Intracellular Vesicle Trafficking

Transcription

1 Intracellular Vesicle Trafficking Chi-Kuang Yao (IBC, Academia Sinica)

2 Compartmentalization makes difference between bacteria and yeast 1. More compartments with specific functions 2. Function in the Efficient way % of proteins are integral membrane proteins, 10-20% of proteins are membrane-associated proteins 2

3 Importance of intracellular vesicle trafficking 1. Inside network: material exchange and compartment maturation 2. Exterior to interior: Cells eat (nutrients), communicate and respond to environmental cues (signaling). 3. It failure is associated with diseases. 3

4 4

5 Outline 1 Molecular components of vesicular transport 2 ER to Golgi 3 Golgi to endosomal system 4 Plasma membrane to endosomal system 5 Secretory pathways Figure 13-3 Molecular Biology of the Cell ( Garland Science 2008) 5

6 1 Molecular components of vesicular transport 1. Cargo recognition 2. Assembly of coat proteins 3. Vesicle pinch off 4. Targeting, Docking, and fusion Figure 13-2 Molecular Biology of the Cell ( Garland Science 2008) 6

7 Coat proteins participate in distinct transport steps 1. Concentrate cargo 2. Stabilize vesicular integrity Figure 13-5 Molecular Biology of the Cell ( Garland Science 2008) 7

8 Major coat proteins Figure 13-4 Molecular Biology of the Cell ( Garland Science 2008) 8

9 Formation of COPII-coated vesicles 1. Recruit and activate Sar1 by Sar1-GEF 2. Sar1-GTP anchors to membrane and recruit Sec 23/24 (COPII subunits) Note: Arf1 mediated Clathrin-coated vesicle formation is very similar. 4. Sar1 GTP hydrolysis may lead to membrane scission 3. Adding Sec 13/31 to induce bud formation Figure 13-13a Molecular Biology of the Cell ( Garland Science 2008) 9

10 The assembly and disassembly of a Clathrin coat: cytosolic proteins Cytosolic proteins required for clathrin-mediated endocytosis (CME): Clathrin adaptor proteins for cargo selection Small GTPases for coat assembly Dynamin (GTPase) for vesicle pinch off Auxillin & Hsp70 (ATPase) to uncoat clathrin Figure 13-8 Molecular Biology of the Cell ( Garland Science 2008) 10

11 The assembly of Clathrin coat Triskelion: 3 HCs + 3 LCs The very first coat protein discovered by Barbara Pearse in Clathrin coat: 36 triskelions form 12 pentagons and 6 hexagons Figure 13-6, -7 Molecular Biology of the Cell ( Garland Science 2008) 11

12 Clathrin adaptor proteins bridge membrane, cargo and Clathrin coat Major cargo recognition complex at PM: AP-2 Binds PI(4,5)P 2 located at the inner leaflet of the plasma membrane. Binds cargo, brings in clathrin, AAK1 kinase phosphorylates m2 subunit, promotes clathrin assembly. Other adaptor proteins (CLASPs): GGAs, epsins, AP180/CALM, amphiphysin, disabled 2, b-arrestin, Numb, Stonin 2 Traub LM., Nat Rev Mol Cel Biol (2009) 12

13 Adaptor proteins in Clathrin-mediated vesicular transport lysosome AP-3 AP-2 AP-1 AP-1 AP-1 AP1: from TGN and endosome AP2: from plasma membrane AP3: to lysosome AP4:?? 13

14 Traub LM., Nat Rev Mol Cel Biol (2009) 14

15 Small GTPases for coat assembly 15

16 Vesicle pinch off: Dynamin (Clathrin coat vesicle) Dynamins: pinch-off GTPase, GTP hydrolysis generates twisting force that constricts the membrane Dynamin I: expressed in neurons and neuroendocrine cells Dynamin II: expressed in all cell types Dynamin III: testis, heart, brain and lung tissue 16

17 shibire encodes the sole Drosophila Dynamin (Koenig and Ikeda, 1989) Figure 13-12b Molecular Biology of the Cell ( Garland Science 2008) 17

18 Phosphoinositides mark organelles and membrane domains Chemistry: negatively charged phospholipids. Synthesis: in ER from cytidine diphosphate diacylglycerol with inositol. Delivery: via vesicular transport or specific transport proteins. Localization: cytosolic side of eukaryotic membrane. Interaction: Proteins containing PH domain, non-specific electrostatic interactions. Figure Molecular Biology of the Cell ( Garland Science 2008) 18

19 Structure and synthesis of phosphoinositides Red: kinase reaction Green: phosphatase reaction Figure Molecular Biology of the Cell ( Garland Science 2008) 19

20 Phosphoinositide binding motifs 20

21 Vesicle targeting guided by Rab GTPases 21

22 Rab GTPases have >60 members in the family * Rab GTPase: functioning like SarI and Arf in vesicular transport Cytosolic GDP-Rab-> membrane bound GTP-Rab. * Different Rab targeting to specific donor organelles membranebound GEFs and target organelles Rab effectors (various type of proteins) Table 13-1 Molecular Biology of the Cell ( Garland Science 2008) 22

