1. Molecular Cell Biology
Harvey Lodish • Arnold Berk • Paul Matsudaira •
Chris A. Kaiser • Monty Krieger • Matthew P. Scott •
Lawrence Zipursky • James Darnell
Molecular Cell Biology
Fifth Edition
Chapter 16:
Moving Proteins into Membranes
and Organelles
Copyright © 2004 by W. H. Freeman & Company

2. Protein sorting in different organelles are governed by the same basic mechanisms with variations

3. Questions for protein sorting
Sequence context of the signal sequence
Identity of the receptor for the signal sequence
Translocation channel structure and how protein pass it
Energy source for protein translocation

4. Signal sequence or Uptake-targeting sequence
Carrying the information for a protein to target to a particular organelle destination.
Signal sequences are often removed from the mature protein by specific proteases.

5. Receptor proteins
Each organelle carries a set of receptor proteins that bind only to specific kinds of signal sequences.

6. Translocation channel
Translocation channel will allow the protein to pass through the membrane once a protein containing a signal sequence has been recognized by its receptor protein.

7. Energy
Protein translocation will only go unidirectionally because it is energy-consuming.

8. Topics for this chapter
ER protein targeting
Bacterial protein export
Targeting of proteins to mitochondria, chloroplast, and peroxisomes

9. Translocation of secretory proteins across the ER membrane
In this book, secretory proteins are proteins that will be secreted or will become luminal proteins in the ER, Golgi, and lysosomes.
Cotranslational translocation
Post-translational translocation

10. Co-translational translocation of secretory proteins across the ER membrane
Signal sequence: 16~30 residues with 6-12 hydrophobic residues (the core) and one or more positively charged amino acids adjacent to them. It will direct the ribosome to the ER membrane and initiates translocation of the growing peptide across the ER membrane.

11. Co-translational translocation of secretory proteins across the ER membrane
Receptor: SRP (signal-recognition particle) and SRP receptor
SRP is consisted of six proteins and a 300 nt RNA
SRP receptor is consisted of two subunits, a and b (a is larger)

12. SRP (signal-recognition particle)
It use its hydrophobic binding groove to interact with ER signal sequence.

13. Co-translational translocation of secretory proteins across the ER membrane
Translocation channel: Sec61 complex
Sec61 is consisted of three subunit, a, b and g. a is the largest, containing 10 membrane-spanning a helices.
Energy source: GTP hydrolysis

14. Co-translational translocation of secretory proteins across the ER membrane
Synthesis starts on free ribosome first.
The whole complex must be translocated to ER before the 70th residue was added. Without ER, the synthesis will come to a halt.

15. Co-translational translocation of secretory proteins across the ER membrane
GTP hydrolysis is required to increase the fidelity of this process. If the protein does not have correct signal sequence, GTP hydrolysis will occur and protein will leave the complex.

16. Co-translational translocation of secretory proteins across the ER membrane
If the protein is correct, GTP hydrolysis will trigger translocon (Sec61) to open and protein will enter ER lumen.

17. Sec61 complex 5-6 nm (h) x 8.5 nm (diameter)
The pore is 2 nm in diameter

18. Co-translational translocation of secretory proteins across the ER membrane
By recognizing its C-terminal sequence, signal peptidase will cleave the ER signal sequence after it enters the ER lumen.

19. Co-translational translocation of secretory proteins across the ER membrane
Signal sequence: residues containing 6-12 residues of hydrophobic core with one or more positively charged residues adjacent to them
Receptor: SRP and SRP receptor
Translocon: Sec61 complex
Energy requirement: GTP hydrolysis

20. Post-translational translocation of secretory protein across ER membrane
Some yeast proteins are translocated into ER by this pathway.
Signal sequence context is the same as co-translational translocation.
Receptor: Sec63 complex and Bip
Translocon: Sec61 complex
Energy requirement: ATP hydrolysis

21. Post-translational translocation of secretory protein across ER membrane
Translocating polypeptide chain will only slide back and forth without Bip, a member of Hsp70 family.

22. Post-translational translocation of secretory protein across ER membrane
Bip hydrolyzes ATP and Bip-ADP binds translocating polypeptide so it can enter ER.

