1. How proteins get out of the Golgi apparatus? TGN

2. Proteins that exit the Golgi apparatus are directed to three possible destinations

4. Lysosomes are ’bags’ filled with hydrolytic enzymes

5. 1. Acid phosphatase 2. Deoxiribonuclease 3. Ribonuclease 4. Galattosidase 5. Mannosidase 6. Esosaminidase 7. Carboxipeptidase 8. Amminopeptidase 9. Elastase 10. D-cathepsin Many different enzymatic activities are present in lysosomes

6. Acid phosphatase can be demonstrated by hystochemistry  -glycerophosphate glycerol P + lead nitrate P +lead phosphate ACID PHOSPHATASE

7. Enzymes can be visualized by hystochemical reactions Acid phosphatase

8. DIFFERENT INTRACELLULAR COMPARTMENTS HAVE DIFFERENT pH pH CAN BE MEASURED BY THE USE OF pH- SENSITIVE FLUORESCENT PROBES LYSOSOMES HAVE A LOW pH OF ≈5-red ENDOSOMES pH5.5-6.5 blue-green

9. Lysosome are heterogeneous

10. Mannose 6-phosphate is a signal that directs a lysosomal hydrolase to lysosomes

11. MANNOSE phosphotransferase UDP-GlcNAc UMP MAN-6-P-GlcNAc MAN-6-P phosphoglycosidase GlcNAc PROTEIN Lysosome targeting signal forms in 2 steps

12. Mannose-6-phosphate signal formation

13. A phosphotransferase recognizes lysosomal hydrolases in the Golgi apparatus

14. LYSOSOMAL HYDROLASE PHOSPHOTRANSFERASE UDP-GlcNAc UMP catalytic site recognition site A phosphotransferase recognizes lysosomal hydrolases in the Golgi apparatus

15. Newly synthesized lysosomal hydrolases are delivered from Golgi apparatus to endosomes

16. M6P E Clathrin M6P receptor + GOLGI APPARATUS ENDOSOME Lysosomal enzymes are transported to endosomes

17. What happens if the phosphotransferase is lacking?

18. Lysosomal diseases are caused by a defect in the enzymes that degrade a variety of molecules within the cell. Non-degraded compounds accumulate in the lysosomes and alter their function. MUCOPOLYSACCHARIDOSIS SPHINGOLIPIDOSES OLIGOSACCHARIDOSIS They are progressive diseases that lead to severe functional alterations. Some of them are lethal. Autosomal recessive. Frequency ≈ 1/5000. Lysosomal diseases are rare and severe, genetic diseases CLASSIFICATION

19. Multiple pathways deliver materials to lysosomes

20. Autophagy degrades proteins and organelles, when needed

22. Secretory pathways can be constitutive or regulated

23. proteina di membrana lipide di membrana segnale TGN proteina solubile SECREZIONE COSTITUTIVA SECREZIONE REGOLATA Secretory pathways can be constitutive or regulated

24. CONSTITUTIVEREGULATED all cells continuous vescicles do not accumulate short-lived vesicles signal not necessary no concentration specialized cells non-continuous vesicles accumulate long-lived vesicles signal necessary concentration Secretion can be either constitutive or regulated

25. We can envisage 4 steps in protein secretion 1.Vesicle formation from the TGN (BUDDING) 2.Vesicle maturation 3.Vesicle transport to the plasma membrane 4.Vesicle fusion to the plasma membrane

26. 1 VESICLE FORMATION (BUDDING)

27. 2 Secretory granules undergo maturation

28. Some proteins undergo processing during secretory granule maturation

29. Secretory granules move along microtubules 3

30. DIC MICROSCOPY 3 Secretory granules move along microtubules

31. 3

32. Two families of molecular motors move along microtubules

33. Kinesin motors ‘walk’ on microtubules

34. Exocytic vesicles and granules fuse to the plasma membrane 4

35. When a vesicle fuse to the plasma membrane: 1.Its content is discharged in the extracellular space 2.The vesicle membrane fuse with the plasmamembrane 3.Proteins of the vesicle membrane are delivered to the plasmamembrane 4

36. Ca++ IONS PLAY A KEY ROLE IN THE EXOCYTIC PROCESS Ca++ pump Ca++, Na+

37. Exocytosis of a mast cell is very rapid after the signal histamine secretion

38. Figure 13-69 Molecular Biology of the Cell (© Garland Science 2008) Exocytosis can be polarized

39. Synaptic vesicle fusion occurs very rapidly

40. For fusion to occur membranes should be closer than 1.5 nm

41. SNARE proteins play a crucial role in the fusion process

42. In some cases the fusion step can be very short KISS AND RUN

43. Rapid formation of synaptic vesicles occurs in a nerve cell

44. Figure 13-70 Molecular Biology of the Cell (© Garland Science 2008) Exocytosis is needed for plamamembrane recycling

45. Protein sorting in the secretory pathway requires signals No signal needed Aggregation:  pH e  Ca++ Disulfide bond: CgB, POMC Protease cleavage site: Prorenin Carboxypeptidase E: Proinsulin and Proenkephalin Carboxi terminal: PC2 CONSTITUTIVEREGULATED

46. Many cell types, like epithelial cells, are polarized Apical membrane Basolateral membrane Tight junction

47. Epithelia cells can be cultured on filters in bicameral systems They form polarized monolayers TER BIOTIN

48. Protein secretion in polarized cells requires sorting signals Ap Bl cytosolic sequence transmembrane domain GPI anchor? (N or O) sugars cytosolic sequences similar to endocytic signals

49. In a polarized epitelial there are two ways to sort proteins to the plasma membrane

50. Figure 13-71 Molecular Biology of the Cell (© Garland Science 2008) Nerve cells are also well polarized

51. There are similarities in the polarity of nerve cells and epithelial cells

52. THE END