1. Intracellular Compartments and Protein Sorting
The compartmentalization of cells
The transport of molecules between the nucleus and the cytosol
The transport of proteins into mitochondria and chloroplasts (Discussion topic)
Peroxisomes
The endoplasmic reticulum
Chapter 12 Molecular Biology of the Cell
Copyright © Garland Science 2008

2. The compartmentalization of cells
All eucaryotic cells have the same basic set of membrane-
enclosed organelles.
Evolutionary origins explain the topological relationships of
organelles.
Proteins can move between compartments in different
ways.
Signal sequences direct proteins to the correct cell
address.
Most organelles cannot be constructed de novo: they require
information in the organelle itself.
各胞器內含有不同的蛋白質，各有所司。這些蛋白質如何運送到正確的胞器內，且保證其方位正確？

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

4. Table 12-1 Molecular Biology of the Cell (© Garland Science 2008)

5. Table 12-2 Molecular Biology of the Cell (© Garland Science 2008)

6. Topologically equivalent spaces are shown in red.
Blue arrows indicate the extensive vesicular traffic.
Mitochondria and chloroplasts do not participate in this vesicular transport.
Figure Molecular Biology of the Cell (© Garland Science 2008)

7. Vesicular transport (Ch. 13)
What does “topologically equivalent” mean?
Vesicular transport
(Ch. 13)
Figure Molecular Biology of the Cell (© Garland Science 2008)

8. Each newly synthesized organelle protein must find its way from a ribosome in the cytosol, where the protein is made, to the organelle where it functions.
It does so by following a specific pathway, guided by sorting signals in its amino acid sequence that function as signal sequences or signal patches. Sorting signals are recognized by complementary sorting receptors, which deliver the protein to the appropriate target organelle. Proteins that function in the cytosol do not contain sorting signals and therefore remain there after they are synthesized.
During cell division, organelles such as the ER and mitochondria are distributed intact to each daughter cell. These organelles contain information that is required for their construction, and so they cannot be made de novo.

9. A protein traffic “roadmap.”
Figure Molecular Biology of the Cell (© Garland Science 2008)

10. Red: positively charged amino acids
Green: negatively charged amino acids
Blue: hydroxylated amino acids
White: hydrophobic amino acids
Table Molecular Biology of the Cell (© Garland Science 2008)

11. The gated transport of molecules between the nucleus and the cytosol
Nuclear pore complexes perforate the nuclear envelope.
Nuclear localization signals direct nuclear proteins to the nucleus.
Nuclear import receptors binds to both nuclear localization
signals and NPC proteins.
The Ran GTPase imposes directionality on transport through
NPCs.
Transport through NPCs can be regulated by controlling access
to the transport machinery.
During mitosis the nuclear envelope disassembles.

12. Page 704 Molecular Biology of the Cell (© Garland Science 2008)

13. Figure Q12-1 Molecular Biology of the Cell (© Garland Science 2008)

14. Figure 12-8 Molecular Biology of the Cell (© Garland Science 2008)

15. Figure 12-9 Molecular Biology of the Cell (© Garland Science 2008)

16. < 5000 Da
Figure Molecular Biology of the Cell (© Garland Science 2008)

17. Figure 12-11 Molecular Biology of the Cell (© Garland Science 2008)

18. Colloidal gold spheres coated with peptides containing nuclear localization signals enter the nucleus through NPCs.
Figure Molecular Biology of the Cell (© Garland Science 2008)

19. How can nuclear transport be uni-directional?
The RAN-GTPase imposes directionality in transport through NPC.
Figure Molecular Biology of the Cell (© Garland Science 2008)

20. Nuclear import receptors binds to both nuclear localization signals and NPC proteins.
Nuclear transport receptor
(import signal)
Figure 12-16a Molecular Biology of the Cell (© Garland Science 2008)

21. Ran-GDP in cytosol Ran-GTP in nucleus
(GTPase-activating protein)
This picture should be viewed from the other side (counter-clock-wise).
(Guanine exchange factor)
Ran-GTP in nucleus
Figure Molecular Biology of the Cell (© Garland Science 2008)

22. Next page Low affinity to unload cargo High affinity to import cargo
High affinity to export cargo
Low affinity to unload cargo
Next page
Figure Molecular Biology of the Cell (© Garland Science 2008)

23. Cytosol
Nucleus
Figure 12-16b Molecular Biology of the Cell (© Garland Science 2008)

24. T cell activation  nuclear transport and gene transcription.
Ca2+-activated phosphatase
Resting state
Shuttling proteins contain both import and export signals; they can shuttle back and forth between the nucleus and the cytosol. The [Ca2+] i level controls relative rates of transport and thus determines the preferred location of these proteins.
Activated state
T cell activation  nuclear transport and gene transcription.
Figure Molecular Biology of the Cell (© Garland Science 2008)

