Secretion in mammlian cells - 2

Question 1

Componants of budding?

Answer 1

Sar1a/Sar1b - GTPase
Sec23/24 - adaptor protein
Sec13/31 - coat

Question 2

What was shown with regards to ER localisation in Sec13/31 in presence of Sec23 mutants?

Answer 2

The protein wasn’t localised to the ER by the adaptor protein – showed a problem in recruitment of Sec13/31 DUE TO mutations in Sec23

Question 3

What were shown by defects in association of the coat with COPII vesicles?

Answer 3

Reduced GTPase activity, causing problems in association of the coat (Sec13/31) and GTPase enzyme (Sar1)

Question 4

Is cell morphology impacted ? How can this be investigated?

Answer 4

• GOLD LABELLING CAN BE USED
• embed cells with antibodies that are specific to SAR1 - these antibodies are coupled to GOLD so when GOLD PARTICLES are seen SAR1 is present
• showed that there was a presence of SAR1 at exit sites but LESS BUDDING SEEN

Question 5

Only certain tissues are impacted by defective budding. Why is this the case?
What was shown by levels of Sec23A and Sec23B in many tissues?

Answer 5

• In the case of defective proteins involved in budding, in some tissues REDUNDANCY allows other protein paralogues to carry out the function of proteins that are mutated.
• In an assay of many tissues there were comparable levels of Sec23 paralogues —- either may compensate for mutated forms in different tissues?

Question 6

ER morphology in Cranio–lenticulo–sutural dysplasia (CLSD) —- what is seen in budding?

Answer 6

• ER is distended
• In mutants budding is seen but the buds don’t pinch off to allow separate COPII vesicle structures from the ER
• Intermediate structures are seen - budding sites form but no coat is recruited
• COPII formation defect
• Less Sec31 antibodies seen (by gold labelling)

Question 7

What is seen regarding affinity of Sec23 and Sar1b in mutants?

Answer 7

Lower affinity between the GTPase and adaptor – causes the composition of vesicles to be different

Question 8

What can be used to investigate cargo sorting and what did studies show?

Answer 8

• ERGIC53 (ER-Golgi intermediate compartment 53 kDa protein) is the cargo that can be used in an in vitro assay to investigate cargo sorting
• Cargo sorting doesn’t appear to be impacted

Question 9

Examples of cargo transported by COPII vesicles?

Answer 9

SNARES, Secreted molecules, recycling components, transmembrane proteins, p23/24 family

Question 10

How is budding impacted by the COPII components in mutants?

Answer 10

• Sec13/31 isn’t recruited so no coat forms
• Sec23/24 is still recruited and recognises cargo
• SAR1a/1b are highly homologous but there are cargo-specific mutations that are present in different tissues

Question 11

BUDDING - what occurs to initiate budding and the process?

Answer 11

Sar1 activated by SEC12 (a guanine nucleotide exchange factor)
Sec23/24 recognises cargo and recruits Sec13/31

Question 12

2 models proposed to accomodate large cargo?

Method for how this is investigated?

Answer 12

MODEL 1 - larger vesicles and vesicular carriers are used to accomodate more cargo
MODEL 2 - non vesicular intermediate is used between the ER and Golgi to accomodate more cargo

Investigated using MICROSCOPY

Question 13

How is collagen used in the interest of investigating transportation of large cargo?

Answer 13

Pro-collagen localises with Sec23A (adaptor)

Able to show that pro collagen is packed INTO LARGE VESICLES

Question 14

What happens with regards to budding during the UPR?

Answer 14

• Cell as a response to stress undergoes the UPR to cope with the stress put on the ER.
• In presence of the UPR signal transducer, a downregulation of COPII proteins is seen

Question 15

What is the Coatamer? WHat is it made up of?

Answer 15

• Describes the complex of adaptor protein and coat of COPII vesicle and GTPase
• 7 protein complex forms it – can build sub complexes one of which relates to the adaptor and one relates to the coat

Question 16

What direction are COPII vesicles transported in in the secretory pathway?

Answer 16

both ANTERO and RETROGRADE directions

Question 17

Examples of cargo transported ANTEROGRADE

Answer 17

Pro-insulin and VSVG protein

Question 18

Examples of cargo transported RETROGRADE

Answer 18

RxR motifs, Dilysine motifs, KDEL receptors and KDEL

Question 19

What is the TGN and why do we need it?

Answer 19

TGN - trans golgi network. a MAJOR PROTEIN SORTING STATION

• establishes planar cell polarity
• sorts proteins to apical and basolateral membranes
• sorts proteins and cargo to consituative and secreted pathways
• sorts newly synthesized lysosomal proteins

Question 20

How does sorting occur?

Answer 20

SORTING SIGNAL - m-p-6 signal needed to sort lysosomal proteins.

Question 21

Outline process of sorting to the lysosome (via m-p-6)

Answer 21

• m-p-6 (a sugar modification molecule)is added to lysosomal hydrolase enzyme (which cycles between the TGN and endocytic pathway)
• this allows packaging and delivery of the protein to endosomal compartments
• once it arrives at the endosomal compartment this causes a drop in pH therefore increase in [H+]
• this dissociates the lysosomal receptor from the enzyme and the receptor is packaged into a recycling vesicle and sent back to the TGN
• the lysosomal enzyme has a phosphate removed and becomes active again in the lysosome

Question 22

EXAMPLES OF LYSOSOMAL SORTING DISEASE?

These involve mutations in lysosomal hydrolases and so proteins and lipids cannot be digested properly.

Answer 22

1) GLcNAc phosphotransferase recessive - - no m-p-6 is added to lysosomal hydrolases so proteins aren’t packaged into clathrin coated vesicles. These are then secreted into the blood stream.
2) Icell disease - inclusion cell disease - fibroblasts contain no lysosomal hydrolases and so are found in the blood as they aren’t properly packaged.