1. Chapter 12
Intracellular Compartments and Protein Sorting

2. Figure 12-1 Molecular Biology of the Cell (© Garland Science 2008)

3. Protein Movement between Compartments
Most proteins are synthesized on cytoplasmic ribosomes and must be delivered to their ultimate compartment of residence.
Proteins contain sorting signals that direct their movement throughout the cell.
These sorting signals are recognized by specific receptors that mediate delivery to the appropriate organelle.
There are three major types of protein traffic between compartments:
1) Gated transport
2) Transmembrane translocation
3) Vesicular transport

4. Figure 12-6 Molecular Biology of the Cell (© Garland Science 2008)

5. Gated Transport (Nuclear Import/Export)
Figure Molecular Biology of the Cell (© Garland Science 2008)

6. Transmembrane Translocation
Proteins are directly translocated across the membrane bilayer.
Translocation is performed by a membrane protein complex that forms a translocation pore.
Proteins pass through the membrane bilayer as unfolded chains.
Two major strategies are used to accomplish this feat: co-translational & post-translational import.

7. Vesicular Transport
Figure 12-7

8. Protein sorting signals

9. Signal sequences direct protein delivery
Destination SS
Mitochondria
Cytoplasm
1. Deletion of signal sequence (SS)
Cytoplasm
2. Addition of a signal sequence
Mitochondria SS

10. Mitochondrial Protein Import
Mitochondria utilize the energy from electron transport and oxidative phosphorylation to synthesize the majority of the cell's ATP.
Most mitochondrial proteins are synthesized on cytoplasmic ribosomes and are post-translationally imported into this organelle.
Because of the double membrane surrounding this organelle, there are four targets for mitochondrial proteins:
1. Outer membrane 3. Inner membrane
2. Intermembrane space 4. Matrix space
Mitochondrial proteins usually contain an N-terminal targeting sequence that is capable of forming an amphipathic -helix; positively-charged residues are clustered on one side of the helix and uncharged residues are present on the other.
The mitochondrial outer membrane contains specific receptor proteins that bind to the mitochondrial targeting signal.

11. The four compartments within mitochondria
Figure Molecular Biology of the Cell (© Garland Science 2008)

12. Signal sequence for mitochondrial protein import.
Note the amphipathic nature of the -helix.
Figure 12-22

13. (Matrix/Inner Membrane)
Protein translocators in mitochondrial membranes
(Matrix/Inner Membrane)
(Inner Membrane)
Figure 12-23

14. Mitochondrial Protein Import (cont’d)
Translocation into the mitochondrial matrix requires both ATP hydrolysis and an electrochemical gradient across the inner mitochondrial membrane.
Translocation occurs at sites where the inner and outer membrane are in close apposition. These regions are known as contact sites.
Proteins are imported into the mitochondria in an unfolded state.
Maintenance in an unfolded state is mediated by hsp70 proteins that act as molecular chaperones.
Protein transport into the inner membrane or intermembrane space requires additional targeting signals.
Much of our current knowledge of mitochondrial protein import has come from in vitro studies with isolated mitochondria.

15. Protein import into mitochondria
Figure Molecular Biology of the Cell (© Garland Science 2008)

16. The role of energy in mitochondrial protein import
Figure Molecular Biology of the Cell (© Garland Science 2008)

17. The hsp70 family of molecular chaperones
Figure Molecular Biology of the Cell (© Garland Science 2008)

18. The role of energy in mitochondrial protein import
Figure Molecular Biology of the Cell (© Garland Science 2008)

19. Figure 12-28 Molecular Biology of the Cell (© Garland Science 2008)

20. Figure 12-28b Molecular Biology of the Cell (© Garland Science 2008)

21. Figure 12-28 Molecular Biology of the Cell (© Garland Science 2008)

22. Studying mitochondrial protein import in vitro
Isolated mitochondria are mixed with the radioactively-labeled protein to be studied
IMPORT ?

