ER + Golgi + Lysosome + Vesicle Transport

Question 1

What does the nucleolus do

Answer 1

• Area in nucleus with condensed chromosomes
• Site for synthesis and processing of rRNA and
  production/assembly of ribosomes

Question 2

What is the structure of the ER

Answer 2

• Rough ER – cisternae
• Smooth ER – connected via tubules
• Tubules and sacs interconnect, and membrane is continuous with the outer nuclear membrane.

Question 3

What is the central roles of of ER

Answer 3

• biosynthesis of both lipids and proteins

Question 4

What does the RER and SER do

Answer 4

• Rough – lysosomal proteins (digestive enzymes),
  membrane proteins, proteins for export from cell
• Smooth – make and store proteins, chy, phospholipids & steroids, detox

Question 5

If proteins neeed to stay in the RER how does this happen

Answer 5

• Stop-transfer signal halts translocation and proteins remains embedded in ER membrane
• These are either delivered to other membranes (e.g. cell membrane) or stay in ER (role in ER function)

Question 6

How do drugs get detoxified

Answer 6

• On the SER in hepatocytes of the liver
• Lipophilic drugs are modified to increase water solubility

Question 7

Where do phospholipids get made

Answer 7

• New phospholipids are assembled in the outer leaflet of the ER membrane by membrane bound enzymes which combine their components
• Scramblase is a phospholipid translocator which causes the two leaflets to equilibrate
• New phospholipids are carried to the Golgi, plasma membrane, lysosomes and endosomes by vesicular transport

Question 8

What is the ER in muslces cells called

Answer 8

THE SARCOPLASMIC RETICULUM (SR)

Question 9

What is the golgi’s job

Answer 9

• Sorting and dispatching of products from the
  ER
• Builds and attaches oligosaccharide chains to many proteins and lipids that the ER sends to it.
• Oligosaccharides can act as tags for transport
  to endosomes.

Question 10

What is the Cis Golgi Network (CGN)

Answer 10

The cis face lies nearest the ER and is the site at which vesicles from the ER dock

Question 11

What is the Trans Golgi Network (TGN)

Answer 11

The trans face is the site from which vesicles depart for the cell surface or other compartments

Question 12

Name 2 functions fo the golgi apparatus

Answer 12

1. Proteins destined for secretion and for a variety of organelles/vesicles within the cell are sorted, modified and dispatched from the golgi
2. It is also a major site of carbohydrate synthesis
   in the form of glycoproteins and proteoglycans (glucose chains)

Question 13

What are the 7 steps proteins take to fold correctly in ER

Answer 13

1. Almost all proteins in the ER lumen are glycosylated and this acts as a folding tag
2. During the initial phases of folding, 2 of the 3 terminal glucose molecules are removed
3. Calnexin is a chaperone which recognises the single glucose of incompletely folded proteins and prevents their export to Golgi
4. The final glucose is removed then…
5. If partially folded glucosyl transferase adds another glucose and it tries again
6. continually misfolded proteins are chaperoned back to the ER protein translocator and are sent to the cytoplasm for degradation (proteosome)
7. Correctly folded proteins go on to be exported to the Golgi

Question 14

What are the 8 steps folded proteins take to go from ER to golgi

Answer 14

1. Fully folded proteins are targeted to ER exit sites
2. For soluble proteins this involves interactions with transmembrane receptors
3. Proteins need “exit” signals for efficient export: non-cargo proteins are packaged at a much lower rate
4. The coat protein COPII interacts with the cytosolic tail of the transmembrane receptors causing a vesicle to bud off (covering the whole vesicle, giving it its shape)
5. Once COPII coated vesicles bud from the ER they rapidly shed their coat
6. The vesicles then undergo homotypic fusion (join together)
7. The resulting vesicular tubular cluster (VTC) moves along microtubules to deliver its contents to the Golgi
8. Once the cargo reaches the Golgi it is
   released from its transmembrane receptor, due to a decrease in pH

Question 15

Why would the change in pH cause the cargo
to be released from its receptor?

