intracellular membrane transport, protein sorting and secretion Flashcards
what is retrograde transport?
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
what types of proteins are secreted?
- extracellular matrix proteins
- serum proteins
- hormones
- digestive enzymes
- plasma membrane proteins
- ER resident proteins
- golgi resident proteins
- lysosomal enzymes
give an example of a secreted extracellular matrix protein and give its cell type
collagen. found in fibroblasts and is secreted constitutively
give an example of a secreted serum protein and state its cell type
immunoglobulins. found in B-cells
give an example of a secreted digestive enzyme and state its cell type
trypsin. pancreatic acinar cells
give an example of a secreted plasma membrane protein
EGF receptor
what did labelling experiments using secreted proteins find?
secretory proteins are localised to the ER lumen shortly after synthesis
how was it discovered that secreted proteins are localised to the ER?
(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
why are pancreatic exocrine cells used for experiments investigating protein transfer?
these cells are loaded with ER and produce a lot of secretory proteins in the form of enzymes
what are pulse-chase experiments used for?
to define kinetics and pathways of transport for newly synthesised proteins
explain how pulse-chase experiments can be used to investigate secreted proteins
- 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
what were the key findings of pulse-chase experiments into secreted proteins?
- 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
how has GFP been used to investigate protein transport?
- 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
what is co-translational insertion?
where transport of secretory proteins into the ER occurs whilst the nascent protein is still bound to the ribosome
how do the ribosomes assist protein sorting?
- if the synthesised protein is secretory, ribosomes attach to the ER
- ribosomes remain cytosolic if the protein is set to remain in the cytosol
what are the requirements for protein sorting?
- 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
what is the role of the translocation machinery in protein sorting?
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
how is energy provided to transport proteins during protein sorting?
unidirectional transport of a protein is achieved by coupling translocation to an energetically favourable process such as GTP/ATP hydrolysis
how are signal sequences utilised to facilitate protein secretion?
- 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
what is the signal recognition particle?
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