23 Rabs and their effectors regulate vesicular trafficking 23

24 Stenmark H., Nat Rev Mole Cel Biol (2009) 24

25 Vesicle fusion by SNARE complexes 25

26 SNAREs mediate membrane fusion SNARE: stands for Soluble NSF Attachment REceptor" are a large protein superfamily consisting of more than 60 members in yeast and mammalian cells. v-snare: v: vesicle (1 polypeptide) t-snare: t: target (2-3 polypeptides) Figure Molecular Biology of the Cell ( Garland Science 2008) 26

27 SNAREs bring two layers of membrane in close proximity trans-snare complex membrane fusion cis-snare complex (complex in a single membrane) 27

28 SNARE disassembly: requires NSF cycle NSF:N-ethylmaleimide sensitive factor (an ATPase). The role of NSF is to loose these SNARE complexes once membrane fusion has occurred, using the hydrolysis of ATP as an energy source. Figure Molecular Biology of the Cell ( Garland Science 2008) 28

29 Specificity of SNARE assembling Jahn and Scheller, Nature reviews,

30 Ca 2+ dependent synaptic transmission 30

31 31

32 1 Molecular components of vesicular transport 2. Rab proteins (Targeting and Docking) 1. Coat proteins (Cargo recognition vesicle formation) 3. SNARE proteins (Fusion) Figure 13-2 Molecular Biology of the Cell ( Garland Science 2008) 32

33 2 Transport from ER to Golgi 33

34 ER: cargo selection ER exit: 1. Default: in the lack of retention signal Selective: Some proteins have transport signals that are bound by corresponding receptors to facilitate ER exit. ( e.g. ERGIC53 a receptor for blood clotting factors, Factors V and VIII) 2. Bud from ER exit sites that lack of ribosome and dispersed throughout whole ER network. Figure 13-20, -21 Molecular Biology of the Cell ( Garland Science 2008)

35 ER resident protein retrieval 1. ER retrieval (retention) signal ER membrane proteins have KKXX motif at the extreme C-terminal end. -- interact directly with COPI coat ER soluble proteins have KDEL motif at the C-terminal end. -- interact with KDEL receptor at lower ph 6 and release in the ER at the neutral ph 2. Kin recognition Proteins function together, aggregate together to prevent the package into transport vesicles. Figure Molecular Biology of the Cell ( Garland Science 2008)

36 COPII-coated vesicles fused via Homotypic fusion after exiting from ER 36

37 Unfolded protein response UPR: quality control 1. initially to restore normal function of the cell by halting protein translation, while signaling to increase production of the molecular chaperones involved in removing misfolded proteins. Stringent UPR response may not be a good thing! Example: cystic fibrosis. 2. When this is not achieved within a certain time or the disruption is prolonged, the UPR aims to initiate the programmed cell death (apoptosis) of the cell. Figure 13-20, -21 Molecular Biology of the Cell ( Garland Science 2008)

38 Golgi apparatus Observed by Camillo Golgi in 1897 Received Nobel Prize in Physiology or Medicine together with Santiago RamÓn y Cajal for their work on nervous system in 1906 Figure Molecular Biology of the Cell ( Garland Science 2008) 38

39 Not all cells have organized Golgi stack Figure Molecular Biology of the Cell ( Garland Science 2008) Otsuga et al., JCB (1998) 39

40 N-glycosylation of proteins occurs in successive stages across the stack Figure Molecular Biology of the Cell ( Garland Science 2008) 40

41 Why add sugar groups on proteins? 1. Make the folding intermediates more soluble to prevent aggregation. 2. Establish the glyco-code that marks the progression of protein folding and mediates the binding with chaperones. 3. Prevent from protease degradation 4. Require for signaling ER N-linked glycosylation Golgi Figure Molecular Biology of the Cell ( Garland Science 2008) 41

42 N-linked and O-linked glycosylation in Golgi And serine O-linked glycosylation occurs on serine or threonine which can compete with phosphorylation. Figure Molecular Biology of the Cell ( Garland Science 2008) O-glycosylation enzymes also present in cytosol and nucleus. 42

43 Models for cargo transport in Golgi At the heart of this controversy is the COPI-coated vesicles. These vesicles bud from Golgi, and nobody knows what cargo they carried and which direction they go. In yeast, some secretory proteins can be secreted in the absence of COPI, suggesting that both models may coexist. Figure Molecular Biology of the Cell ( Garland Science 2008) 43

44 3 Transport from TGN to lysosome 44

45 Lysosome: the principle site of intracellular digestion The lysosomal hydrolases mainly come from ER-Golgi anterograde transport The lysosomal digestive targets mainly come from endocytosis Figure Molecular Biology of the Cell ( Garland Science 2008) 45