25. Ran-GTP
Ran-GEF
Figure Molecular Biology of the Cell (© Garland Science 2008)

26. The extensive traffic of materials between the nucleus and cytosol occurs through nuclear pore complexes (NPCs), which provide a direct passageway across the nuclear envelope. Proteins containing nuclear localization signals are actively transported inward through NPCs, while RNA molecules and newly made ribosomal subunits contain nuclear export signals, which direct their outward active transport through NPCs. Some proteins, including the nuclear import and export receptors, continually shuttle between the cytosol and nucleus.
Ran-GTPase provides both the free energy and the directionality for nuclear transport. Cells regulate the transport of nuclear proteins and RNA molecules through the NPCs by controlling the access of these molecules to the transport machinery. Because nuclear localization signals are not removed, nuclear proteins can be imported repeatedly, as is required each time that the nucleus reassembles after mitosis.

28. The endoplasmic reticulum (I)
The ER is structurally and functionally diverse.
Signal sequences were first discovered in proteins imported into
the rough ER.
A signal-recognition particle (SRP) directs ER signal sequences
to a specific receptor in the rough ER membrane.
The polypeptide chain passes through an aqueous pore in the
translocator.
Translocation across the ER membrane does not always require
ongoing polypeptide chain elongation.
In single-pass transmembrane proteins, a single internal ER
signal sequence remains in the lipid bilayer as a membrane-
spanning a helix.
Combinations of the start-transfer and stop-transfer signals
determine the topology of multipass transmembrane proteins.

29. The endoplasmic reticulum (II)
Translocated polypeptide chains fold and assemble in the lumen of
the rough ER.
Most proteins synthesized in the rough ER are glycosylated by the
addition of a common N-linked oligosaccharide.
Oligosaccharides are used as tags to mark the state of protein
folding.
Improperly folded proteins are exported from the ER and degraded
in the cytosol.
Misfolded proteins in the ER activate an unfolded protein
response.
Some membrane proteins acquire a covalently attached
glycosylphosphatidylinositol (GPI) anchor.
The ER assembles most lipid bilayers.

30. Page 723 Molecular Biology of the Cell (© Garland Science 2008)

31. The ER extends as a network throughout the entire cytosol of a cultured animal cell, so that all regions of the cytosol are close to some portion of the ER membrane.
Figure 12-34a Molecular Biology of the Cell (© Garland Science 2008)

32. ER (mostly) Mitochondria, chloroplasts, nuclei, and peroxisomes
Figure Molecular Biology of the Cell (© Garland Science 2008)

33. Figure 12-36c Molecular Biology of the Cell (© Garland Science 2008)

34. Figure 12-37b Molecular Biology of the Cell (© Garland Science 2008)

35. Purified microsomal fraction
Figure 12-37a, 41b Molecular Biology of the Cell (© Garland Science 2008)

36. The Signal Hypothesis by G. Blobel
The easiest way to test this hypothesis is to find the common part of ER proteins, which would intuitively serve as the ER signal peptide. However, it was not easy to do so when the genes encoding these proteins were not identified yet.
Figure Molecular Biology of the Cell (© Garland Science 2008)

37. Figure 12-39b Molecular Biology of the Cell (© Garland Science 2008)

38. 此圖與前圖左右對稱
Figure 12-39a Molecular Biology of the Cell (© Garland Science 2008)

39. ER signal sequence and SRP direct ribosomes to the ER membrane.
Q: What are the advantages of having SRP and its receptor in this protein translocation machinery?
Figure Molecular Biology of the Cell (© Garland Science 2008)

40. Figure 12-41a Molecular Biology of the Cell (© Garland Science 2008)

41. The structure of a Sec61 complex, part of the central potion of a protein translocator.
Gray 接縫 塞子
Blue/Red
Gray
To be released into the membrane
When the ER signal binding site is occupied, the translocator opens up its seam and remove the plug to allow the translocation of a growing polypeptide.
Figure Molecular Biology of the Cell (© Garland Science 2008)

42. Side view
Bottom view
Figure Molecular Biology of the Cell (© Garland Science 2008)

43. Protein translocation across ER can be driven by 3 ways.
Figure Molecular Biology of the Cell (© Garland Science 2008)

44. The translocation of a soluble protein with a cleaved signal peptide (start) .
Figure Molecular Biology of the Cell (© Garland Science 2008)

45. The translocation of a single-pass transmembrane protein with a cleaved signal peptide (start) and an internal signal peptide (stop).
Figure Molecular Biology of the Cell (© Garland Science 2008)