Answer 15

• Cargo in the VTC is made up of proteins
• The proteins draw in H+ ions making it acidic
• The acidity makes the aa sequence in the proteins change shape, changing the cargos shape
• Therefore the receptors don’t fit anymore and as a result release the cargo

Question 16

Why would stuff have to go back to ER from golgi and how

Answer 16

• Receptors and COPII get recycled
• Also proteins which have escaped the ER by mistake need retrieving
• COPI coated vesicles bud from VTC / Golgi, are uncoated & transported back to ER carrying escaped proteins and receptors
• Escaped ER proteins contain thencytosolic sequence which interacts directly with COPI

Question 17

2 things

What happens to a complex oligosaccharide when it passes through the ER and golgi

Answer 17

1. 14-sugar oligosaccharide added in the ER can be processed in many ways:
   - there are >200 modifying enzymes
   - their availability & the sequence of the protein itself tells the cell how to process it
2. Oligosaccharide processing is ordered:
   - sugars are removed and added in turn, each step relies on previous one
   - In part achieved by the localisation (cis, medial, trans) of the enzymes in particular compartments

Question 18

Name 4 modifications that happen in the golgi

Answer 18

1. Oligosaccharide processing
2. O-linked glycosylation (in the cisternae)
3. Phosphorylation of oligosaccharides on lysosomal proteins (in the CGN)
4. Sulphation of tyrosine and carbohydrates (in the TGN)

Question 19

4 points

What is the point of glycosylation (Oligosaccharides) ?

Answer 19

1. As a protein marker - for folding, transport between ER to golgi, sorting in golgi
2. As a protector - from proteases
3. As a cellular marker - for cell-cell recognition and adhesion mechanism
4. Regulatory Roles - Cell surface signalling receptors may be glycosylated, so if there are changes to the glycosylation pattern it can change the activity of the protein

Question 20

What is the lysosome

Answer 20

1. Membrane bound organelle
2. Major site of intracellular digestion
3. Contain around 40 types of hydrolysis enzymes
4. Maintained at about pH5 by a H+ pump in the lysosomal membrane

Question 21

How is the lysosome cell protected by nits own digestive enzymes

Answer 21

1. The lysosomal membrane keeps the enzymes of of the cytosol
2. The acid hydrolases don’t work at cellular pH (7.2)
3. Enzymes are protected by each other by glycosylation

Question 22

What are the ways the lysosome is formed

Answer 22

1. Hydrolase enzymes made in the ER then transported to create the late endosome —> lysosome
2. Extracellular things that need to be digested enter the cell via endocytosis, then form the early endosome —> late endosome —> lysosome
3. Engulfed things via phagocytosis or dead cells due to autophagy that need digesting turn into lysosome

Question 23

What are the 3 things that can happen to extracellular contents sent to lysosome by endocytosis

Answer 23

1. Recycling - Membrane & many receptors sent to “recycling endosome” then vesicles return to plasma membrane
2. Transcytosis - Vesicles return to different part of PM; transports material across cell
3. Degradation - Cargoes (& some receptors) sent to late endosomes which mature into lysosomes

Question 24

What are they 3 steps that happen in the golgi to transport things to lysosome

Answer 24

1. Signal peptide directs synthesis of lysosomal proteins into the endoplasmic reticulum. N-linked Glycosylation
2. Cis Golgi network: Signal patch directs phosphorylation of terminal mannose on N-linked oligosaccharide
3. In the trans Golgi network:
   - M6P receptors in the membrane of the trans Golgi network bind M6P- labelled lysosomal hydrolases on the luminal side
   - and to adaptins on the cytosolic side…
   - packaging the hydrolases into clathrin coated transport vesicles which bud from the Golgi

Question 25

What happens after vesicles for lysosome leave the golgi

Answer 25

1. Transport vesicles fuse with the late endosome
2. Acidic pH (~ 6) causes the M6P receptor to dissociate from M6P- labelled hydrolase, releasing it into the lumen
3. A signal in the cytoplasmic tail of the M6P receptor targets it into a transport vesicle which takes it back to the trans Golgi network
4. In the late endosome phosphate is removed from mannose sugars on the hydrolases to ensure that they are not returned to the Golgi
5. As the H+ pump lowers the pH the acid hydrolases begin to digest macromolecules delivered from the early endosomes
6. and the endosome matures into a lysosome

