Vesicle Transport. Vesicle pathway: many compartments, interconnected by trafficking routes 3/17/14

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1 Vesicle Transport Vesicle Formation Curvature (Self Assembly of Coat complex) Sorting (Sorting Complex formation) Regulation (Sar1/Arf1 GTPases) Fission () Membrane Fusion SNARE combinations Tethers Regulation by Rabs SM Proteins Vesicle pathway: many compartments, interconnected by trafficking routes 1

2 Two Key Steps: 1. sorting during vesicle formation 2. targeting during vesicle fusion Morphological Experiments Suggest Role of Vesicle Coats 2

3 Each type acts at distinct locations Components that participate in budding of coated vesicles" Figure 17-51! 3

4 Clathrin coated vesicles Clathrin cycle 4

5 Dynamin pinches of the vesicles GTPase Shibire mutant has coated pits but no budding off of synaptic vesicles GTP hydrolysis by dynamin is required for pinching off of clathrin-coated vesicles " Figure 17-55! 5

6 Clathrin adaptins and dynamin Figure 7.22 Phagocytosis Pinocytosis Receptor-Mediated Endocytosis EXTRACELLULAR FLUID Solutes Pseudopodium Plasma membrane Ligand Receptor Coat proteins Food or other particle Coated pit Coated vesicle Food vacuole Vesicle CYTOPLASM 6

7 Figure 7.22a Phagocytosis Pseudopodium of amoeba EXTRACELLULAR FLUID Solutes Pseudopodium Bacterium Food vacuole 1 µm An amoeba engulfing a bacterium via phagocytosis (TEM). Food or other particle Food vacuole CYTOPLASM Figure 7.22b Pinocytosis 0.5 µm Plasma membrane Pinocytosis vesicles forming in a cell lining a small blood vessel (TEM). Vesicle 7

8 Figure 7.22c Receptor-Mediated Endocytosis Plasma membrane Coat proteins Ligand Receptor Coat proteins 0.25 µm Top: A coated pit. Bottom: A coated vesicle forming during receptor-mediated endocytosis (TEMs). Coated pit Coated vesicle The macromolecules that are degraded in the lysosome arrive by endocytosis, phagocytosis, or autophagy. 8

9 Transport from the ER to the Golgi When the protein is properly folded, COPII coated vesicles transport the proteins via the vesicular tubular cluster (vtc) to the cis-golgi network. The COPII coating is removed (Sar1 hydolyzes GTP) and the vesicles fuse with each other to form the vtc. The vtc is motored along microtubules that function like railroad tracks. The vtc fuses with the cis- Golgi network. Some proteins exiting the ER are returned to the ER by COPI coated vesicles. These proteins are identified by the presence of specific signal sequences that interact with the COPI vesicles or associate with specific receptors. Example of retrieved protein: ER chaperones like BiP that are mistakenly transported. This example describes the situation of BiP. BiP has the signal sequence, KDEL. When BiP escapes the ER, it associates with the KDEL receptor. The slightly acid environment of the vtc and Golgi favor this association. When the returning vesicle fuses with the ER, the neutral ph of the ER causes BiP to dissociate from the receptor. 9

10 Proteins exiting the ER join the Golgi apparatus at the cis Golgi network. The Golgi apparatus consists of a collection of stacked compartments. nucleus Cell membrane The Golgi Apparatus has two major functions: 1. Modifies the N-linked oligosaccharides and adds O-linked oligosaccharides. 2. Sorts proteins so that when they exit the trans Golgi network, they are delivered to the correct destination. 10

11 Modification of the N-linked oligosaccharides is done by enzymes in the lumen of various Golgi compartments. While N-linked glycosylation appears to function in protein-folding in the lumen of the ER, the function of the oligosaccharide modifications occurring in the Golgi is largely unknown. One ultimate destination of some proteins that arrive in the TGN is the lysosome. These proteins include acid hydrolases. Lysosomes are like the stomach of the cell. They are organelles surrounded by a single membrane and filled with enzymes called acid hydrolases that digest (degrade) a variety of macromolecules. A vacular H+ ATPase pumps protons into the lysosome causing the ph to be ~5. 11

12 The macromolecules that are degraded in the lysosome arrive by endocytosis, phagocytosis, or autophagy. The acid hydrolases in the lysosome are sorted in the TGN based on the chemical marker mannose 6-phosphate. The phosphate is added in the Golgi This was first attached in the ER. 12

13 Adaptins bridge the M6P receptor to clathrin. Hydrolases are transported to the late endosome which later matures into a lysosome. Acidic ph causes hydrolase to dissociate from the receptor. Guidance of vesicular transport SNAREs: specificity and fusion Rab GTPases: initial docking and tethering of vesicles to target membranes and matching of v- and t- SNAREs 13

14 SNARE proteins guide vesicular transport 20 SNAREs, vesicle-snares, target-snares SNAREs specify compartment identity and control specificity 4 α helices in trans-snare complexes 14

15 Rab proteins ensure the specificity of vesicle docking >30 Rabs On cytosolic surface C-terminal regions are variable: Bind to other proteins, including GEFs After docking SNAREs may mediate membrane fusion SNARE complex 15

16 The entry of enveloped viruses into cells HIV Similar to SNAREs Proteins leave the ER in COPII-coated transport vesicles ER exit sites (no ribosomes) Selective process 16

17 Only properly folded and assembled proteins can leave the ER Chaperones cover up exit signals Homotypic membrane fusion to form vesicular tubular clusters 17

18 Vesicular tubular clusters Lacks many of the ER proteins Short-lived COPI-coated Retrograde transport: carry back the ER resident proteins that leaked out ER retrieval signals: KKXX in ER membrane proteins, KDEL sequence in soluble ER resident proteins ph controls affinity of KDEL receptors Membrane proteins in Golgi and ER have shorter TM domains (15 aa) Cholesterol 18

19 Ordered series of Golgi compartments Cisternae, tubular connections Plant cell Two main classes of N-linked glycosilation core complex oligosaccharides high-mannose oligosaccharides 19

20 Oligosaccharide processing in the ER and the Golgi Histochemical stains: biochemical Compartmentalization of the Golgi Functional compartmentalization 20

21 Transport through the Golgi may occur by vesicular transport or cisternal maturation (not mutually exclusive) Collagen rods- SUBJECT OF PAPER Scales in algae Two Key Steps: sorting during vesicle formation & targeting during vesicle fusion 21

22 Summary 1. Vesicular transport, biosynthetic-secretory and endocytic pathways; 2. Coated vesicles; 3. Coat assembly and disassembly, budding, dynamin, coat-recruitment GTPases; 4. Targeting and fusion by Rab GTPases, SNAREs; 5. ER to Golgi: COPII, folding, fusion (cluster), retrograde; 6. Golgi apparatus structure and polarity; 7. Continuation of glycosylation; 8. Compartmentalization of Golgi cisternae. 22