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Biomed 2 > Protein targeting and Export > Flashcards

Protein targeting and Export Flashcards

1
Q

how can other colours of protein be obtained

A

mutating the β-barrel structure
or using proteins from other organisms

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2
Q

feature of chimera

A

can make fusions with your protein of interest

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3
Q

disadvantages of chimera protein

A

may not fold properly or act normally due to large FP

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4
Q

what does DAPI and Hoechst stain bind to and what does it emit

A

minor groove of DNA and emits blue light when exposed to UV light

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5
Q

what does phalloidin toxin bind to

A

F-actin
can be conjugated to different fluorophores

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6
Q

procedure of immunofluorescence

A

specific antibodies labelled with fluorescent dye (primary antibody) attach to the antigen

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7
Q

procedure of secondary immunofluorescence

A

same as primary IF except primary antibodies aren’t tagged with fluorescent dye
secondary antibodies tagged with fluorescent dye bind to primary antibodies

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8
Q

disadvantages of immunofluorescence

A

cell have to be permeabilised for antibodies to bind to them - dead
antibodies may give false signals via non-specific binding

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9
Q

when does cell migration occur

A

wound healing
movement of WBC’s to sites of infection
metastasis

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10
Q

what is required for migration

A

synthesis of actin filament

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11
Q

what does zip code protein recognise

A

specific secondary structure in 3’UTR of β-actin mRNA

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12
Q

what does ZBP1 bind to

A

β-actin mRNA in nucleus as part of a larger mRNP complex

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13
Q

what gets transported through the nuclear pore complex with the aid of
Ran GTPase

A

mRNP’s

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14
Q

what does organelle specific targeting require

A

a nuclear localisation signal
not removed following transport

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15
Q

name a basic NLS and its features

A

SV40 large 40 antigen
rich in lysine (K) and arginine (R)
can be bipartite - can be split in two

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16
Q

name a non-basic NLS and its features

A

hnRNPA1
hydrophobic

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17
Q

what does indirect immunofluorescence of HeLa cells show

A

some endogenous protein is in the nucleus
most is in the cytoplasm

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18
Q

what can passively diffuse into through a nuclear pore

A

20-40KDa

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19
Q

function of Ran GTPase

A

transports bigger proteins and mRNA’s (in mRNP) through the NPC

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20
Q

structure of the nuclear import receptor

A

heterodimer of importin-α and importin-β

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21
Q

function of importin-α/β

A

α - recognises NLS in cargo
β - interacts with nucleoporins in NPC

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22
Q

what do nucleoporins contain and what is the function of it

A

FG-rich repeats - hydrophobic
help transport the complex into the nucleus

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23
Q

what is step 4 of the nuclear import process

A

nuclear Ran GTPase interacts with importin
releases cargo

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24
Q

step 5 of nuclear import

A

importin exits to cytoplasm

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25
Q

step 6 of nuclear import

A

Ran-GAP simulates Ran to hydrolyse GTP

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26
Q

step 7 and 8 of nuclear import

A

7 - importin is now free for another round of import
8 - Ran-GDP enters the nucleus to be recycled by Ran-GEF

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27
Q

what family do importins and exportins belong to

A

karyopherins

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28
Q

what detects green actin

A

FISH

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29
Q

what does DAPI and phalloidin detect

A

DAPI - blue actin
phalloidin - red actin

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30
Q

features to exit the nucleus of organelle specific targeting

A

requires nuclear export signals
can be anywhere in the sequence
rich in hydrophobic residues - leucine/isoleucine

31
Q

outline the composition of the mitochondrial genome

A

13 proteins
22 tRNA
2 rRNA

32
Q

what are the 13 mitochondrial gene encoded proteins sub-units of

A

the oxidative phosphorylation system

33
Q

what is an import assay used for

A

can mix together cellular components to determine wether a protein has been imported into a membrane bound organelle

34
Q

what is mitochondrial targeting peptide mTP

A

binds to receptor on mitochondrial outer surface and brought towards translocase of outer membrane

35
Q

what needs to happen to precursor protein before it can pass to through translocase pore

A

unfolded

36
Q

step 1 of translocation

A

unfolding helped by chaperones - requires ATP
chaperone - Hsc70

37
Q

step 2 of translocation

A

mTP binds to receptor

38
Q

steps 3 and 4 of translocation

A

3- mTP brings the protein to the outer membrane
4 - the protein begins to pass through

39
Q

step 5 of translocation and what drives it

A

the proximity of the inner membrane allow the protein to pass through the IM
driven by ATP turnover which is driven by HSc70

