1. Chapte r 8 Protein localization

2. 8.1 Introduction 8.2 Chaperones may be required for protein folding 8.3 Post-translational membrane insertion depends on leader sequences 8.4 A hierarchy of sequences determines location within organelles 8.5 Signal sequences initiate translocation 8.6 How do proteins enter and leave membranes? 8.7 Anchor signals are needed for membrane residence 8.8 Bacteria use both co-translational and post- translational translocation 8.9 Pores are used for nuclear ingress and egress 8.10 Protein degradation by proteasomes

3. Leader of a protein is a short N-terminal sequence responsible for passage into or through a membrane. 8.1 Introduction

4. Figure 8.1 Overview: proteins that are localized post-translationally are released into the cytosol after synthesis on free ribosomes. Some have signals for targeting to organelles such as the nucleus or mitochondria. Proteins that are localized cotranslationally associate with the ER membrane during synthesis, so their ribosomes are "membrane-bound". The proteins pass into the endoplasmic reticulum, along to the Golgi, and then through the plasma membrane, unless they have signals that cause retention at one of the steps on the pathway. They may also be directed to other organelles, such as endosomes or lysosomes. 8.1 Introduction

5. Figure 8.2 Proteins synthesized on free ribosomes in the cytosol are directed after their release to specific destinations by short signal motifs. 8.1 Introduction

6. Figure 8.3 Membrane- bound ribosomes have proteins with N-terminal sequences that enter the ER during synthesis. The proteins may flow through to the plasma membrane or may be diverted to other destinations by specific signals. 8.1 Introduction

7. Figure 8.4 A protein is constrained to a narrow passage as it crosses a membrane. 8.2 Chaperones may be required for protein folding

8. Figure 8.5 Chaperone families have eukaryotic and bacterial counterparts (named in parentheses). 8.2 Chaperones may be required for protein folding

9. Figure 8.6 DnaJ assists the binding of DnaK (Hsp70), which assists the folding of nascent proteins. ATP hydrolysis drives conformational change. GrpE displaces the ADP; this causes the chaperones to be released. Multiple cycles of association and dissociation may occur during the folding of a substrate protein. 8.3 The Hsp70 family is ubiquitous

10. Figure 8.7-1 A protein may be sequestered within a controlled environment for folding or degradation. 8.4 Hsp60/GroEL forms an oligomeric ring structure

11. Figure 8.7-2 GroEL forms an oligomer of two rings, each comprising a hollow cylinder made of 7 subunits. 8.4 Hsp60/GroEL forms an oligomeric ring structure

12. Figure 8.8 Two rings of GroEL associate back to back to form a hollow cylinder. GroES forms a dome that covers the central cavity on one side. Protein substrates bind to the cavity in the distal ring. 8.4 Hsp60/GroEL forms an oligomeric ring structure

13. Figure 8.9 Protein folding occurs in the proximal GroEL ring and requires ATP. Release of substrate and GroES requires ATP hydrolysis in the distal ring. 8.4 Hsp60/GroEL forms an oligomeric ring structure

14. Figure 8.10 Leader sequences allow proteins to recognize mitochondrial or chloroplast surfaces by a post- translational process. 8.5 Post-translational membrane insertion depends on leader sequences

15. Figure 8.12 The leader sequence of yeast cytochrome c oxidase subunit IV consists of 25 neutral and basic amino acids. The first 12 amino acids are sufficient to transport any attached polypeptide into the mitochondrial matrix. 8.5 Post-translational membrane insertion depends on leader sequences

16. Figure 8.13 TOM proteins form receptor complex(es) that are needed for translocation across the mitochondrial outer membrane. 8.5 Post-translational membrane insertion depends on leader sequences

17. Figure 8.14 Tim proteins form the complex for translocation across the mitochondrial inner membrane. 8.5 Post-translational membrane insertion depends on leader sequences

18. Figure 8.15 Tim9-10 takes proteins from TOM to either TIM complex, and Tim8- 13 takes proteins to Tim22-54. 8.5 Post-translational membrane insertion depends on leader sequences

19. Figure 8.16 A translocating protein may be transferred directly from TOM to Tim22-54. 8.5 Post-translational membrane insertion depends on leader sequences

20. Figure 8.17 Mitochondria have receptors for protein transport in the outer and inner membranes. Recognition at the outer membrane may lead to transport through both receptors into the matrix, where the leader is cleaved. If it has a membrane- targeting signal, it may be re-exported. 8.6 A hierarchy of sequences determines location within organelles

21. Figure 8.18 The leader of yeast cytochrome c1 contains an N-terminal region that targets the protein to the mitochondrion, followed by a region that targets the (cleaved) protein to the inner membrane. The leader is removed by two cleavage events. 8.6 A hierarchy of sequences determines location within organelles