Question 26

How do things leave the lysosome

Answer 26

• Semi-permeable membrane
  1. Passive diffusion – small hydrophobic molecules can pass out of the lysosome by diffusion
  2. Membrane transporters – digestion products (sugars, amino acids and nucleotides) are transported out of the lysosome by transporters in the membrane
  3. Secretory Lysosomes – Lysosomal secretion allows cells to eliminate indigestible debris

Question 27

What is clathrin

Answer 27

Coats the vesicles which transport molecules between the golgi, the lysosomes and the plasma membrane
Like COPI and COPII but for after the golgi

Question 28

What is the structure of clathrin

Answer 28

• Each clathrin subunit contains 3 large and 3 small polypeptide chians
• together form a three legged triskelion
• the triskelions form a basket-like convex framework of pentagons and hexagons

Question 29

How does clathrin attach to the membrane

Answer 29

• Protein complex called adaptin is needed to attach clathrin coat to the membrane
• Adaptins bind with transmemrabnous receptors (which capture soluble cargo for transport) on the outside of the membrane
• There are 4 different adaptins, each recognising a different set of cargo receptors

Question 30

How do clathrin coated vesicles leave

Answer 30

• The vesicle is pinched off by another protein, dynamin
• Which binds in a ring to the neck of the budding vesicle
• The coat is rapidly lost and recylced and the naked vesicle is ready for transport

Question 31

How do vesicles know where to dock

Answer 31

Complimentary SNARES

Question 32

What are SNARES

Answer 32

• Surface markers that identify vesicles according to origin and cargo
• Complimentary SNARES are displayed on target membranes
• vesicular v-SNARE is complimentary to target membrane t-SNARE
• When SNARES meet they wrap around each other forming a stable trans-SNARE complex, which locks the two membranes together (docking)

Question 33

How do membranes fuse

Answer 33

• Need to be very close proximity so that water is excluded from between
• The formation of the trans-SNARE complex brings the two membranes closer

Question 34

What is constitutive secretory pathway

Answer 34

• Exocytosis
• A type of vesicular transport
• The default pathway
• Any protein which is not either returned to ER, STaying in golgi, going to lysosomes or regulated secretion is transported this way

Question 35

What are secretory vesicles stored in some cells called

Answer 35

Secretory granules

Question 36

How do secretory proteins get transported from the golgi

Answer 36

• Secretory proteins aggregate
• Leave the trans golgi network as loosely bound immature secretory vesicles
• loosely bound meaning there is lots of space in the vesicle due to extra membrane around it
• The concentration of secretory proteins increases as the vesicle matures due to:
• excess membrane goes back to golgi
• increased acidity in maturing vesicle causing tigher aggregation

Question 37

How do secretory vesicles get processed

Answer 37

• By proteolysis
• produces very short peptides (neuropeptides)
• also protects cell from its own hydrolytic enzymes

Question 38

What causes secretory vesicles to be secreted from/taken into cells

Answer 38

• Triggers
• Vesicles can dock with the plasma membrane but fusion only occurs following a specific trigger

Question 39

Examples of release of secretory vesicles in response to a trigger

Answer 39

• Digestive enzymes released in response to food in the gut
• nerve cell vesicles which hold neurotransmitters in response to action potential
• pancrease release insulin in response to raised blood sugar

Question 40

Why does there need to be balance between exocytosis and endocytosis in a cell

Answer 40

Endocytosis results in increase in surface area of plasma membrane
So balance is needed with exocytosis

Question 41

What are the 3 types of endocytosis

Answer 41

1 Pinocytosis
2 Receptor mediated endocytosis
3 Phagocytosis

Question 42

What is pinocytosis

Answer 42

Type of endocytosis
Clathrin coated vesicles containing molecules for digestion for the lysosome

Question 43

What is receptor-mediated endocytosis

Answer 43

e.g. low density lipoproteins (LDLs) containing cholesterol interact with LDL-receptors on animal cells. LDLs gets delivered to lysosome and cholesterol released

Question 44

What is phagocytosis

Answer 44

• Specialized white blood cells (macrophages, neutrophils and dendritic cells) can take up large particles like microorganisms and dead cells
• Particles get recognized by cell surface receptors on phagocytes
• This triggers pseudopods to engulf the particle creating a large endocytic vesicle (phagosome)
• Phagosome fuses with lysosome to get digested