23. Post-translational translocation of secretory protein across ER membrane
Bip-ADP pull translocating polypeptide chain into ER.

24. Insertion of proteins into ER membrane
Main questions:
How integral proteins can interact with membranes?
How topogenic sequences direct the insertion and orientation of various classes of integral proteins into the membrane?
Integral membrane proteins
GPI-anchored proteins

25. Topogenic sequences
Membrane-spanning segments determine a protein’s topology.
20-25 hydrophobic amino acids
Form a-helix

26. Type I integral membrane proteins
Single pass with cleaved N-terminal signal sequence
C-terminal region on the cytosolic side, N-terminal region on the ER/Golgi lumen or cell exterior

27. Type I integral membrane proteins
Sequence context is the same as the signal sequence for ER targeting
Has stop-transfer anchor sequence
Receptor: SRP, SRP receptor

28. Hydropathy profile
Hydropathy profile can be used to search for topogenic sequences.

29. Type II and III integral membrane proteins
Single pass with no cleavable signal sequence
The orientations are opposite

30. How type II and III membrane proteins anchor differently?
Positively charged residues located on one side of the hydrophobic sequences tend to stay on the cytosolic side that determines the orientation of protein.

31. Type IV integral membrane proteins
Multi-pass
Can be further categorized into IV-A and IV-B, depending on the orientation

32. Positively-charged segment determines the orientation of integral membrane proteins

33. Glycosylphosphatidylinositol (GPI)

34. GPI-anchored protein
Some cell-surface proteins are anchored to the phospholipid bilayer by a covalently attached amphipathic molecule, GPI.
Proteins with GPI anchors can diffuse in the plane of the phospholipid bilayer membrane. On the contrary, many proteins anchored by membrane-spanning a helices are immobilized in the membrane.

35. Synthesis of GPI-anchored proteins
They are synthesized and initially anchored to the ER membrane exactly like type I transmembrane proteins.
A short sequence adjacent to the membrane-spanning domain is recognized by a transamidase.

36. Synthesis of GPI-anchored proteins
Transamidase cleaves off the original stop-transfer anchor sequence and transfers the remainder of the protein to a preformed GPI anchor in the membrane.

37. Export of bacterial proteins
Bacterial proteins will be exported to two destinations:
Inner membrane and periplasmic space
Outer membrane

38. Export of bacterial proteins to the inner membrane and periplasmic space
Signal sequence: N-terminal hydrophobic sequence (will be cleaved by signal peptidease later)
Receptor: Ffh (similar to SRP) and FtsY (similar to SRP receptor)
Translocon: SecY, SecE and SecG; similar to Sec61 complex
Energy requirement: ATP hydrolysis

39. Inner membrane and periplasmic space transport is post-translational only
SecA (similar to Bip) push the protein into translocon.

40. Translocation across inner and outer membrane to extracellular space
Four types
Type I and II: two-step translocation
Substrate will be folded and acquire disulfide bonds in the periplasmic space first
Periplasmic proteins (spanning inner and outer membranes) translocate folded proteins outside
Energy requirement: ATP hydrolysis

41. Translocation across inner and outer membrane to extracellular space
Four types
Type III and IV: single-step translocation
Large protein complexes spanning both membranes
Pathogenic bacteria use type III for protein secretion and injection
Agrobacterium tumefaciens (膿桿菌) use type IV to transport T-DNA into plant cells.

43. Protein sorting to mitochondria and chloroplast
Mitochondrial protein targeting
Mitochondrial matrix proteins
Inner-membrane proteins
Intermembrane space proteins
Chloroplast protein targeting
Stromal protein targeting
Thylakoid protein targeting

44. Targeting for mitochondrial matrix
Matrix-targeting sequence: residues, amphipathic helix with Arg and Lys residues on one side and hydrophobic residues on the other
Receptor: import receptor
Translocon: general import pore (Tom40, Tim23/17 and Tim44, passive channel)
Energy requirement: ATP hydrolysis

45. Hsc60

46. Mitochondrial inner membrane protein targeting
Energy comes from proton motive force
Three pathways:
Path A use the same machinery used for targeting of matrix proteins.
Tom20/22 recognize their matrix targeting sequence

47. Mitochondrial inner membrane protein targeting
Precursors of path B contain both a matrix targeting sequence and internal hydrophobic domains recognized by an inner-membrane protein named Oxa1.

