2 in the reaction
RH2 + O2
R + H2O2
Catalase utilizes the hydrogen peroxide to oxidize a variety of other substrates by the
“peroxidative” reaction detoxifying various toxic molecules

51. Plasmalogen

52. Two types of peroxisomes found in plant cells

53. A model that explains how peroxisomes proliferate and
how new peroxisomes may arise

54. Import of peroxisomal matrix proteins directed by PTS1 targeting sequence
Step 1: Catalase and most other peroxisomal matrix proteins contain a C-terminal PTS1 uptake-targeting sequence
(-Ser-Lys-Leu-COO¯ or a related sequence) that binds to the cytosolic receptor Pex5. PTS2 is an amino-terminal targeting sequence contained in a protein leader sequence, which is cleaved (?) by a peroxisome-specific protease after import of the proteins into the matrix. Peroxisomal 3-ketoacyl-CoA thiolases are the best example of proteins with PTS2 sequences. The PTS2 consensus sequence in plants is: R XXX (I/L) XXHL.
Step 2: Pex5 with the bound matrix protein interacts with the Pex14 receptor located on the peroxisome membrane.
Step 3: The matrix protein-Pex5 complex is then transferred to a set of membrane proteins that are necessary for
translocation into the peroxisomal matrix by an unknown mechanism. Translocation across the peroxisomal
membrane depends on ATP hydrolysis and the proteins traverse the membrane in a folded conformation.
Step 4: At some point, either during translocation or in the lumen, Pex5 dissociates from the matrix protein and returns
to the cytosol.

56. The ER has a central role in lipid and protein synthesis

57. Co-translational and post-translational protein translocation

58. The rough and smooth ER

59. Membrane-bound ribosomes define the rough ER
The ER captures transmembrane and selected water-soluble proteins
from the cytosol as they are being synthesized

60. The smooth ER, sometimes called transitional ER contain exit
sites from which transport vesicles carrying newly synthesized
proteins and lipids bud off for transport to the Golgi apparatus

61. Rough and smooth regions of ER can be separated by centrifugation

62. The signal hypothesis
Signal sequences were first discovered in proteins imported into the rough ER

67. The signal-recognition particle (SRP)

69. SRP bound to the ribosome

71. How ER signal sequences and SRP direct ribosomes to the ER membrane

72. Free and membrane-bound ribosomes

73. The SRP receptor in the ER membrane is composed of two different polypeptides

75. Model for SRβ-Mediated Regulation of SRP-Dependent Protein Targeting to the Endoplasmic Reticulum
SRP binds to the signal sequence, emerging from the ribosome exit site via SRP54, and protein translation is delayed. This ribosome-nascent chain (RNC)-SRP complex is targeted to the ER membrane by SR. SRβ (cyan) resides in the membrane and has to be loaded with GTP by an exchange factor in order to bind SRα (binding domain SRX in magenta). The exchange factor for SRβ is not known, and the translocon is a potential candidate. In the assembled SR-SRP-RNC complex, all three GTPases are loaded with GTP and the complex is now destined for translocation. The signal sequence is then transferred to the translocon, SRP and SR dissociate, and protein elongation resumes. GTP hydrolysis of SRP54, SRα, and SRβ can occur after nascent chain transfer in a yet poorly understood fashion.
(Schwartz and Blobel, 2003, Cell 112: )

76. Structure of the Sec61 complex
The polypeptide chain passes through an aqueous pore in the translocator
Structure of the Sec61 complex

77. A ribosome bound to the eucaryotic protein translocator

78. Translocation across the ER membrane does not always require ongoing
polypeptide chain elongation

79. The ER signal sequence is removed from most soluble proteins
after translocation

80. Integration into the ER membrane of a single-pass transmembrane
protein with a cleaved signal sequence

81. Integration into the ER membrane of a single-pass transmembrane
protein with an internal signal sequence

82. Integration into the ER membrane of a single-pass transmembrane
protein with an internal signal sequence

83. Integration into the ER membrane of a double-pass transmembrane
protein with an internal signal sequence

84. Integration into the ER membrane of a multipass transmembrane protein

85. Translocated polypeptide chains fold and assemble in the lumen of the rough ER
Mutation Research 569 (2005) 29-63

86. addition of a common N-linked oligosaccharide
Most proteins synthesized in the rough ER membrane are glycosylated by the
addition of a common N-linked oligosaccharide

87. Protein glycosylation in the rough ER
A membrane-bound oligosaccharyl transferase catalyzes the transfer of the oligosaccharide to an asparagine in the polypeptide chain as it is being synthesized

88. Synthesis of the lipid-linked precursor oligosaccharide in the rough ER membrane

89. Oligosaccharides are used as tags to mark the state of protein folding
The glucosyl transferase determines whether the protein is folded properly or not

91. Improperly folded proteins are exported from the ER and degraded in the cytosol

92. Misfolded proteins in the ER activate an unfolded protein response

94. The attachment of a GPI anchor to a protein in the ER

95. The ER assembles most lipid bilayers
The synthesis of phosphatidylcholine

96. The role of phospholipid translocators in lipid bilayer synthesis