1. Molecular Biology of the Cell
Alberts • Johnson • Lewis • Raff • Roberts • Walter
Molecular Biology of the Cell
Fifth Edition
Chapter 12
Intracellular Compartments and Protein Sorting
Copyright © Garland Science 2008

2. Membrane Enclosed Organelles in a Eukaryotic cell
Figure Molecular Biology of the Cell (© Garland Science 2008)

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

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

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

6. Evolution of membranes  specialization of function
Figure 12-3a Molecular Biology of the Cell (© Garland Science 2008)

7. Development of thylakoid in chloroplasts
Figure 12-3b Molecular Biology of the Cell (© Garland Science 2008)

8. Origin of compartments in Eukaryotic cells
Figure 12-4a Molecular Biology of the Cell (© Garland Science 2008)

9. Origin of compartments in Eukaryotic cells
Figure 12-4b Molecular Biology of the Cell (© Garland Science 2008)

10. Topological relationship between compartments
Figure Molecular Biology of the Cell (© Garland Science 2008)

11. Proteins can move between compartments in different ways
Figure Molecular Biology of the Cell (© Garland Science 2008)

12. Proteins can move between compartments in different ways
synthesis of all proteins begins on ribosomes in the cytosol of Eukaryotic cells (except on mitochondria)
their fate depends on their amino acid sequence, which can contain a sorting sequence that directs their delivery to locations outside the cytosol.
If no sorting sequence is present the protein stays in the cytosol.

13. How proteins move from one compartment to another
Gated transport : between nucleus and cytoplasm; via nuclear pore complex (selective gate for macromolecules).
transmembrane transport : from cytosol into a space that is topologically distinct. Via protein translocators (proteins unfold in translocators).
vesicular transport : transport vesicles ferry proteins from one compartment to the next. Between topologically equivalent areas.

14. How proteins move from one compartment to another:
VESICULAR TRANSPORT
Figure Molecular Biology of the Cell (© Garland Science 2008)

15. Sorting Sequences Direct Proteins to the Correct Cell Address
signal sequences (ss):
15-60 amino acids long
often found at the N-terminus of the protein
signal peptidase enzyme removes the ss after the protein arrives to its destination.
can also be internal stretches of amino acids, these remain part of the protein.
sometimes they are made of a 3D arrangement of amino acids called a signal patch.
Each ss specifies a particular destination in the cell
are necessary and sufficient for protein targeting
recognized by sorting receptors that guide them to appropriate location. Receptors are reused (like a bus).

16. Sorting Sequences Direct Proteins to the Correct Cell Address
Table Molecular Biology of the Cell (© Garland Science 2008)

17. Panel 12-1 important

18. Most organelles can not be formed de novo, they need information from the organelle itself
During cell division, most organelles become tiny vesicles, so that when the cell divides in two it has in it some of each organelle that reforms. So the information to make the organelle is not in the DNA but it is in the organelle itself.

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

20. Nuclear envelop organization
Figure Molecular Biology of the Cell (© Garland Science 2008)

21. Nuclear pore complexes in the nuclear envelop
Figure Molecular Biology of the Cell (© Garland Science 2008)

22. Nuclear pore complexes proteins are nucleoporins
Figure 12-9a Molecular Biology of the Cell (© Garland Science 2008)

23. Nuclear pore complexes (NPCs)
In a cell there are NPCs
Transports up to 500 macromolecules per second
Can transport in both directions at the same time
Each contain one or more aqueous passages : small water soluble molecules can pass by passive diffusion. (5000 daltons or less) (each aa is ~110 daltons

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

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

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

27. Nuclear pore complexes: size cut-off to free diffusion
Figure Molecular Biology of the Cell (© Garland Science 2008)

28. Nuclear Localization Signals (NLS) directs nuclear proteins to the nucleus
Figure Molecular Biology of the Cell (© Garland Science 2008)

29. Visualizing active import through the NPC: coating gold particles with NLS signal.  Electron microscopy
Figure Molecular Biology of the Cell (© Garland Science 2008)

30. Nuclear import receptor bind to both NPCs and to NLS
Figure Molecular Biology of the Cell (© Garland Science 2008)

31. Nuclear import receptors
The import receptors are soluble cytosolic proteins
The import receptors bind to the NPC at FG repeats
The receptor-cargo complexes move along the transport path by repeatedly binding, dissociating and re-binding to adjacent FG-repeats.
Once inside the nucleus the receptor dissociates from cargo and return to the cytosol.

32. Nuclear export works like nuclear import, but in reverse
Nuclear Export Signals and Nuclear Export Receptors are needed for export.
The Nuclear Export Receptors are called Karyopherins.

