Protein targeting, the secretory pathway and endocytosis Flashcards
(37 cards)
What are the types of signal peptides
Membrane topology stop signals – decide if protein is membrane bound or soluble
Routing signals – decide where the protein goes
Sorting signals – once on route decides further routes
Retention signal – decides how long the protein stays on route
No signal – remain in the cytosol and have no need for a signal (lactate dehydrogenase etc)
The protein signal is a genetic code which is translated into the primary structure
Terminal signal peptide
Unique amino acid sequence
What happens to signal peptides after use
Can be cleaved off and the protein will still function as normal – once the protein is in position a signal sequence is no longer required
What is a signal patch
Signal revealed in correctly folded protein
They require correct tertiary folding and removal of the patch will destroy the protein
These patches remain a permanent fixture of the protein
If the protein is made incorrectly due to damage these signal patches can become disrupted – this means the protein has no signal and becomes sequestered
What is the transfection approach to research signal peptides
reate retroviral plasmid construct with the signal genetic code in the ORF
Clone in protein genetic sequence of a protein that normally resides in the cytosol
Signal peptide will direct the protein to the desired organelle
Subcellular fractionation and immuno-blot for the presence of protein in the desired organelle fraction
What is the biochemical approach to research signal peptides
Radioactive labelled protein (using C14 etc)
Specific signal sequence previously identified through bioinformatics
Protein is transported to specific organelle (in vitro)
Organelles will become radioactive if the protein has been taken up
What is the genetic approach to research signal peptides
Yeast cells contain an enzyme called histidinol dehydrogenase which converts histidinol into histidine in the cytoplasm
Yeast can be genetically modified where histidinol dehydrogenase contains a signal which directs it to the ER
When these cells are cultured without histidine they die due to the accumulation of histidinol in the cytoplasm
What is the secretory pathway and what is the order of it
Each step is highly coordinated due to the signal sequences found in proteins
If secretory pathway signals are known for a particular species (sheep) ; human proteins can be genetically tagged to sheep signal peptides – this will then be translated and secreted in the milk which can then be harvested
Cytosol –> ER –> Golgi –> cell surface
What happens in the cytosol
All proteins begin here – nascent polypeptide chain during translation
Secretory pathway signal peptides are translated first – receptors in ER membrane recognise this – proteins that lack this will remain in the cytosol
What happens in the endoplasmic reticulum (ER)
Explains why the rough ER is encursted with ribosomes
SRP (signal recognition protein) associated with N-terminal of nascent polypeptide —> complex formed : nascent protein, ribosome and SRP (translocon)
What does the SRP have affinity for and what does it cause upon binding
SRP has affinity for the SRP receptor on the ER membrane
Binding to SRP receptor opens the channel and allows for ribosome docking
Nascent peptide feed into ER lumen
N-terminal ER signal translated first (5’ –> 3’) starting with Met
What are the characteristics of the N-terminal ER signal peptide
These N-terminal ER signal peptides are 15-35 amino acids long and all contain the same general chemistry
Have a positive region 2-10 residues long from the N-terminal
Central hydrophobic region alpha helix known as the core region
Next to the core is a turn inducing residue (proline or glycine) around 6 residues from the cleavage site
The cleavage site is preceded by the small polar residues (like alanine) and is recognised by the signal peptidases
What are the 2 things that happen once in the ER
depending on whether the protein is soluble or not
Insoluble – remain membrane bound (cell receptor)
Soluble – N-terminal sequence is cleaved (free in the lumen)
What are ER retention signals
Once in the ER, depending on the signal, proteins can move anywhere in the cell
ER retention signals
Proteins are destined to leave the ER
KDEL (amino acid sequence) relocates protein to the ER; protein that “bud off” ER that contain this sequence are recognise by the cis golgi membrane receptors which cause the protein to return to the ER – this is due to the N-terminal ER signal being cleaved
KKXX is an ER retention signal for membrane bound ER proteins – the return pathway involves the COP-I protein complex (coat protein complex)
KDEL binds to KDELR (KDEL-receptor) in cis golgi membrane – this activates GTPase which initiates the return process
What faces does the Golgi complex have
Has 2 faces : cis and trans
What are the functions of both faces
Budding ER vesicles approach the cis golgi face —> move through the golgi sacs and if appropriate leave at the trans face
What differs between membrane sacs in the Golgi
Enzymes
What is the function of the Golgi complex
Post-translational modification of proteins occur in the golgi
Glycosilation
Proteolytic activity
The golgi sorts, packs and modifies proteins – proteins that are destined for secretion or expression on the cell membrane are usually modified at some point
Proteolytic cleavage – insulin is produced as a single polypeptide (pre-pro-insulin)
Pre-pro-insulin –> pro-insulin –> insulin (active form)
What is glycosylation
Addition of carbohydrate (glycosylation) - is important for proteins that are expressed on the cell membrane
This can be N linked or O linked
Signals with this primary structure of the protein dictated glycosylation sites (NXS = Ans-x-Ser/Thr where x is any amino acid)
Why is N-glycosylation important
Protein must be in the correct 3D structure
Asn (asparagine) is involved with the N-glycosylation
What happens before a glycosylated protein is added to the cell membrane
This is held together in a complex with dolichol before it is added to the membrane protein
What happens at each layer of the Golgi
As protein moves through the golgi compartments, a new sugar layer is added or modified in each
In the golgi suagrs are modified – mannose is removed and N-acetyl-D-mannosamine (NANA) etc is added
What happens to proteins that progress to the trans Golgi membrane
Some proteins progress through the golgi and reach the trans golgi membrane network (TGN)
Vesicles leave the TGN and move through the plasma membrane (unregulated, continuous secretion)
Some vesicles that leave the TGN can be stored and release on activation (TNFα or insulin)
After leaving the TGN, soluble proteins will be secreted into the extracellular matrix (exocytosis)
Membrane proteins will remain attached to plasma membrane with glycosylation facing the extracellular matrix
What are the associated coat proteins involved in vesicle formation
Vesicle formation has different mechanisms depending on whether the pathway is constitutive or regulated
Vesicles are membranes formed using associated coat proteins
Clathrin
COP-I – 7 subunits
COP-2 – 4 subunits
Both COP proteins are from constitutive secretion
What is clathrin
Has 3 large polypeptides (heavy) and 3 small polypeptides (light)
They are joined together by creating a triskeleton structure
They join together to form a very large multisubunit clathrin structures
Takes shape as a clathrin sphere
These spheres form spontaneously in vitro
During vesicle formation, clathrin forms the vesicle coat
