Lecture 3: The Golgi and Lysosomes Flashcards
Learning Objectives
- What is the structure and the functions of the Golgi apparatus?
- What is the structure and the functions of lysosomes?
Is the Golgi body bigger or smaller than the ER?
Smaller
What is the structure of the Golgi body?
The Golgi body is composed of layers of cisternae, which have vesicles budding off and joining them. There is a cis face and a trans face. The cis face receives vesicles from the ER, whilst the trans face buds off secretory vesicles which leave the Golgi.
The arrangement of the layers of cisternae in the Golgi body is as follows: cis Golgi network (CGN) cis cisterna medial cisterna trans cisterna trans Golgi network (TGN)
Discuss the transport of proteins and lipids in vesicles from the ER to the Golgi.
After transport vesicles budded from the ER have shed their COPII coats, they begin to fuse together using t- and v-SNARES to make vesicular tubular clusters. These clusters move along microtubules to the cis face of the Golgi. As they are going, they bud off vesicles to return proteins which shouldn’t have left the ER (proteins with the ER-retaining KDEL sequence), a correctional mechanism called the retrieval pathway. These vesicles have COPI coats. The ER membrane is constantly being replaced to counter the pinching off.
What are the functions of the Golgi apparatus?
The Golgi body accepts proteins and lipids from the ER and covalently modifies, labels and sorts them before sending them off to lysosomes, secretory vesicles or out of the cell.
One of the main forms of modification involves carbohydrates and trimming/building up of N-linked oligosaccharides which were attached in the ER, and attaching new oligosaccharides through O-linked glycosylation.
How are the different functions of the Golgi split up between its cisternae?
Cis Golgi Network (CGN) - SORTING phosphorylation of oligosaccharides on lysosomal proteins
cis cisterna - removal of Man
medial cisterna - removal of Man, addition of GlcNAc
trans cisterna - addition of Gal and NANA
Trans Golgi network (TGN) - SORTING sulfation of tyrosines and carbohydrates
The enzymes are kept in their respective cisternae too. This is partly by kin recognition - the proteins aggregate so that they are too large to fit into a vesicle to move to the next cisterna. The enzymes in the Golgi body are all transmembrane/membrane bound proteins (ER proteins are soluble and lumenal).
Discuss the modifications to N-linked olgiosaccharides on proteins which occur in the Golgi,
N-linked oligosaccharides can become complex oligosaccharides or high-mannose oligosaccharides from modifications in the Golgi. Remember that N-linked oligosaccharides are attached to an Asn-X-Ser/Thr (X can’t be proline). Asn = asparagine.
Complex oligosaccharides are generated when the original N-linked oligosaccharide added in the ER is trimmed and further sugars added. They still contain the original two N-acetyl glucosamine residues and some mannose residues. Galactose, sialic acids and fucose can be added.
High-mannose sugars are trimmed but have no extra sugars added in the Golgi.
If an oligosaccharide is exposed on the outside of the protein, it is likely to become a complex oligosaccharide, but if an oligosaccharide is hidden within a folded protein, it is inaccessible and likely to be a high-mannose oligosaccharide.
Describe O-linked glycosylation.
O-linked glycosylation occurs in the Golgi body and involves the addition of an oligosaccharide to the OH group of a Serine or a Threonine in the protein. The first sugar to be added is N-acetylglactosamine.
Heavily O-linked glycoproteins are mucins, the glycoproteins in mucus secretions and proteoglycan core proteins, the Golgi modifies to become proteoglycans. These are secreted and become components of the extracellular matrix.
Goblet cells in the gut secrete mucus and have lots of Golgi and large vesicles on the trans face (for secretion).
What is the first sugar added to the protein in a) N-linked glycosylation and b) O-linked glycosylation?
a) N-acetylglucosamine
b) N-acetylglactosamine
How do proteins move through the Golgi body?
There are two theories/models:
1) Vesicular transport model: the cisterna remain stationary and vesicles move forwards and backwards between them. This model is supported by electron microscopy.
2) Cisternal maturation model: the cisternae mature from cis to medial to trans, taking up the new enzymes which characterise their new identity. This model is supported by fluorescence and new microscopes.
How do proteins and lipids leave the Golgi after they have been modified?
They are packed into vesicles and leave via the specialised exit sires. They are sorted so as to go to the lysosome, secretory vesicles or to outside the cell.
Describe the important features of a lysosome.
Lysosomes are present in all eukaryotes and are full of soluble hydrolytic enzymes, which can digest the macromolecules of the cell (proteases, nucleases, glycosidases, etc).
These enzymes are all acid hydrolases: they require a proteolytic cleavage and an acid environment. Therefore the lumen of the lysosome has a pH of 4.5-5.0, which is maintained by the H+ ATPase, which pumps H+ across the membrane using energy from the hydrolysis of ATP. (This is the opposite of the chloroplast/mitochondria ATPases, but has a similar structure). This H+ concentration gradient also provides the energy to drive metabolite transport across the membrane.
The lysosomal membrane contains membrane proteins which are highly glycosylated, which protects them from the proteases in the lysosome.
Since the hydrolytic enzymes in the lysosomes are acid hydrolyases, they work best in acidic conditions. This means the cell is protected if these enzymes do escape from a lysosome, because they do not work efficiently in the cytosolic pH of 7.2.
What are the differences between early endosomes, late endosomes and lysosomes?
Early endosomes are small, irregularly shaped intracellular organelles, which are not acidic. They are the first destination of material that is taken into the cell by endocytosis, which is transported to the early endosomes in transport vesicles. Early endosomes contain some hydrolytic enzymes. Some of the materials they receive through endocytosis are removed and recycled in the cell.
Early endosomes then become late endosomes. Late endosomes are slightly acidic (pH 6) and hydrolytic digestion begins.
Late endosomes then mature into lysosomes with a further drop in pH to become more acidic (pH 4.5-5.0). During the maturation process, membrane proteins specialised for endosomes are removed from the membrane and taken to endosomes or the Trans Golgi Network.
How are proteins targeted for the lysosome?
Proteins which need to be targeted for the lysosome are lysosomal hydrolyases and membrane proteins of lysosomes (which are heavily glycosylated to protect them from the hydrolyases in lysosomes).
Lysosomal hydrolyases carry a mannose-6-phosphate (M6P) tag, which is added to their N-linked oligosaccharides in the Golgi. Transmembrane M6P receptors in the Trans Golgi network bind the M6P tag on the lumenal side and adaptor proteins for assembling clathrin coats on the cytosolic side. This means the lysosomal hydrolyases are packaged into vesicles and transported to the endosome (which becomes a lysosome).
Once the vesicle has lost its clathrin coat it fuses with an early endosome. In this non-acidic environment the M6P tags are still bound to the M6P receptors. As the early endosome matures into a late endosome, the lower (more acidic) pH allows the lysosomal hydrolases to remove the M6P from the receptor. Then an acid phosphatase removes the phosphate from mannose, and the signal is gone.
What are the three ways in which material can enter the lysosome for digestion?
- Endocytosis - transport vesicles transport material to an early endosome which matures into a lysosome
- Autophagy - cell is in starvation mode, recognises organelles which don’t work and should be recycled. The organelle is enclosed in a bilayer, forming an autophagosome which fuses with a lysosome and is digested
- Phagocytosis - phagocytes engulf objects in a phagosome which fuses with a lysosome and is digested
