Protein targeting and Export Flashcards
how can other colours of protein be obtained
mutating the β-barrel structure
or using proteins from other organisms
feature of chimera
can make fusions with your protein of interest
disadvantages of chimera protein
may not fold properly or act normally due to large FP
what does DAPI and Hoechst stain bind to and what does it emit
minor groove of DNA and emits blue light when exposed to UV light
what does phalloidin toxin bind to
F-actin
can be conjugated to different fluorophores
procedure of immunofluorescence
specific antibodies labelled with fluorescent dye (primary antibody) attach to the antigen
procedure of secondary immunofluorescence
same as primary IF except primary antibodies aren’t tagged with fluorescent dye
secondary antibodies tagged with fluorescent dye bind to primary antibodies
disadvantages of immunofluorescence
cell have to be permeabilised for antibodies to bind to them - dead
antibodies may give false signals via non-specific binding
when does cell migration occur
wound healing
movement of WBC’s to sites of infection
metastasis
what is required for migration
synthesis of actin filament
what does zip code protein recognise
specific secondary structure in 3’UTR of β-actin mRNA
what does ZBP1 bind to
β-actin mRNA in nucleus as part of a larger mRNP complex
what gets transported through the nuclear pore complex with the aid of
Ran GTPase
mRNP’s
what does organelle specific targeting require
a nuclear localisation signal
not removed following transport
name a basic NLS and its features
SV40 large 40 antigen
rich in lysine (K) and arginine (R)
can be bipartite - can be split in two
name a non-basic NLS and its features
hnRNPA1
hydrophobic
what does indirect immunofluorescence of HeLa cells show
some endogenous protein is in the nucleus
most is in the cytoplasm
what can passively diffuse into through a nuclear pore
20-40KDa
function of Ran GTPase
transports bigger proteins and mRNA’s (in mRNP) through the NPC
structure of the nuclear import receptor
heterodimer of importin-α and importin-β
function of importin-α/β
α - recognises NLS in cargo
β - interacts with nucleoporins in NPC
what do nucleoporins contain and what is the function of it
FG-rich repeats - hydrophobic
help transport the complex into the nucleus
what is step 4 of the nuclear import process
nuclear Ran GTPase interacts with importin
releases cargo
step 5 of nuclear import
importin exits to cytoplasm
step 6 of nuclear import
Ran-GAP simulates Ran to hydrolyse GTP
step 7 and 8 of nuclear import
7 - importin is now free for another round of import
8 - Ran-GDP enters the nucleus to be recycled by Ran-GEF
what family do importins and exportins belong to
karyopherins
what detects green actin
FISH
what does DAPI and phalloidin detect
DAPI - blue actin
phalloidin - red actin
features to exit the nucleus of organelle specific targeting
requires nuclear export signals
can be anywhere in the sequence
rich in hydrophobic residues - leucine/isoleucine
outline the composition of the mitochondrial genome
13 proteins
22 tRNA
2 rRNA
what are the 13 mitochondrial gene encoded proteins sub-units of
the oxidative phosphorylation system
what is an import assay used for
can mix together cellular components to determine wether a protein has been imported into a membrane bound organelle
what is mitochondrial targeting peptide mTP
binds to receptor on mitochondrial outer surface and brought towards translocase of outer membrane
what needs to happen to precursor protein before it can pass to through translocase pore
unfolded
step 1 of translocation
unfolding helped by chaperones - requires ATP
chaperone - Hsc70
step 2 of translocation
mTP binds to receptor
steps 3 and 4 of translocation
3- mTP brings the protein to the outer membrane
4 - the protein begins to pass through
step 5 of translocation and what drives it
the proximity of the inner membrane allow the protein to pass through the IM
driven by ATP turnover which is driven by HSc70
steps 6 and 7 of translocation
6 - mTP is cleaved from the protein
7 - chaperones help fold protein into an active state
what would occur if you added valinomycin to mitochondria