46 Digestive enzymes are compartmentalized in the lysosome * > 40 different kind of digestive enzymes * Requires ph for optimal activity (so, leaky hydrolases cannot work well) * Lysosomal transmembrane proteins are heavily glycosylated to avoid being digested by hydrolases. Figure Molecular Biology of the Cell ( Garland Science 2008) 46

47 M6P receptor mediates the transport of lysosomal hydrolases Figure Molecular Biology of the Cell ( Garland Science 2008) 47

48 Retromer: Another coat involved in retrograde of M6P receptor to Golgi Retromer- a multiprotein complex that assembles into a coat on endosomal membranes only when 1. It can binds to the cytoplasmic tails of the cargo receptors. 2. It can interact directly with a curved phospholipid bilayer 3. It can bind to a specific phosphorylated phosphatidylinositol lipid (acts as an endosomal marker) SNX: sorting nexin Induce or sense curvature Phox homology domain: binding to PI(3)P VPS: vaculolar protein sorting Cargo: e.g. mannose-6-phopsphate receptor acid hydrolase receptor

49 Selective autophagy 49

50 4 Transport from the plasma membrane: endocytosis 50

51 Endocytosis (nutrient uptake, signaling turnover) Cellular eating - Macrophage, Neutropils - Pathogens - Triggered process - Receptor-mediated Cellular drinking -Almost all cells -Constitutive process -Could be both Clathrin & caveolin coated -Uptake or recycle macromolecules - Signaling regulation -Receptor mediated -clathrin coat -a common signal peptide on the receptor 51 YXXy y: hydrophobic

52 Phagocytosis By professional phagocytes Macrophage Neutrophil Macrophages can also eat glass and latex beads. Live animal cells have do-not-eat-me signals on PM. Figure Molecular Biology of the Cell ( Garland Science 2008) 52

53 Pinocytosis: Clathrin-dependent vs. Clathrin-independent Clathrin-coated vesicle caveolae Uncoating: auxillin & Hsp70 (ATPase) Lipid raft-dependent endocytosis is in large part defined as the cholesterol-sensitive, clathrinindependent internalization of ligands and receptors from the plasma membrane. It encompasses the endocytosis of caveolae, smooth vesicles that form a subdomain of cholesterol. Dynamin-dependent Dynamin-dependent 53

54 Receptor-mediated endocytosis: LDL receptor, an example LDL particle (1 in 500) LDL receptor: endocytosis signal peptide is NPXY, interacting with adaptor protein AP2 EGFR also has NPXY motif but this motif also use non-ap2 clathrin-adaptor mediated endocytosis 54

55 Traub LM., Nat Rev Mol Cel Biol (2009) 55

56 Nobel Prize in Physiology or Medicine in 1985 For their discoveries concerning the regulation of cholesterol metabolism Joseph L. Goldstein Michael S. Brown UT Southwestern Medical Center, Dallas, TX USA 56

57 Fate of endocytosed receptor proteins EGFR LDLR, TfR Figure Molecular Biology of the Cell ( Garland Science 2008) 57

58 Multivesicular body sequesters proteins destined to be degraded by lysosome Figure Molecular Biology of the Cell ( Garland Science 2008) 58

59 ESCRTs sort endocytosed proteins into MVB ESCRT (endosomal sorting complex for transport) proteins exist in cytosol and recruit to endosomal membrane by phos-pi and ubiquitin-tag (could be one or multiple on the cytosolic site of receptor as well as K63-polyubiquitination) The active domain of receptor is isolated. Figure 13-57, -58 Molecular Biology of the Cell ( Garland Science 2008) 59

60 5 Transport from TGN to the cell exterior: exocytosis 60

61 Figure Molecular Biology of the Cell ( Garland Science 2008) Hormones, neurotransmitter, digestive enzymes 61

62 3 protein sorting pathways in TGN Figure Molecular Biology of the Cell ( Garland Science 2008) 62

63 The formation of secretory vesicles Figure Molecular Biology of the Cell ( Garland Science 2008) 63

64 Importance of intracellular vesicle trafficking 1. Inside network: material exchange and compartment maturation 2. Exterior to interior: Cells eat (nutrients), communicate and respond to environmental cues (signaling). 3. It failure is associated with diseases. 64

65 Synaptic vesicle recycling sustains neurotransmitter release in response to repetitive neuronal activity Why Endocytosis? Synaptic vesicle depletion - Vesicle replenishment Plasma membrane extension - Membrane equilibration Protein traffic jam - Active zone Clean up (Nobel lecture by Thomas Sudhof, 2014)

66 Clathrin-mediated endocytosis is one of major routes to recycle exocytic lipids and proteins Occurs at MILD stimulation Retrieves SINGLE synaptic vesicle Endocytosis immediately occurs upon exocytosis : A Coupling Mechanism Voltage-gated Ca 2+ channel (Adapted from Wu et al., elife, 2014) (Adapted from Milosevic et al., Neuron, 2011)

67 Modes of synaptic vesicle endocytosis