40
Q

steps 6 and 7 of translocation

A

6 - mTP is cleaved from the protein
7 - chaperones help fold protein into an active state

41
Q

what would occur if you added valinomycin to mitochondria

A

the disruption to the mitochondrial membrane potential would cause inhibition of mitochondrial import

42
Q

difference between direct and indirect mitochondrial import disease

A

direct - deafness - dystonia syndrome
indirect - neurodegenerative proteinopathies

43
Q

average number of genes of cpDNA and proteins needed for it to function

A

120 genes
2-3,000 proteins

44
Q

what is the difference between mTP and cTP

A

unlike mTP, cTP has no well defined sequence or structure

45
Q

what do mTP and cTP have in common

A

serine rich
rarely have amino acids
range from 20 to 100 residues in length

46
Q

what might proline be important for in terms of cTP

A

keeping the cTP unfolded for certain proteins

47
Q

what is the core of ER-localised proteins composed of

A

6-12 hydrophobic amino acids preceded by one or more basic amino acids (R/K)

48
Q

where in mature ER proteins are residues with small side chains found

A

at -3 and -1 positions upstream of the cleavage site (of the signal sequence)

49
Q

step 1 of translocation into the ER

A

ribosome pauses protein synthesis and docks to the signal recognition particle in the ER membrane

50
Q

step 2 of translocation into the ER

A

the SRP receptor facilitates binding of the ribosome to the translocon pore

51
Q

what is the structure of the translocon in the ER

A

3 Sec 61 protein sub-units α,β,γ
single α-helix forms the base of the structure
heterotrimeric complex

52
Q

step 3 of translocation into the ER

A

an α-helix in one of the translocon proteins acts as a plug
GTP hydrolysis opens the pore

53
Q

step 4 of translocation into the ER

A

translation elongation is restored and the nascent protein is fed into the ER lumen
signal sequence is cleaved off

54
Q

what does the structure of the translocon allow it to do

A

able to hinge to allow proteins to pass through laterally into the membrane

55
Q

what is a stop-transfer sequence

A

stop-transfer sequence is 22 amino acids long and hydrophobic
stops translocation through the channel

56
Q

what 2 sequences do type II/III membrane proteins contain

A

signal-anchor sequence
stop-transfer sequence

57
Q

when are proteins N-glycosylated

A

when they pass through the translocon in the ER

58
Q

when can proteins be O-glycosylated

A

after they are fully synthesised

59
Q

what is the structure of the N-linked glycosylation sugar and where is it transferred from

A

preformed oligosaccharide - 14 residues long
transferred from a novel membrane lipid called dolichol phosphate

60
Q

how does N-glycosylation occur

A

oligosaccharide is linked to asparagine (N/Asn) residues in the protein at sequences containing Asn-X-Ser/Thr

61
Q

function of glycosidase enzyme in terms of N-glycosylation

A

the oligosaccharide is further processed by removing glucose and mannose
one of the glucose may be re-added later to aid with folding

62
Q

function of mannosidases

A

prevents further addition of glucose to incorrectly folded proteins

63
Q

what is ER associate protein degradation (ERAD)

A

incorrectly folded protein is retro translocated to the cytoplasm and degraded by a proteasome

64
Q

what gets targeted to the lysosome

A

mannose-6-phosphate

65
Q

what is the function of N-acetylglucosamine phosphotransferase

A

recognises a signal patch and catalyses the reaction of the terminal mannose and UDP-N acetylglucosamine forming 6P-N-acetylglucosamine

66
Q

function of phosphodiesterase

A

cleaves N-acetylglucosamine from the intermediate leaving a protein-glycosyl-mannose 6P

67
Q

what recognises mannose-6P

A

mannose-6-phosphate receptors in the trans golgi

68
Q

where does the protein bound to mannose-6P relocate to and what happens to that place

A

to a clathrin coated vesicle that form specialised transport vehicles
the clathrin disassembles

69
Q

what happens to the transport vesicle after the clathrin is disassembled

A

it is targeted, with the lysosomal protein inside to an early endosome

70
Q

what happens after the vesicle fuses with the early endosome

A

the acidic pH causes the lysosomal protein to dissociate from the mannose-6-phosphate receptor

71
Q

once the lysosomal protein is inside the early endosome, what occurs

A

the phosphate from mannose-6P is hydrolysed creating the mature enzyme, then the late endosome fuses with a lysosome

72
Q

how does O-glycosylation occur

A

involves linking sugars to the OH group of serine/threonine and takes place exclusively in the Golgi

73
Q

what determines your blood group

A

O-glycosylation
wether you have the transferase for N-acetylgalactosamine

Biomed 2 (18 decks)

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