the disruption to the mitochondrial membrane potential would cause inhibition of mitochondrial import
difference between direct and indirect mitochondrial import disease
direct - deafness - dystonia syndrome
indirect - neurodegenerative proteinopathies
average number of genes of cpDNA and proteins needed for it to function
120 genes
2-3,000 proteins
what is the difference between mTP and cTP
unlike mTP, cTP has no well defined sequence or structure
what do mTP and cTP have in common
serine rich
rarely have amino acids
range from 20 to 100 residues in length
what might proline be important for in terms of cTP
keeping the cTP unfolded for certain proteins
what is the core of ER-localised proteins composed of
6-12 hydrophobic amino acids preceded by one or more basic amino acids (R/K)
where in mature ER proteins are residues with small side chains found
at -3 and -1 positions upstream of the cleavage site (of the signal sequence)
step 1 of translocation into the ER
ribosome pauses protein synthesis and docks to the signal recognition particle in the ER membrane
step 2 of translocation into the ER
the SRP receptor facilitates binding of the ribosome to the translocon pore
what is the structure of the translocon in the ER
3 Sec 61 protein sub-units α,β,γ
single α-helix forms the base of the structure
heterotrimeric complex
step 3 of translocation into the ER
an α-helix in one of the translocon proteins acts as a plug
GTP hydrolysis opens the pore
step 4 of translocation into the ER
translation elongation is restored and the nascent protein is fed into the ER lumen
signal sequence is cleaved off
what does the structure of the translocon allow it to do
able to hinge to allow proteins to pass through laterally into the membrane
what is a stop-transfer sequence
stop-transfer sequence is 22 amino acids long and hydrophobic
stops translocation through the channel
what 2 sequences do type II/III membrane proteins contain
signal-anchor sequence
stop-transfer sequence
when are proteins N-glycosylated
when they pass through the translocon in the ER
when can proteins be O-glycosylated
after they are fully synthesised
what is the structure of the N-linked glycosylation sugar and where is it transferred from
preformed oligosaccharide - 14 residues long
transferred from a novel membrane lipid called dolichol phosphate
how does N-glycosylation occur
oligosaccharide is linked to asparagine (N/Asn) residues in the protein at sequences containing Asn-X-Ser/Thr
function of glycosidase enzyme in terms of N-glycosylation
the oligosaccharide is further processed by removing glucose and mannose
one of the glucose may be re-added later to aid with folding
function of mannosidases
prevents further addition of glucose to incorrectly folded proteins
what is ER associate protein degradation (ERAD)
incorrectly folded protein is retro translocated to the cytoplasm and degraded by a proteasome
what gets targeted to the lysosome
mannose-6-phosphate
what is the function of N-acetylglucosamine phosphotransferase
recognises a signal patch and catalyses the reaction of the terminal mannose and UDP-N acetylglucosamine forming 6P-N-acetylglucosamine
function of phosphodiesterase
cleaves N-acetylglucosamine from the intermediate leaving a protein-glycosyl-mannose 6P
what recognises mannose-6P
mannose-6-phosphate receptors in the trans golgi
where does the protein bound to mannose-6P relocate to and what happens to that place
to a clathrin coated vesicle that form specialised transport vehicles
the clathrin disassembles
what happens to the transport vesicle after the clathrin is disassembled
it is targeted, with the lysosomal protein inside to an early endosome
what happens after the vesicle fuses with the early endosome
the acidic pH causes the lysosomal protein to dissociate from the mannose-6-phosphate receptor
once the lysosomal protein is inside the early endosome, what occurs
the phosphate from mannose-6P is hydrolysed creating the mature enzyme, then the late endosome fuses with a lysosome
how does O-glycosylation occur
involves linking sugars to the OH group of serine/threonine and takes place exclusively in the Golgi
what determines your blood group
O-glycosylation
wether you have the transferase for N-acetylgalactosamine