23. ? IMPORT Import may be detected by one of the following methods:
1) Density gradient centrifugation; if imported, proteins will fractionate with the organelle.
2) SDS-PAGE analysis to determine if the signal sequence was removed during the import reaction.
3) Protease protection assays; imported protein will be protected from the action of added proteases.
By adding or removing different components from the import reaction, one can determine the requirements for protein import.

24. in vitro studies of mitochondrial protein import
Figure Molecular Biology of the Cell (© Garland Science 2008)

25. Secretory Pathway
Proteins enter into the secretory pathway at the ER where they are co-translationally inserted into the ER membrane.
Proteins then travel to successive organelles via membrane-bound intermediates.

26. Endoplasmic Reticulum

27. Functions of the ER
The entry point for proteins that proceed through the secretory pathway.
Modification of proteins: a predominant modification is the glycosylation of specific asparagine residues (N-linked sugars).
Quality control: proteins must be properly folded before they are allowed to leave the ER. Proteins that fail to achieve a native state are degraded.
Sequestration of Ca++ from the cytoplasm.
Primary site of lipid biosynthesis.

28. Figure 12-35 Molecular Biology of the Cell (© Garland Science 2008)

29. Abundant smooth ER in steroid-hormone-secreting cell
A 3-D reconstruction of ER in liver cell
Figure 12-36c Molecular Biology of the Cell (© Garland Science 2008)

30. Rough ER in pancreatic exocrine cell

31. Free
Membrane-bound
Figure 12-41a

32. Separating the Smooth & Rough ER
Figure 12-37b Molecular Biology of the Cell (© Garland Science 2008)

33. The Signal Hypothesis George Palade: 1974 Nobel Prize in Medicine
Figure 12-38

34. Protein Import into the ER
Step 1: Establishing a tight interaction with the ER membrane
A hydrophobic signal peptide, usually at the N-terminus of the protein, directs entry into the ER.
The signal peptide is recognized by the Signal Recognition Particle (SRP) as soon as it emerges from the ribosome. This interaction arrests translation.
The ER membrane contains an SRP receptor that mediates the initial association of the SRP-ribosome complex with the cytoplasmic face of the ER.
The ribosome subsequently associates with a translocation complex (the Sec61 complex) in the ER membrane and the SRP is released back into the cytosol.

35. Table 12-3 Molecular Biology of the Cell (© Garland Science 2008)

36. Signal-recognition particle (SRP)
Figure 12-39a

37. Two functions for the signal sequence:
Targets protein/ribosome complex to the ER membrane.
Serves as a start-transfer sequence that opens translocation pore.
Figure Molecular Biology of the Cell (© Garland Science 2008)

38. Protein Import into the ER
Step 2: Co-translational translocation of the polypeptide
Upon association with the ER, the ribosome resumes translation and co-translationally inserts the polypeptide chain into the ER lumen through a translocation pore.
The protein is passed through the membrane as a single, unfolded chain and folds into its native conformation within the ER lumen. This folding process requires protein chaperones.
The N-terminal signal peptide is removed by Signal Peptidase, a protease present in the lumen of the ER.
Integral membrane proteins contain "stop transfer" sequences that result in a block to the translocation process.

39. Structure of the Sec61 translocation complex
Figure Molecular Biology of the Cell (© Garland Science 2008)

40. A ribosome bound to the Sec61 protein translocator
Figure Molecular Biology of the Cell (© Garland Science 2008)

41. Translocation of a soluble protein
Figure Molecular Biology of the Cell (© Garland Science 2008)

42. A single-pass transmembrane protein
Figure Molecular Biology of the Cell (© Garland Science 2008)

43. NOTE: Orientation across membrane bilayer.
Integration of a single-pass membrane protein with an internal signal sequence
NOTE: Orientation across membrane bilayer.
Figure Molecular Biology of the Cell (© Garland Science 2008)

