Protein Sorting, Intracellular Trafficking, and Vesicular Transport

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1 Protein Sorting, Intracellular Trafficking, and Vesicular Transport Noemi Polgar, Ph.D. Department of Anatomy, Biochemistry and Physiology Phone:

2 Outline Part 1- Trafficking within the cell Intracellular compartments and protein and vesicular trafficking Nuclear import/export ER-Golgi transport Part 2- Trafficking to and from the cell membrane The endocytic pathway Endolysosomal degradation The exocytic pathway Article discussion (Chapters 12 and 13 in Molecular Biology of the Cell)

3 Cellular compartments Molecular Biology of the Cell ( Garland Science 2008)

4 Secretory and endocytic pathways Molecular Biology of the Cell ( Garland Science 2008)

5 Protein trafficking Molecular Biology of the Cell ( Garland Science 2008)

6 How do newly synthesized proteins find their proper location? Mechanisms involve: Signal peptides (zip codes) Gated protein translocation channels Organelle specific receptors Translocases Chaperones Soluble escorting factors Energy dependence (ATP, GTP, membrane potential)

7 Typical Signal Sequences (Zip codes) Molecular Biology of the Cell ( Garland Science 2008)

8 Cell Sorting Gated transport (between topographically similar compartments - nucleus to cytosol) Transmembrane transport (between topographically different compartments - cytosol to lumen) Vesicular transport (between topographically similar compartments - lumens)

9 Transport between the nucleus and the cytosol Molecular Biology of the Cell ( Garland Science 2008)

10 Nuclear pore complexes in the nuclear envelope

11 Gated diffusion barrier of the Nuclear Pore Complexes Molecular Biology of the Cell ( Garland Science 2008)

12 Nuclear import receptors Molecular Biology of the Cell ( Garland Science 2008)

13 Ran-mediated directionality of nuclear transport Molecular Biology of the Cell ( Garland Science 2008)

14 Ran-mediated directionality of nuclear transport Molecular Biology of the Cell ( Garland Science 2008)

15 Ran-GTP: nuclear import receptor: cargo complex Molecular Biology of the Cell ( Garland Science 2008)

16 Cycles of loading and unloading of nuclear import receptors Molecular Biology of the Cell ( Garland Science 2008)

17 Early study of nuclear import signals Immunofluorescence of the cellular location of the SV40 T-antigen Kalderon et al, 1984, Cell

18 Metaphase requires breakdown and reassembly of nuclear envelope

19 Endoplasmatic reticulum - and its targeted transport - Molecular Biology of the Cell ( Garland Science 2008)

Endoplasmic Reticulum (ER) functions Protein synthesis Protein modification oligosaccharide modification Quality control: protein folding

20 Endoplasmic Reticulum (ER) functions Protein synthesis Protein modification oligosaccharide modification Quality control: protein folding Secretory pathway: where all plasma membrane proteins and secreted proteins begin their journey Lipid synthesis Ca2+ storage Detoxification reactions

21 What distinguishes the rough and smooth ER? Different Functions Rough ER: contains ribosomes and is involved in protein synthesis and folding. Contains sites for vesicle budding and export of cargo to the Golgi Smooth ER: lacks ribosomes and is implicated in drug metabolism, steroid synthesis, and Ca2+ storage

22 Figure 12-36a Molecular Biology of the Cell ( Garland Science 2008) Rough ER

23 Figure 12-36b Molecular Biology of the Cell ( Garland Science 2008) Smooth ER

24 Smooth and rough ER in 3D Figure 12-36c Molecular Biology of the Cell ( Garland Science 2008)

25 Co-translational and post-translational protein translocation Figure Molecular Biology of the Cell ( Garland Science 2008)

26 Study of Co-translational translocation in the ER via Microsomes

27 Co-translational translocation Transmembrane proteins embedded in the ER membrane. Water-soluble proteins fully translocated into the ER lumen

28 How do ER signal sequences and SRP direct ribosomes to the ER membrane? Molecular Biology of the Cell ( Garland Science 2008)

29 Molecular Biology of the Cell ( Garland Science 2008)

Sec61 and the ER translocon 3 subunits, multiple hydrophobic transmembrane helices protein conducting channel Sec61 associates with several TRAPs, (translocon associated protein),

