How proteins get out of the Golgi apparatus? TGN

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

Lysosomes are ’bags’ filled with hydrolytic enzymes

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

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

Enzymes can be visualized by hystochemical reactions Acid phosphatase

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 pH blue-green

Lysosome are heterogeneous

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

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

Mannose-6-phosphate signal formation

A phosphotransferase recognizes lysosomal hydrolases in the Golgi apparatus

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

Newly synthesized lysosomal hydrolases are delivered from Golgi apparatus to endosomes

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

What happens if the phosphotransferase is lacking?

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

Multiple pathways deliver materials to lysosomes

Autophagy degrades proteins and organelles, when needed

Secretory pathways can be constitutive or regulated

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

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

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

1 VESICLE FORMATION (BUDDING)

2 Secretory granules undergo maturation

Some proteins undergo processing during secretory granule maturation

Secretory granules move along microtubules 3

DIC MICROSCOPY 3 Secretory granules move along microtubules

3

Two families of molecular motors move along microtubules

Kinesin motors ‘walk’ on microtubules

Exocytic vesicles and granules fuse to the plasma membrane 4

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

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

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

Figure Molecular Biology of the Cell (© Garland Science 2008) Exocytosis can be polarized

Synaptic vesicle fusion occurs very rapidly

For fusion to occur membranes should be closer than 1.5 nm

SNARE proteins play a crucial role in the fusion process

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

Rapid formation of synaptic vesicles occurs in a nerve cell

Figure Molecular Biology of the Cell (© Garland Science 2008) Exocytosis is needed for plamamembrane recycling

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

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

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

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

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

Figure Molecular Biology of the Cell (© Garland Science 2008) Nerve cells are also well polarized

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

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