46. The translocation of a single-pass transmembrane protein with an internal ER signal peptide (start).
+ a.a. is always located at the cytosolic side, while – a.a. is always located at the ER luminal side.
Figure Molecular Biology of the Cell (© Garland Science 2008)

47. The translocation of a double-pass transmembrane protein with two internal ER signal peptides (start and stop).
Figure Molecular Biology of the Cell (© Garland Science 2008)

48. The translocation of a multi-pass transmembrane protein with multiple internal ER signal peptides.
Rhodopsin
Figure Molecular Biology of the Cell (© Garland Science 2008)

49. The endoplasmic reticulum (II)
Translocated polypeptide chains fold and assemble in the lumen of
the rough ER.
Most proteins synthesized in the rough ER are glycosylated by the
addition of a common N-linked oligosaccharide.
Oligosaccharides are used as tags to mark the state of protein
folding.
Improperly folded proteins are exported from the ER and degraded
in the cytosol.
Misfolded proteins in the ER activate an unfolded protein
response.
Some membrane proteins acquire a covalently attached
glycosylphosphatidylinositol (GPI) anchor.
The ER assembles most lipid bilayers.

50. N-linked core oligosaccharide (GlcNAc)2(Man)3
Translocator polypeptide chains are further processed in the lumen of the rough ER: folding, assembling, and glycosylation.
N-linked core oligosaccharide
(GlcNAc)2(Man)3
Figure Molecular Biology of the Cell (© Garland Science 2008)

51. Protein glycosylation in the rough ER (1st step).
Dolichols function as a membrane anchor for the formation of the oligosaccharide Glc3-Man9-GlcNAc2.
Figure Molecular Biology of the Cell (© Garland Science 2008)

52. Synthesis of the lipid-linked precursor oligosaccharide in the rough ER membrane
Figure Molecular Biology of the Cell (© Garland Science 2008)

53. Oligosaccharides are used as tags to mark the state of protein folding in the ER.
Incorrect Correct
Calnexin, calretilculin, and ERp57 are chaperones.
Figure Molecular Biology of the Cell (© Garland Science 2008)

54. Improperly folded proteins are exported from the ER and degraded in the cytosol.
“ER stress”  Activates an unfolded protein response. (輔導老師教官刑警)
Figure Molecular Biology of the Cell (© Garland Science 2008)

55. Different cell types may use different ways.
Transported to Golgi
Kinase
Ribonuclease
(next page)
Protease
Figure 12-55a Molecular Biology of the Cell (© Garland Science 2008)

56. (Details of pathway 1, previous page)
Figure 12-55b Molecular Biology of the Cell (© Garland Science 2008)

57. Another role of membrane carbohydrates
Some membrane proteins are attached with a glycosylphosphatidylinositol (GPI) anchor.
GPI PI
This protein remains membrane-bound and eventually locates on the cell exterior (see Fig ).
Figure Molecular Biology of the Cell (© Garland Science 2008)

58. Most membrane lipids are synthesized in the cytosolic half of ER.
Figure Molecular Biology of the Cell (© Garland Science 2008)

59. Phospholipid translocators help lipid bilayer synthesis.
Figure Molecular Biology of the Cell (© Garland Science 2008)

60. The ER produces almost all of the cell’s lipids and a major portion of cell’s proteins. In the ER lumen, the proteins fold and oligomerize, disulfide bonds are formed, and oligo-saccharides are added. The pattern of glycosylation is used to indicate the extent of protein folding. Proteins that do not fold or oligomerize correctly are translocated back into the cytosol, where they are deglycosylated, ubiquitylated, and degraded in proteasomes. If misfolded proteins accumulate in excess in the ER, they trigger an unfolded protein response, which activates appropriate genes in the nucleus to help the ER to cope.
Only proteins that carry a special ER signal sequence, which can be recognized by a signal recognition particle (SRP), are imported into the ER. SRP binds to both the growing peptide chain and a ribosome, directs them to a receptor protein on the cytosolic surface of the rough ER membrane, initiates the protein translocation process through a transmembrane protein translocator.

61. Soluble proteins pass completely into the ER lumen, whereas transmembrane proteins are translocated partway across the ER membrane and remain anchored there by one or more membrane-spanning a-helical regions in their polypeptide chains. These hydrophobic portions of the protein can act either as start-transfer or stop-transfer signals during translocation process. When a polypeptide contains multiple, alternating start-transfer and stop-transfer signals, it will pass back and forth across the bilayer multiple times as a multipass transmembrane protein.
The asymmetry of protein insertion and glycosylation in the ER establishes the sidedness of the membranes of all the other organelles that the ER supplies with membrane proteins.

62. Thank You!