46. A double-pass transmembrane protein
Figure Molecular Biology of the Cell (© Garland Science 2008)

47. Insertion of a multipass membrane protein into the ER
Figure Molecular Biology of the Cell (© Garland Science 2008)

48. Genetic approaches for studying the mechanism of protein translocation
Wild-type
Engineered Cell
Enzyme in cytosol: cell lives without histidine
Enzyme targeted to ER: cell dies without histidine
Mutant Engineered Cell
Not all enzyme targeted to ER: cell lives without histidine
Panel 12-1

49. Most proteins in the secretory pathway are glycosylated; modified by the addition of sugar residues.
A precursor oligosaccharide unit is added to particular asparagine residues (N-linked carbohydrate).
Figure Molecular Biology of the Cell (© Garland Science 2008)

50. Protein glycosylation in the rough ER
Figure 12-51

51. Synthesis of the lipid-linked precursor oligosaccharide in the rough ER membrane
Figure 12-52

52. Possible functions for the N-linked oligosaccharide chains?
Promoting protein folding & stability.
Protecting the protein from proteolysis.
Serving as a targeting determinant.
Facilitating or directing anterograde (forward) transport.
Promoting cell-to-cell adhesion.

53. The role of N-linked glycosylation in ER protein folding
Figure 12-53

54. The export & degradation of misfolded ER proteins
The ER functions as a quality control organelle.
Proteins that are not properly folded are exported from the ER and degraded in the cytosol.
Figure 12-54

55. Quality control in the ER and Cystic Fibrosis
A particular deletion that removes three nucleotides in the Cftr gene is the most common mutation responsible for this disease.
This deletion results in the removal of a phenylalanine residue, F508.
The encoded protein is recognized by the ER quality-control machinery and is ultimately targeted for degradation (in the cytoplasm).
However, the encoded protein would be FUNCTIONAL if it was allowed to go to the plasma membrane. *
Knowing the above, how might you try to treat CF patients that possess this cftr allele? M *
Degradation
Plasma membrane

56. Lipid synthesis in the ER
The cytoplasmic half of the ER bilayer is the primary site of phospholipid synthesis. The enzymes that catalyze these reactions are ER membrane proteins whose active sites face the cytosol.
Phospholipid translocators function to "flip" specific phospholipids from one half of the bilayer to the other.
Specific phospholipid transfer proteins (PLTPs) transport phospholipids from the ER to mitochondria and peroxisomes.

57. Synthesis of phosphatidylcholine
Figure Molecular Biology of the Cell (© Garland Science 2008)

58. The role of phospholipid translocators in lipid bilayer synthesis
SCRAMBLASE
FLIPPASE
Figure 12-58

59. Phospholipid exchange/transfer proteins

60. Ch. 13: Intracellular Vesicular Traffic
Figure Molecular Biology of the Cell (© Garland Science 2008)

61. Biosynthetic-Secretory/Endocytic Pathway
Figure Molecular Biology of the Cell (© Garland Science 2008)

62. Figure 13-3b Molecular Biology of the Cell (© Garland Science 2008)

63. Vesicular Transport
The lumen of each compartment communicating by way of vesicular traffic is topologically equivalent.
The two primary pathways for vesicular traffic are known as the biosynthetic-secretory and the endocytic pathways.
A transport vesicle must select the cargo to be transported to the next compartment and exclude that which is to remain behind.
To ensure compartment identity, a transport vesicle must fuse only with the appropriate target organelle.

64. Protein coats facilitate multiple steps of vesicular transport

65. Coated Vesicles
Most transport vesicles form from specialized "coated" regions of the membrane and bud off as coated vesicles.
These coats are protein structures that form on the cytosolic face of a membrane region that will form the transport vesicle.
Several coat structures have been identified in eukaryotic cells and each appears to perform a distinct transport function.
Coated vesicles generally mediate the directional flow of specific types of membranes.
The assembly of a coat structure on a membrane may be the driving force in bud formation.
Coat proteins play an important role in the selection of vesicle cargo.

66. Three examples of coated vesicles
Figure Molecular Biology of the Cell (© Garland Science 2008)

67. Generation of membrane curvature
Membrane deformation by proteins that exert mechanical force.
Curvature generation by scaffolding proteins (coat proteins).
Curvature generation by a hydrophobic insertion (wedging) mechanism.

