Hettema - Cell destination

Question 1

where are the majority of proteins synthesised?

Answer 1

cytosol

Question 2

define targeting

Answer 2

movement of newly synthesised proteins from the cytosol to the membrane of an organelle

Question 3

define translocation

Answer 3

process of moving a protein across a membrane

Question 4

define sorting

Answer 4

portein travelling from one membrane bound compartment to another

Question 5

explain the signal sequence (SS) hypothesis

Answer 5

proteins containing a short a/a sequence that directs a protein to a specific cell location

Question 6

describe Gunther Blobel’s experiment that led to the SS hypothesis

Answer 6

label a/a’s in secretory proteins with a radioactive label
homogenise cells (leads to microsome formation)
protease protection assay (use detergent to remove microsome membrane - protease degrades secretory protein)

Question 7

what did Cesar Milstein’s experiment prove?

Answer 7

evidence for signal sequence and co-translational import

Question 8

describe Cesar Milstein’s experiment

Answer 8

cell-free protein synthesis with one half having microsomes added at the beginning and one with microsomes added later on
when the microsomes are added later there is no incorporation into proteins and the signal seq isn’t removed
when microsomes are added at the beginning the proteins are incorporated and the signal seq is removed

Question 9

describe the cotranslational import of a soluble protein

Answer 9

signal sequence recognition particle (SRP) binds to the signal sequence
SRP blocks translation temporarily
SRP binds specific receptor on the ER membrane
Transfer of polypeptide/ribosome complex to translocon leads to translocon opening.
When SRP and SRP receptor dissociate from translocon they hydrolyse each of their GTP.
SS enters ER lumen and is cleaved off polypeptide chain by signal peptidase and rapidly degraded.
Transl. begins again and polypeptide enter ER lumen through translocon.

Question 10

describe the insertion of a type I transmembrane protein

Answer 10

• Same as cotranslational import of soluble proteins until ribosome/nascent protein complex binds to translocon. The signal peptidase removes the SS. Nascent chain elongated until hydrophobic stop-transfer sequence. The nascent chain can no longer elongate into ER lumen. Stop-transfer sequence moved laterally into membrane and translocon closes. The chain begins to elongate again until stop codon is reached. (N-terminus in ER and C-terminus in cellular env)

Question 11

describe the insertion of a type II transmembrane protein

Answer 11

• Same as cotranslational import of soluble proteins until the ribosome/nascent protein complex binds to translocon. NO SS PRESENT – hydrophobic signal-transfer sequence acts as a ER signal and membrane-anchor sequence. At the N-terminus end of the nascent protein there is a positive charge (just before the stop-transfer sequence), this orients the N-terminal end into the cellular env and not the ER lumen. As the chain is elongated the signal-anchor sequence is moved laterally out of the translocon and into the membrane. (N-terminus in cellular env and C-terminus in ER)

Question 12

describe the post-translational import of secretory protein

Answer 12

signal sequence binds translocon and is cleaved by signal peptidase
Hsp70 pulls the unfolded polypeptide chain in by ATP hydrolysis
chaperones help fold the nascent mRNA

Question 13

name 3 types of protein modification and where and when they occur

Answer 13

when: happen to newly synthesised proteins
- glycosylation: in ER and Golgi
- disulphide bond formation: in ER
- proper folding and multimeric complex formation: in ER

Question 14

why do proteins undergo glycosylation?

Answer 14

• may promote folding
• stabilises some secreted proteins
• involved in cell to cell interactions

Question 15

name 2 pieces of evidence that suggest glycosylation promotes protein folding

Answer 15

tunicamycin inhibits glycosylation (and secondary protein folding in ER)
mutation of glycosylation site in haemagglutinin (HA) = build up of unfolded HA in ER

Question 16

describe n-linked glycosylation

Answer 16

added to nascent chain in RER
linked to Asn residue
complex oligosaccharide

Question 17

describe o-linked glycosylation

Answer 17

added to nascent chain in RER
linked to ‘OH’ group of Thr/Ser residue
simple oligosaccharide

Question 18

describe the processing and folding of n-linked oligosaccharides in the RER

Answer 18

1) glycosylation occurs in translocation
2) glucosidases trim core glycans
3) if the protein isnt folded correctly UGGT (UDP-glucose: glycoprotein glucosyltransferase) adds glucose again
4) mannose is removed and the oligosaccharide is transferred to the Golgi

Question 19

what happens to incorrectly folded proteins?

Answer 19

transferred to cytosol to be degraded by proteosome

Question 20

describe the target sequence and location for ER-bound peptides

Answer 20

N-terminus

6-12 h.phobic a/a’s preceded by +ve a/a

Question 21

describe the target sequence and location for mitochondrial-bound peptides

Answer 21

N-terminus

amphipathic helix, 20-50 a/a’s. Arg and Lys on one side and h.phobic on the other side

Question 22

describe the target sequence and location for chloroplast-bound peptides

Answer 22

N-terminus

usuallly rich in Ser, Thr and smalll h.phobic a/a’s. Usually low in Glu and Asp

Question 23

describe the target sequence and location for peroxisome-bound peptides

Answer 23

PTS1 signal (Ser-Lys-Leu) for C-terminal
PTS2 signal for N-terminus

Question 24

describe the post-translational uptake of precursor proteins into the mitochondria

Answer 24

type of protease protection assay

1) uptake-targeting seq &mitochondrial protein made in cell-free system
2a) add yeast mitochondria (to half of solution)
3a) add trypsin - no degradation as there has been uptake into mitochondria
2b) add trypsin (to half of solution) - degradation as there has been no uptake into mitochondria

Question 25

describe protein import into the mitochondrial matrix

Answer 25

1) unfolded precursor binds receptor 2) protein inserts into outer membrane channel 3) threads through TOM and TIM complexes pulled by Hsp70 4) protein folded into active conformation (this requires inner and outer membrane contact)

Question 26

describe the interaction between methotrexate (MTX) and chimeric DHFR

Answer 26

MTX binds DHFR which folds it, meaning it cannot pass through the translocon and only the signal sequence is removed DHFR would otherwise enter the mitochondrial matrix and be folded (this requires inner and outer membrane contact)

Question 27

describe peroxisomal import

Answer 27

PTS1 binds receptor receptor which binds to membrane | receptor is recycled after PTS1 dissociation (requires ATP hydrolysis)

Question 28

what is hyperoxaluria type I?