33. The Ran GTPase Imposes Directionality on Transport Through the NPCs
Figure Molecular Biology of the Cell (© Garland Science 2008)

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

35. Binding of Ran-GTP causes the import receptor to release their cargo
Figure 12-16a Molecular Biology of the Cell (© Garland Science 2008)

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

37. There is a tight regulation in the proteins localization in/out of the nucleus
Figure Molecular Biology of the Cell (© Garland Science 2008)

38. Cells conrol transport by regulating NL an NE signals; these can be turned on/off by phosphorylation of residues near the signal sequence
Figure Molecular Biology of the Cell (© Garland Science 2008)

39. The nuclear lamina is located on the nuclear side of the inner nuclear membrane: a meshwork of interconnected proteins called nuclear lamins: lamins are intermediate filaments.
Figure Molecular Biology of the Cell (© Garland Science 2008)

40. During mitosis the nuclear envelope breaks down
Figure Molecular Biology of the Cell (© Garland Science 2008)

41. When nucleus disassembles during mitosis, the nuclear lamina depolymerizes.
The disassembly is a result of direct phosphorylation of the nuclear lamins by a kinase called cyclin dependent kinase (cdk) that is activated at mitosis.
later in mitosis the nuclear envelope reassembles on the surface of chromosomes.
NLS are not cleaved off after import presumably because they need to be imported repeatedly once after cell division.

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

43. The endoplasmic reticulum
membrane constitutes more than half of the total membrane in animal cells.
It consists of branching tubules and flattened sacs
Its membrane is continuous with the nuclear envelope
ER lumen is called cisternal space
It serves as calcium storage
All proteins that are sent outside the cell, or to the golgi or to lysosomes pass by the ER first.

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

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

46. ER functional specialization
Rough ER
In mammalian cells proteins are imported into the ER before complete synthesis of the polypeptide chain – co-translational translocation (whereas in mitochondria and peroxisomes it is post-translational).
Rough ER has ribosomes on it
Smooth ER does not have ribosomes; involved in lipid metabolism, streroid.
Transitional ER are areas of SER where vesicles bud off

47. Co- and post- translational translocation into the ER
Figure Molecular Biology of the Cell (© Garland Science 2008)

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

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

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

51. ER functional specialization – liver cells
In the liver most cells are hepatocytes, have ;large amount of smooth ER. This makes lipoprotein particles which carries lipid in the blood to other parts of the body.
Also present on ER membranes are enzymes that detoxify lipid soluble drugs, the enzyme called cytochrome P450 makes drugs that are water insoluble, water soluble, so now they are excreted in the urine.
Also the ER functions in Calcium storage; in response to extracellular signals calcium is released in the cytoplasm (ex. Muscle cells sacroplasmic reticulum)

52. ER derived vesicles = microsomes are used to study the ER
Figure 12-37b Molecular Biology of the Cell (© Garland Science 2008)

53. Purified Rough ER microsomes by electron microscopy
Figure 12-37a Molecular Biology of the Cell (© Garland Science 2008)

54. Protein translocation into the ER
Transmembrane proteins partially translocate across the ER membrane.
Water soluble proteins fully translocate and are released in the lumen of the ER
 The proteins are directed by the ER Signal Sequence

55. Signal hypothesis
The experiment that showed the function of ER signal sequence:
mRNA translated in vitro to make a normally secreted protein; without microsomes this protein is larger than normal (secreted); it was found that it has an N-terminal leader peptide, which gets cleaved off by signal peptidase in the ER after translocation…

56. Signal hypothesis
Figure Molecular Biology of the Cell (© Garland Science 2008)

57. ER Signal Sequence and SRP
Guided by SRP: Signal Recognition Particle which cycles between the ER membrane and the cytosol;
SRP binds to the ER SS and to a receptor on the ER membrane.
SRP consists of 6 proteins + 1 RNA molecule
ER SS vary greatly in aa sequence; but has 8 or more non-polar aa in its center
So how can the SRP be so specific to all the different SS?
Has a hydrophobic pocket.
SRP is rod-like; one end it binds ER SS another end bonds between the large and small ribosomal subunits  blocks elongation, stoping translation; this ensures that the protein is not sent to the cytoplasm
(this is VERY important for lysosomal enzymes as they can be deadly if the cell puts them in the cytoplasm)

58. SRP
Figure 12-39a Molecular Biology of the Cell (© Garland Science 2008)

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

60. SRP binds to ER SS and to SRP receptor followed by targeting to translocator in the ER membrane
Figure Molecular Biology of the Cell (© Garland Science 2008)

61. Two pools of ribosomes (cytoplasmic and ER associated)
Figure 12-41a Molecular Biology of the Cell (© Garland Science 2008)

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

63. The translocator (sec61 complex) forms a water filled pore in the membrane: polypeptide chain passes through this pore
Figure Molecular Biology of the Cell (© Garland Science 2008)

64. In Eukaryotes four sec61 complexes assemble to form a large translocator that can be visualized on ribosomes
Figure Molecular Biology of the Cell (© Garland Science 2008)