30 Sec61 and the ER translocon 3 subunits, multiple hydrophobic transmembrane helices protein conducting channel Sec61 associates with several TRAPs, (translocon associated protein), oligosaccharyltransferases, and signal peptidases Anchors to ribosome Binding of ribosome and signal sequence by Sec61 allows pore opening across the membrane of 4-6 nm Signal sequence forms a loop in the pore with the N-terminus exposed to the cytoplasm

31 Structure of the Sec61 complex Molecular Biology of the Cell ( Garland Science 2008)

32 Molecular Biology of the Cell ( Garland Science 2008) Protein translocation variations on a theme

33 Model to explain how a soluble protein is translocated across the ER membrane Figure Molecular Biology of the Cell ( Garland Science 2008)

34 Stop-Transfer Sequences how a single-pass transmembrane protein integrates into the ER Molecular Biology of the Cell ( Garland Science 2008)

35 Stop-Transfer Sequences Double-pass transmembrane proteins

36 Stop-Transfer Sequences Multipass transmembrane protein

37 Intracellular membrane traffic Secretory and endocytic pathways Molecular Biology of the Cell ( Garland Science 2008)

38 Molecular Biology of the Cell ( Garland Science 2008) Vesicular Transport

39 Molecular Biology of the Cell ( Garland Science 2008) Coated Vesicles

40 Molecular Biology of the Cell ( Garland Science 2008) EM of coated vesicles

41 Clathrin-coated vesicles Molecular Biology of the Cell ( Garland Science 2008)

42 The assembly and disassembly of a clathrin coat Molecular Biology of the Cell ( Garland Science 2008)

43 Molecular Biology of the Cell ( Garland Science 2008) Phosphoinositides

44 Molecular Biology of the Cell ( Garland Science 2008) Adaptor proteins

45 Dynamin pinches off vesicles from the membrane Dynasore Molecular Biology of the Cell ( Garland Science 2008)

46 Accumulation of clathrin-coated vesicles in Dynamin mutant Molecular Biology of the Cell ( Garland Science 2008)

47 The assembly and disassembly of a clathrin coat Molecular Biology of the Cell ( Garland Science 2008)

48 Model for COPII-dependent cargo selection and vesicle formation Dancourt and Barlowe, Annual Reviews of Biochemistry 2010, 80:71-99

COPII proteins Sec13 and Sec31 form the outer shell of the coat Membrane invagination, fission Cargo-containing COPII-coated transport container is released into the cytoplasm Vesicle diameter ~

49 COPII proteins Sec13 and Sec31 form the outer shell of the coat Membrane invagination, fission Cargo-containing COPII-coated transport container is released into the cytoplasm Vesicle diameter ~ nm Un-coating is also mediated by Sec23/24 (both coat assembly and dis-assembly factor) Un-coating is regulated by GTPase-Activating factor (GAP) Molecular Biology of the Cell ( Garland Science 2008)

50 Exit and Return: COPII and COPI Molecular Biology of the Cell ( Garland Science 2008)

51 Model for COPI-dependent cargo selection and vesicle formation Beck et al. FEBS Letters 583 (2009)

52 Coated vesicles

53 Vesicular tubular clusters transport proteins between ER and Golgi Molecular Biology of the Cell ( Garland Science 2008)

54 Transport from the ER via the Golgi apparatus Molecular Biology of the Cell ( Garland Science 2008)

55 Cargo recruitment of ER vesicles Major checkpoint proteins to exit must be properly folded, complexes fully assembled Others are retained by chaperones eventually tarnsported to cytosol for degradation Molecular Biology of the Cell ( Garland Science 2008)

Vesicular tubular clusters Heterotypic fusion: - Fusion of pre-golgi intermediates, fusion between different compartments - Trans-SNARE pairing Homotypic fusion: -

56 Vesicular tubular clusters Heterotypic fusion: - Fusion of pre-golgi intermediates, fusion between different compartments - Trans-SNARE pairing Homotypic fusion: - Used to built larger vesicular structures- vesicular tubular cluster - Clusters mediate transport between ER and Golgi Molecular Biology of the Cell ( Garland Science 2008)

57 Vesicular tubular clusters bud off COPI-coated transport vesicles of their own serve as a retrieval pathway Molecular Biology of the Cell ( Garland Science 2008)

Retrieval of ER resident proteins KDEL receptor captures the soluble ER resident proteins and carries them in COPI-coated transport vesicles back to the ER