68. Different coated vesicles mediate distinct transport steps within the secretory pathway
Figure Molecular Biology of the Cell (© Garland Science 2008)

69. Clathrin-coated Vesicles
The primary component of one membrane coat is clathrin, a large protein complex composed of three subunits each of a heavy chain and a light chain.
Clathrin-coated vesicles mediate Golgi-to-lysosome protein delivery and plasma membrane receptor- mediated endocytosis.
Clathrin coat assembly provides the mechanical force necessary for bud emergence and vesicle formation.
Other proteins in this coat, known as adaptins, mediate the binding and sequestration of specific transmembrane receptors and their bound cargo.

70. Clathrin-coated pits & vesicles on the inner surface of the p. m
Clathrin-coated pits & vesicles on the inner surface of the p.m. in cultured fibroblasts
Figure Molecular Biology of the Cell (© Garland Science 2008)

71. The structure of a clathrin coat
36 triskelions (12 pentagons, 6 hexagons)
Normal coat has 12 pentagons and variable no. of hexagons
Figure 13-7

72. Clathrin coat assembly & disassembly
Emphasize that coat comes first, before the cargo receptors.
The coat is NOT forming around the cargo!
Indicate that uncoating is promoted by an ATPase.
Lipids can contribute to coat assembly (by interactions with sp proteins)
Point them to the PI section in the text.
Figure Molecular Biology of the Cell (© Garland Science 2008)

74. Cargo selection by the clathrin coat

75. Dynamin is a small GTP-binding protein(?).
EM is of shibire (“paralyzed”) flies
The role of dynamin in pinching off clathrin-coated vesicles from the membrane.
Figure Molecular Biology of the Cell (© Garland Science 2008)

76. Monomeric GTPases control coat assembly
Figure 13-13a

77. GTP-binding proteins act as molecular switches
“Inactive”
GTP-binding proteins act as molecular switches
(GTP)
(GDP)
GEF = Guanine nucleotide Exchange Factor
GAP = GTPase-Activating Protein
“Active”
Figure 3-73 Molecular Biology of the Cell

78. Monomeric GTPases control coat assembly
Sar1- COP II
ARF - COP I Clathrin
Figure 13-13a

79. Coat recruitment & cargo selection
Figure 13-13b Molecular Biology of the Cell (© Garland Science 2008)

80. Formation of COPII-coated vesicles
Sec13/31 “cage”
Point out that order was determined from in vitro reconstitution experiments.
Outer and inner layers of coat may not be separate in vivo.
Figure 13-13

81. How does a transport vesicle find its correct destination?
Could emphasize spatial organization;
how it might facilitate target recognition.

82. Vesicle Targeting
To ensure compartment identity, transport vesicles must fuse only with the appropriate target membrane.
This specificity is mediated by two classes of proteins: the Rab family of monomeric GTPases and the transmembrane SNARE proteins that mediate membrane fusion.
The active GTP-bound Rab proteins interact with a diverse set of Ras effector proteins that mediate vesicle transport, tethering and fusion to the appropriate target membrane.
Membrane fusion is facilitated by the pairing of complementary transmembrane receptors present on the vesicle (v-SNAREs) and the target membrane (t-SNAREs).
SNARE complex disassembly after membrane fusion is catalyzed by NSF, a cytoplasmic ATPase.

83. Tethering of a vesicle to a target membrane
Point out that Ras effects are a heterogeneous family/bunch.
We will focus on one particular example here.
Figure Molecular Biology of the Cell (© Garland Science 2008)

84. The structure of a trans-SNARE complex
Figure Molecular Biology of the Cell (© Garland Science 2008)

85. A model for how SNARE proteins may catalyze membrane fusion
Figure Molecular Biology of the Cell (© Garland Science 2008)

86. NSF facilitates the dissociation of SNARE proteins after membrane fusion
Figure Molecular Biology of the Cell (© Garland Science 2008)