65. no energy is needed in co-translational translocation
in post-transational translocation BiP, a chaperone assists proteins in folding in the lumen of the ER, actually providing the pulling force to help enter proteins in the ER. This drives un-directional transport
no energy is needed in co-translational translocation
Figure Molecular Biology of the Cell (© Garland Science 2008)

66. ER SS triggers opening of the translocator: it is recognized twice, once by RSP and second time by the translocator – here it acts as “start-transfer signal”
When the nascent (new) polypeptide chain grows long enough ER signal peptidase cleaves off the signal sequence (that gets degraded by proteases in the ER membrane)

67. Figure 12-45 Molecular Biology of the Cell (© Garland Science 2008)

68. Single pass transmembrane protein
N-ter SS initiates translocation, but an additional hydrophobic segment in polypeptide chain stops the transfer process before the entire chain is translocated: stop-transfer signal anchors the protein n the membrane (12-46)
other cases when SS is internal here is stays as the transmembrane domain (12-47)

69. Single pass transmembrane protein
Figure Molecular Biology of the Cell (© Garland Science 2008)

70. Single pass transmembrane protein
Figure Molecular Biology of the Cell (© Garland Science 2008)

71. Multipass transmembrane proteins
Polypeptide passes back and fourth repeatedly across the lipid bilayer; here there is a start transfer sequence followed by a stop transfer sequence
In more complex proteins they have many start transfer sequences and many stop transfer sequences

72. Multipass transmembrane proteins
Figure Molecular Biology of the Cell (© Garland Science 2008)

73. Multipass transmembrane protein
Figure Molecular Biology of the Cell (© Garland Science 2008)

74. ER resident proteins
Contain ER retention signal (4 amino acids in C-terminus)
One important ER resident protein is protein disulfide isomerase (PDI): catalyzes oxidaion of free –SH groups to form disulfide –S-S- bonds
Another ER resident protein is BiP chaperone: pulls proteins post-translationally into the ER through the translocator.

75. Most proteins synthesizes in the RER are glycosylated by the addition of N-linked oligosacharides
covalent addition of sugars
most soluble proteins in ER are glycoproteins
very few proteins in the cytosol are glycosylated
N-linked (N is asparagine); these sugars are added to asparagine

76. Set of sugars is added en bloc by oligosaccharyl transferase : 2 N-acetylglucoseamine, 9 mannose, 3 glucose
Figure Molecular Biology of the Cell (© Garland Science 2008)

77. Dolichol holds the precursor oligosaccharide in ER membrane
Figure Molecular Biology of the Cell (© Garland Science 2008)

78. Synthesis of the lipid linked precursor oligosaccharide in the ER membrane
Nucleotide-sugar intermediates
High energy Phosphate bonds
Figure Molecular Biology of the Cell (© Garland Science 2008)

79. N-linked oligosaccharides are most common in ER
following this, there is glucose trimming (2 glucose taken out)
Less frequently oligosaccharides are linked to the hydroxyl group on the side chain of a serine, threonine or hydroxylysine : theese are O-linked in the Golgi
Some proteins require N-linked oligosaccharides for proper folding; calnexin and calreticulin are lectins, they are chaperones, they bind to the sugar groups of improperly folded proteins and retain them in the ER.  unfolded proteins have glucose added to them and removed continuously (by glucosidase and glycosyl transferase)

80. Incompletely folded proteins are retained in the ER until they fold properly
Figure Molecular Biology of the Cell (© Garland Science 2008)

81. When a protein does not fold properly and exits the ER, it is degraded by the proteasome; sugar is removed and ubiquitin is added
Figure Molecular Biology of the Cell (© Garland Science 2008)

82. Cells carefully monitor eth amount of misfolded proteins
in the ER misfolded proteins trigger the “unfolded protein response”:
Increase transcription of genes encoding ER chaperones, proteins involved in transport from ER to cytosol (retrotranslocation) and protein degradation in the cytosol.
HOW IS THIS DONE?

83. How a misfolded protein send a signal to make more chaperones
Figure 12-55a Molecular Biology of the Cell (© Garland Science 2008)

84. How a misfolded protein send a signal to make more chaperones
Figure 12-55b Molecular Biology of the Cell (© Garland Science 2008)

85. Some membrane proteins acquire a covalently attached Glycosylphosphatidylinositol (GPI) anchor
GPI is added to the C-ter of proteins destined to the plasma membrane, also into lipid rafts. These can then be released from the GPI anchor by specific phospholipases in the plasma membrane
Figure Molecular Biology of the Cell (© Garland Science 2008)

86. The ER assembles most lipid bilayers: phosphatidylcholine, one major phospholipid
Figure Molecular Biology of the Cell (© Garland Science 2008)

87. Figure 12-58 Molecular Biology of the Cell (© Garland Science 2008)