48. Mitochondrial inner membrane protein targeting
Path C is used by multi-pass proteins containing multiple internal mitochondrial targeting sequences recognized by Tom70.

49. Intermembrane-space proteins targeting

50. Intermembrane-space protein targeting
Path A is similar to pathway A for delivery to the inner membrane, but with intermembrane space-targeting sequence that will be recognized by an inner-membrane protease.

51. Intermembrane-space protein targeting
Path B delivers protein to intermembrane space directly by Tom40.

52. Outer-membrane protein targeting
Sequence context: N-terminal short matrix-targeting sequence followed by a long stretch of hydrophobic amino acids.
Sequence will not be cleaved after translocation.

53. Chloroplast stromal protein targeting
Sequence: no common motifs, generally rich in Ser, Thr, and small hydrophobic residues and poor in Glu and Asp.
Import process is similar to mitochondria matrix protein import but import proteins are different.
Chloroplast stroma does not have proton motive force, so the energy of stromal import comes solely from ATP hydrolysis.

54. Thylakoid protein targeting
Four pathways
SRP-dependent pathway
SecA-related pathway
Oxa1 related protein pathway
ΔPH pathway

55. DPH pathway: unfolded proteins were transported to the stroma first, then a set of thylakoid-membrane proteins will transport them to thylakoid lumen.
Thylakoid-targeting sequence contain two closely spaced Arg.
SRP-dependent pathway: transport of plastocyanin (involved in photophosphorylation)
to thylakoid lumen

56. Oxal related protein pathway
Some proteins are inserted into the thylakoid membrane by this pathway.

57. SecA-related protein pathway
This pathway transport proteins into thylakoid lumen.

58. Peroxisome protein targeting
Sequence: PTS1 (SKL or related sequence) near the C-terminus is required for peroxisome targeting, it will not be cleaved after transport
ATP hydrolysis is required.
Receptor: Pex5, Pex14
Translocon: Pex10, Pex12
Folded proteins can be translocated.

60. Principle modification for proteins
Glycosylation: ER, Golgi
Disulfide bond formation: ER
Polypeptide chain folding and multisubunit assembly: ER
Proteolytic cleavage: ER, Golgi, secretory vesicles

61. Glycosylation
Glycosylation help folding, promote cell-cell adhesion and confer stability.
O-linked glycosylation can be found in collagen and glycophorin, containing only one to four sugar residues.
N-linked glycosylation is more complex but more common.

62. N-linked glycosylation
The 14-residue oligosaccharide precursor for N-linked glycosylation is synthesized in ER and will be modified in ER and Golgi later.

63. The oligosaccharide precursor
The three glucose (glc) residues serves as a signal that this precursor is complete and is ready for transfer.

64. N-linked glycosylation
The 14-residue precursor will be transferred from dolichol to the asparagine residue of the target protein with Asn-X-Ser or Asn-X-Thr sequence by oligosaccharyl transferase.
Then three glc and one man will be removed.

65. Disulfide bond formation
In eukaryotic cells, disulfide bonds are formed only in the lumen of the rough ER; in bacterial cells, disulfide bonds are formed in the periplasmic space between the inner and outer membranes. Thus, disulfide bonds are found only in secretory proteins and in the exoplasmic domains of membrane proteins.

66. Disulfide bond formation
Protein disulfide isomerase (PDI) can catalyzed the formation of disulfide bond and can correct wrongly-formed disulfide bond.

68. PDI will be oxidized by Ero1
How Ero1 become oxidized again is not clear….XD

70. Polypeptide chain folding
Both BiP and PDI can help protein folding.
Calnexin and calreticulin will bind selectively to certain N-linked oligosaccharides on growing nascent chains, preventing aggregation of adjacent segments of a protein as it is being made.
Peptidyl-prolyl isomerases will accelerate protein folding by rotating peptidyl-prolyl bonds.

71. BiP and PDI help protein folding

72. Peptidyl-prolyl isomerase
The rotation of peptidyl-prolyl bonds sometimes are the rate-limiting step in the folding of protein domains.

73. The unfolded protein response