intracellular membrane transport, protein sorting and secretion

Question 1

what is retrograde transport?

Answer 1

where proteins are transported the opposite way to proteins for secretion, via trans -> medial -> cis Golgi. this is a mechanism to ensure that the right proteins are present in the right Golgi cisternae

Question 2

what types of proteins are secreted?

Answer 2

• extracellular matrix proteins
• serum proteins
• hormones
• digestive enzymes
• plasma membrane proteins
• ER resident proteins
• golgi resident proteins
• lysosomal enzymes

Question 3

give an example of a secreted extracellular matrix protein and give its cell type

Answer 3

collagen. found in fibroblasts and is secreted constitutively

Question 4

give an example of a secreted serum protein and state its cell type

Answer 4

immunoglobulins. found in B-cells

Question 5

give an example of a secreted digestive enzyme and state its cell type

Answer 5

trypsin. pancreatic acinar cells

Question 6

give an example of a secreted plasma membrane protein

Answer 6

EGF receptor

Question 7

what did labelling experiments using secreted proteins find?

Answer 7

secretory proteins are localised to the ER lumen shortly after synthesis

Question 8

how was it discovered that secreted proteins are localised to the ER?

Answer 8

(labelling experiments)
1- cells briefly incubated with radiolabelled amino acids
2- cells homogenised, plasma membranes fractured and eER sheared into microsomes
3- microsomes treated with a protease with or without detergent
4- digestion of secretory proteins only in the presence of detergent. evidence that new proteins are localised within the membranes of microsomes

Question 9

why are pancreatic exocrine cells used for experiments investigating protein transfer?

Answer 9

these cells are loaded with ER and produce a lot of secretory proteins in the form of enzymes

Question 10

what are pulse-chase experiments used for?

Answer 10

to define kinetics and pathways of transport for newly synthesised proteins

Question 11

explain how pulse-chase experiments can be used to investigate secreted proteins

Answer 11

• guinea pig pancreas prepared and incubated in ‘cold’ leucine free medium containing radiolabelled leucine
• warmed to 37 degrees for 3 mins
• chase performed for 7-117 mins - medium replaced with cold leucine in excess
• proteins can be visualised on journey through ER -> Golgi -> immature secretory vesicles -> zymogen using autoradiography

Question 12

what were the key findings of pulse-chase experiments into secreted proteins?

Answer 12

• secreted proteins pass through the Golgi on the way out of the cell
• secreted proteins never mix with cellular proteins in the cytosol
• choice of cell type is crucial: if the cell is not specialised for secretion, proteins end up in cytosol

Question 13

how has GFP been used to investigate protein transport?

Answer 13

• using a mutant form of VSV-G protein with a GFP tag
• this mutant protein is retained in ER at 40 degrees but moves through ER at 32
• provided evidence for the time frame which proteins move through organelles

Question 14

what is co-translational insertion?

Answer 14

where transport of secretory proteins into the ER occurs whilst the nascent protein is still bound to the ribosome

Question 15

how do the ribosomes assist protein sorting?

Answer 15

• if the synthesised protein is secretory, ribosomes attach to the ER
• ribosomes remain cytosolic if the protein is set to remain in the cytosol

Question 16

what are the requirements for protein sorting?

Answer 16

• a signal intrinsic to the protein
• a receptor that recognises the signal and directs it to the correct membrane
• a translocation machinery
• energy to transfer the protein

Question 17

what is the role of the translocation machinery in protein sorting?

Answer 17

once the protein has interacted with its corresponding receptor, the protein is transferred to the translocation channel which allows it to pass through the membrane bilayer

Question 18

how is energy provided to transport proteins during protein sorting?

Answer 18

unidirectional transport of a protein is achieved by coupling translocation to an energetically favourable process such as GTP/ATP hydrolysis

Question 19

how are signal sequences utilised to facilitate protein secretion?

Answer 19

• there is a hydrophobic N-terminal region that targets nascent secretory proteins to the ER
• this signal contains at least one positively charged AA next to a continuous stretch of 6-10 hydrophobic amino acids
• the charged AA is the signal peptidase cleavage site

Question 20

what is the signal recognition particle?

Answer 20

a cytosolic ribonucleoprotein particle that transiently binds to both the ER and signal sequence in a nascent protein and large ribosomal subunit to form a complex