Answer 28

heritable kidney stone disease which leads to calcium oxalate

Question 29

why do people get hyperoxaluria type I?

Answer 29

70% lack AGT enzyme 30% have AGT enzyme but with incorrect AGT targeting sequence - double mutation of Pro11Leu and Gly170Arg creates mitochondrial targeting signal and leads to slowed dimerisation and folding of AGT

Question 30

what does the Pro11Leu mutation do in the AGT gene?

Answer 30

has a weak mitochondrial targeting sequence therefore goes mainly to peroxisomes

Question 31

describe the pulse-chase experiment by george palade

Answer 31

labelled slices of tissue with radioactive Leu (pulse) washed out leu incubated for set times - saw proteins moving from ER to Golgi to post-golgi

Question 32

describe video microscopy of GFP labelled VSV-G protein

Answer 32

temp sensitive mutant stays in ER at high tmp at lower temp transporter to golgi and PM lowered temp and say the fluorescence levels increase in golgi first, then PM as they decreased in the ER

Question 33

describe what happens to glycoproteins when they enter the golgi from ER and what happens when theyre treated with endoglycosidase D

Answer 33

mannose sugars trimmed in golgi trimmed in golgi means sensitive to endoglycosidase D untrimmed ER protein not sensitive to endonuclease D used temp sensitive mutants to show how fraction of endonuclease D sensitive proteins increased over time

Question 34

describe the steps in vesicular transport

Answer 34

1) coat assembly and concentration of cargo in bud 2) budding 3) uncoating 4) docking and fusion

Question 35

name 3 coat proteins

Answer 35

clathrin cop I cop II

Question 36

how do coat proteins drive budding

Answer 36

by oligomerisation on the membrane and thereby bending the membrane Coat proteins bind sorting signals vSNAREs direct vesicle to organelles

Question 37

describe vesicle formation at ER membrane

Answer 37

Coat assembly under control of GTPase. Sar1-GDP binds Sec12 on ER membrane, the exchange of GDP for GTP anchors Sar1 into the membrane. Sar1-GTP (in mem) drives polymerisation of soluble coat factors (leads to budding). Sorting signals in cargo receptors are recognised by coat proteins. GTP hydrolysis initiates uncoating. Vesicle type COPII (ER --> cis-Golgi) uses GTPase Sar1// COPI (cis-Golgi --> ER) uses ARF as does clathrin.

Question 38

describe vesicle fusion with PM

Answer 38

- Rab-GTP on vesicles binds complex on PM. 3 helices on PM and vSNARE helix interact and form SNARE complex. The membranes fuse. ATP hydrolysis disassembles the SNARE complex. GTP hydrolysis on Rab results in Rab dissociation. The vSNARES are brought back into the vesicle to be recycled.

Question 39

describe anterograde transport

Answer 39

coat assembly and budding from ER coat is released and vesicle meets golgi membrane specificity factors vesicle membrane and donor membrane pair = fusion

Question 40

describe retrograde transport

Answer 40

KDEL containing peptide bind to specific receptor in golgi stack complex gets incorporated into retrograde vesicle delivered to er retrieval of KDEL bearing peptides to ER

Question 41

describe the two models of transport through the Golgi complex

Answer 41

``` stationary cisternal model transport between stacks by vesciles cisternal maturation model retrograde transport trans turns to medial etc ```

Question 42

describe the transport from the golgi to the lysosomes

Answer 42

Man-6-P added in cis-Golgi. Man-6-P protein binds to receptor in trans-Golgi and is incorporated into clathrin-coated vesicle. After vesicle formation the coat is disassembled and fuses with the late endosome (LE). Protein is released from the receptor and dephosphorylated. The LE then fuses with the lysosome and the receptor and coat proteins are recycled.

Question 43

what causes I-cell disease

Answer 43

formation of mannose-6-P is affected

Question 44

describe clathrin receptor-mediated endocytosis

Answer 44

- Adaptor proteins bind receptors, clathrin, membrane, factors required for vesicle formation and release. Dynamin then pinches off the vesicle (by GTP hydrolysis) forming a vesicle.

Question 45

describe endocytosis of LDL

Answer 45

- Ligand binding of LDL to the LDL-receptor. Incorporation of LDL and receptor into a clathrin-coated vesicle. Vesicle is then uncoated and fused with the LE. LDL dissociates from the receptor (requires pH <5) and the LE fuses with the lysosome The LDL receptor is recycled.

Question 46

what happens at pH 7 and pH 5 in LDL release

Answer 46

pH7 ligand binding receptor to LDL particle (bound on mem) | pH5 beta propellor domain becomes positively charged and ligand binding arm attaches (releases in endosome)

Question 47

what is familial hypercholesterolaemia

Answer 47

LDL high in serum mutations in the LDL receptor fat deposition in certain cell types