22. Figure 8.19 A protein approaches the chloroplast from the cytosol with a ~50 residue leader. The N- terminal half of the leader sponsors passage into the envelope or through it into the stroma. Cleavage occurs during envelope 8.6 A hierarchy of sequences determines location within organelles

23. Signal sequence is the region of a protein (usually N-terminal) responsible for co-translational insertion into membranes of the endoplasmic reticulum. 8.7 Signal sequences initiate translocation

24. Figure 8.20 The endoplasmic reticulum consists of a highly folded sheet of membranes that extends from the nucleus. The small objects attached to the outer surface of the membranes are ribosomes. Photograph kindly provided by Lelio Orci. 8.7 Signal sequences initiate translocation

25. Figure 8.21 The signal sequence of bovine growth hormone consists of the N-terminal 29 amino acids and has a central highly hydrophobic region, preceded or flanked by regions containing polar amino acids. 8.7 Signal sequences initiate translocation

26. Figure 8.22 Ribosomes synthesizing secretory proteins are attached to the membrane via the signal sequence on the nascent polypeptide. 8.7 Signal sequences initiate translocation

27. Figure 8.23 The two domains of the 7S RNA of the SRP are defined by its relationship to the Alu sequence. Five of the six proteins bind directly to the 7S RNA. Each function of the SRP is associated with a particular protein(s). 8.7 Signal sequences initiate translocation

28. Figure 8.24 Does a signal sequence enter an aqueous tunnel created by resident ER membrane proteins? 8.8 The translocon forms a pore

29. Figure 8.25 The translocon consists of SRP, SRP receptor, Sec61, TRAM, and signal peptidase. 8.8 The translocon forms a pore

30. Figure 8.26 BiP acts as a ratchet to prevent backward diffusion of a translocating protein. 8.8 The translocon forms a pore

31. Integral membrane protein is a protein (noncovalently) inserted into a membrane; it retains its membranous association by means of a stretch of ~25 amino acids that are uncharged and/or hydrophobic. Transmembrane protein is a component of a membrane; a hydrophobic region or regions of the protein resides in the membrane, and hydrophilic regions are exposed on one or both sides of the membrane. 8.9 How do proteins enter and leave membranes?

32. Figure 8.27 Group I and group II transmembrane proteins have opposite orientations with regard to the membrane. 8.9 How do proteins enter and leave membranes?

33. Figure 8.28 The orientations of the termini of multiple membrane-spanning proteins depends on whether there is an odd or even number of transmembrane segments. 8.9 How do proteins enter and leave membranes?

34. Figure 8.29-1 Does a signal sequence interact directly with the hydrophobic environment of the lipid bilayer or does it directly enter an aqueous tunnel created by resident ER membrane proteins? 8.9 How do proteins enter and leave membranes?

35. Figure 8.29-2 How does a transmembrane protein make the transition from moving through a proteinaceous channel to interacting directly with the lipid bilayer? 8.9 How do proteins enter and leave membranes?

36. Figure 8.29-3 Proteins may be associated with one face of a membrane by acyl linkages to fatty acids. 8.9 How do proteins enter and leave membranes?

37. Figure 8.30 Proteins that reside in membranes enter by the same route as secreted proteins, but transfer is halted when an anchor sequence passes into the membrane. If the anchor is at the C-terminus, the bulk of the protein passes through the membrane and is exposed on the far surface. 8.10 Anchor signals are needed for membrane residence

38. Figure 8.31 A combined signal- anchor sequence causes a protein to reverse its orientation, so that the N-terminus remains on the inner face and the C- terminus is exposed on the outer face of the membrane. 8.10 Anchor signals are needed for membrane residence

39. Figure 8.32 The signal-anchor of influenza neuraminidase is located close to the N-terminus and has a hydrophobic core. 8.10 Anchor signals are needed for membrane residence

40. Figure 8.33 The Tat and SecYEG ystems are used for proteins that are translocated across the inner membrane. YidC may be used ith or without SecYEG to insert proteins into the inner membrane. 8. 11 Bacteria use both co- translational and post-translational translocation

41. Figure 8.34 SecB is a chaperone that transfers a nascent protein to SecA, which is a peripheral membrane protein associated with the integral membrane protein complex SecYEG. Translocation requires hydrolysis of ATP and a protonmotive force. Leader peptidase is an integral membrane protein that cleaves the leader sequence. 8. 11 Bacteria use both co- translational and post-translational translocation

42. Figure 8.35 Nuclear pores are used for import and export. 8.12 Pores are used for nuclear ingress and egress

43. Figure 8.36 Nuclear pores appear as annular structures by electron microscopy. The bar is 0.5 mm. Photograph kindly provided by Ronald Milligan. 8.13 Nuclear pores are large symmetrical structures