58 Retrieval of ER resident proteins KDEL receptor captures the soluble ER resident proteins and carries them in COPI-coated transport vesicles back to the ER affinity! retrieval begins in vesicular tubular clusters and continues from later parts of the Golgi apparatus Molecular Biology of the Cell ( Garland Science 2008)

59 The Golgi complex One of the first organelles described A collection of flattened, membrane-enclosed compartments Molecular Biology of the Cell ( Garland Science 2008)

60 The Golgi complex cis Face is closest to ER tubular links between cisternae - complex Molecular Biology of the Cell ( Garland Science 2008)

61 The functional compartmentalization of the Golgi apparatus

62 Transport through the Golgi - the cisternal maturation model - Golgi cisternae as dynamic structures that mature from early to late New cis cisternae continually form as vesicular tubular clusters arrive from the ER, mature to become a medial cisterna and then a trans cisterna A cisterna moves through the Golgi stack with cargo in its lumen supported by studies of yeast Golgi complexes

63 Transport through the Golgi - the vesicle transport model - Golgi cisternae long-lived structures cisternae hold characteristic set of resident proteins in place cargo proteins transported by vesicles between cisternae Proteins move through the Golgi stack supported by studies of membrane protein aggregates

64 Transport through the Golgi Likely aspects of both models are true stable core of long-lasting cisternae, while rim regions may undergo continuous maturation Molecular Biology of the Cell ( Garland Science 2008)

65 Intracellular membrane traffic Secretory and endocytic pathways Molecular Biology of the Cell ( Garland Science 2008)

66 Vesicle Delivery at Target Membrane Molecular Biology of the Cell ( Garland Science 2008)

67 Rab proteins guide vesicle targeting Molecular Biology of the Cell ( Garland Science 2008)

68 Nature Reviews Molecular Cell Biology 10, (August 2009)

69 Rab effectors Facilitate vesicle transport, membrane tethering, and fusion by interacting with Rabs Structure of effectors vary highly, which adds specificity in vesicle targeting 2 main classes Long coiled-coil proteins Large multisubunit complexes Rab effectors can be motor proteins, tethering proteins Rab effectors can interact with SNAREs Same Rab protein can interact with multiple effectors

Exocyst = 8-protein complex for polarized exocytosis conserved from yeast to humans Sec15 binds to Rab GTPases on vesicles destined for exocytosis Sec10 connects Sec15 to

70 Exocyst = 8-protein complex for polarized exocytosis conserved from yeast to humans Sec15 binds to Rab GTPases on vesicles destined for exocytosis Sec10 connects Sec15 to the rest of exocyst on plasma membrane necessary prior to V- & T-SNARE mediated membrane fusion Assembly is regulated by Ral and Rho family of GTPases, and phosphorylation

71 Vesicle delivery can be induced by signaling Insulin signaling quickly induces Glut4 exocytosis Molecular Biology of the Cell ( Garland Science 2008)

72 Vesicle delivery can be induced by signaling Anti-diuretic hormone induces AQP2 exocytosis

73 Rab proteins in facilitating the docking of transport vesicles

Rabs in the formation of specialized membrane patches A Rab domain can be disassembled and replaced by a different Rab domain, changing the identity of

74 Rabs in the formation of specialized membrane patches A Rab domain can be disassembled and replaced by a different Rab domain, changing the identity of an organelle. Such ordered recruitment of sequentially acting Rab proteins is called a Rab cascade. Molecular Biology of the Cell ( Garland Science 2008)

75 SNARE proteins mediate membrane fusion Molecular Biology of the Cell ( Garland Science 2008)

76 SNARE proteins SNAp Receptor (SNARE) proteins Key last step for vesicle docking and membrane fusion Contain transmembrane domain, coiled-coiled helical bundles, and small luminal domains v-snare: vesicle membrane SNARE: synaptobrevin t-snare: target membrane SNARE: syntaxin Becomes disassembled by NSF (ATPase)

77 Dissociation of SNARE pairs by NSF after a membrane fusion cycle is completed Molecular Biology of the Cell ( Garland Science 2008)

78 Polarized trafficking in epithelia Molecular Cell Biology. 4th edition. Lodish H, Berk A, Zipursky SL, et al. New York: W. H. Freeman; 2000.

79 QUESTIONS?

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