44. Figure 8.37 A model for the nuclear pore shows 8-fold symmetry. Two rings form the upper and lower surfaces (shown in yellow); they are connected by the spokes (shown in green on the inside and blue on the outside). Photograph kindly provided by Ronald Milligan. 8.13 Nuclear pores are large symmetrical structures

45. Figure 8.38 The outsides of the nuclear coaxial (cytoplasmic and nucleoplasmic) rings are connected to radial arms. The interior is connected to spokes that project towards the transporter that contains the central pore. 8.13 Nuclear pores are large symmetrical structures

46. Figure 8.39 The nuclear pore complex spans the nuclear envelope by means of a triple ring structure. The side view shows two-fold symmetry from either horizontal or perpendicular axes. 8.13 Nuclear pores are large symmetrical structures

47. Figure 8.40 Nuclear localization signals have basic residues. 8.13 Nuclear pores are large symmetrical structures

48. Exportins are transport receptors that bind cargo in the nucleus, and translocate into the cytoplasm where they release the cargo. Importins are transport receptors that bind cargo in the cytoplasm, and translocate into the nucleus where they release the cargo. Nucleoporin was originally defined to describe the components of the nuclear pore complex that bind to the inhibitory lectins, but now is used to mean any component of the basic nuclear pore complex. Translocation of a chromosome describes a rearrangement in which part of a chromosome is detached by breakage and then becomes attached to some other chromosome. 8.15 Transport receptors carry cargo proteins through the pore

49. Figure 8.42 There are multiple pathways for nuclear export and import. 8.15 Transport receptors carry cargo proteins through the pore

50. Figure 8.41 A carrier protein binds to a substrate, moves with it through the nuclear pore, is released on the other side, and must be returned for reuse. 8.15 Transport receptors carry cargo proteins through the pore

51. Figure 8.43 The assay for nuclear pore function uses permeabilized cells. 8.15 Transport receptors carry cargo proteins through the pore

52. Figure 8.44 Nuclear import takes place in two stages. Both docking and translocation depend on cytosolic components. Translocation requires nucleoporins. 8.15 Transport receptors carry cargo proteins through the pore

53. Figure 8.45 The state of the guanine nucleotide bound to Ran controls directionality of nuclear import and export. 8.15 Transport receptors carry cargo proteins through the pore

54. Figure 8.46 Importin-b consists of 19 HEAT repeats organized in a right-handed superhelix. Each HEAT unit consists of two a-helices (A and B) lying at an angle to one another. Importin-b is folded tightly around the IBB domain of importin-a. 8.15 Transport receptors carry cargo proteins through the pore

55. Figure 8.47 Importin-b binds Ran-GDP through close contacts to the N- terminal HEAT repeats and to repeats 7-8. The structure is significantly different from the importin-b importin-a structure. 8.15 Transport receptors carry cargo proteins through the pore

56. Figure 8.48 The common feature in proteins that are exported from the nucleus to the cytosol is the presence of an NES. 8.15 Transport receptors carry cargo proteins through the pore

57. Figure 8.49 The ubiquitin cycle involves three activities. E1 is linked to ubiquitin. E3 binds to the substrate protein. E2 transfers ubiquitin from E1 to the substrate. Further cycles generate polyubiquitin. 8.15 Transport receptors carry cargo proteins through the pore

58. Figure 8.50 An archaeal 20S proteasome is a hollow cylinder consisting of rings of  and  subunits. Photographs kindly provided by Robert Huber. 8.15 Transport receptors carry cargo proteins through the pore

59. Figure 8.50 An archaeal 20S proteasome is a hollow cylinder consisting of rings of  and  subunits. Photographs kindly provided by Robert Huber. 8.15 Transport receptors carry cargo proteins through the pore

60. 1. Synthesis of all proteins starts on ribosomes that are "free" in the cytosol. 2. The N-terminal region of a secreted protein provides a signal sequence that causes the nascent protein and its ribosome to become attached to the membrane of the endoplasmic reticulum. 3. A secreted protein passes completely through the membrane into the ER lumen. 4. Bacteria have components for membrane translocation that are related to those of eukaryotes, but translocation often occurs by a post-translational mechanism. 8.17 Summary

61. 5. Nuclear pore complexes are massive structures embedded in the nuclear membrane, and are responsible for all transport of protein into the nucleus and RNA out of the nucleus. 6. Proteins that are actively transported into the nucleus require specific NLS sequences, which are short, but do not seem to share common features except for their basicity. 7. Proteins that are exported from the nucleus have specific NES sequences, which share a pattern of leucine residues; they may bind to nucleoporins. 8. The major system responsible for bulk degradation of proteins, but also for certain specific processing events, is the proteasome, a large complex that contains several protease activities.