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MBB164 > Hettema (Spring) > Flashcards

Hettema (Spring) Flashcards

1
Q

Where are most proteins synthesised?

A
  • cytoplasm
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2
Q

Where are proteins transported to?

A
  • 50% delivered to specific membrane compartments

- many transported to lumen or inserted into membrane of organelles and further transported to other organelles

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3
Q

What is targeting and where does it start?

A
  • starts in cytosol

- delivers newly synthesised proteins to particular membrane of organelle

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4
Q

What is translocation?

A
  • transporting protein across membrane
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5
Q

What is protein sorting?

A
  • transports protein from 1 membrane-bound compartment to another
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6
Q

How do proteins find the right place in cell?

A
  • signal seq hypothesis

- proteins contain codes in AA seq that direct to specific membrane

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7
Q

What is a signal seq?

A
  • relatively short AA seq that directs to specific location w/in cell
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8
Q

General model for protein targeting

A
  • signal seq
  • receptor
  • docking at target membrane
  • translocation
  • recycling of receptor
  • energy source
  • protein folding
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9
Q

What did studying import of proteins into ER show?

A
  • confirmed signal seq hypothesis

- provided 1st biochem insight into how proteins translocated into lumen of organelle

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10
Q

How was it shown that secretory proteins are localised to ER lumen shortly after/during synthesis?

A
  • label cells w/ radioactive AAs
  • homogenise
  • protease prod assay
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11
Q

What is a microsome?

A
  • ER vesicles formed by homogenisation
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12
Q

What did Milstein’s experiments provide evidence for?

A
  • signal seq

- co-translational import

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13
Q

How does presence of microsomes affect cell-free protein synthesis?

A
  • when microsomes present allows co-translational transport of protein into microsome and removal of signal seq, prod mature protein chain w/o signal seq
  • when no microsomes present no removal of signal seq
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14
Q

Evidence for co-translational import

A
  • ribosomes assoc w/ ER membrane
  • newly synthesised protein assoc w/ ER around time of translation
  • in vitro translation in microsome absence renders proteins import incomplete, even if ER signal peptide present
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15
Q

What is the signal peptide?

A
  • at N terminus
  • removed upon entry into lumen of ER
  • core of 6-12 hydrophobic AAs often preceded by several positive AA residues
  • necessary and sufficient for import into ER
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16
Q

What does signal recognition particle (SRP) bind, block and deliver?

A
  • binds signal seq
  • binds specific receptor on endoplasmic membrane
  • blocks translation temporarily
  • delivers nascent protein complex to ER membrane translocation site
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17
Q

What is the series of events carried out by SRP?

A
  • SRP binds signal and blocks translation
  • SRP binds SRP receptor, GTP stabilises interaction and ribosome docks on membrane
  • transfer of ribosome/nascent polypeptide to translocon, pore opens and polypeptide inserted, SRP and SRP receptor dissociate from translocon, hydrolyse GTP and ready for next round
  • translocon resumes, signal seq cleaved as polypeptide elongates and translocates into ER lumen
  • completed polypeptide released into ER lumen, ribosome released and translocon pore closes
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18
Q

Cotranslational translocation of secretory proteins across ER membrane

A
  • specificity –> signal seq
  • receptor –> SRP
  • docking –> SRP receptor
  • protein conducting channel –> translocon
  • energy –> translation (or pulling by Hsp70 for posttranslational import
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19
Q

How many types of transmembrane protein are there?

A
  • 5 types
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20
Q

How is type I transmembrane protein inserted into membrane?

A
  • cotranslational import of soluble proteins until stop transfer anchor seq, prevents further translocation
  • stop transfer anchor seq moved laterally into membrane
  • elongating chain loops out into cytosol and released when translation of ribosome completed
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21
Q

How is type II single-pass transmembrane protein synthesised and inserted into ER membrane?

A
  • single anchor seq recognised by SRP and delivered to translocon
  • N terminus orientated to cytosol, believed to be mediated by positive charge just N-terminal of signal anchor seq
  • signal anchor seq subsequently moves laterally into lipid bilayer
22
Q

Post translational import

A
  • completed secretory protein targeted to ER membrane by binding of signal seq to translocon
  • unfolded polypeptide chain pulled in by Hsp70
  • req ATP hydrolysis
23
Q

What does GFP mito targeting signal (MTS) consist of?

A
  • amphipathic helix of 20-50 AAs
24
Q

What does post translational uptake of precursor proteins into isolated mito req?

A
  • cytosol
  • ATP
  • respiring mito
25
Q

What happens during post translational uptake of precursor proteins into isolated mito?

A
  • yeast mito proteins made by cyto ribosomes in cell free system
  • protein taken up into mito, uptake-targeting seq removed and degraded
  • proteins isolated w/in mito resistant to trypsin
  • uptake-targeting seq and mito protein degraded
26
Q

What are the steps of protein import into mito matrix?

A
  • unfolded precursor
  • binds receptor
  • inserts in channel outer membrane
  • threads through Tom and Tim complex
  • gets processed and Hsp70 pulls precursor in
  • protein folds into active conformation
27
Q

What provides the energy for steps of protein import into mito matrix?

A
  • Hsp70
28
Q

Import into mito

A
  • post translational
  • protein needs to be unfolded during translocation (kept unfolded by chaperones), import is energy dependent
  • cytosolic Hsp70
  • mito Hsp70
  • pmf (ec grad across MIM)
29
Q

What do some proteins form and assemble in cyto?

A
  • form oligomers

- assemble prosthetic groups

30
Q

Are proteins folded for import?

A
  • yes, unfolding not req
31
Q

What targeting signal do most proteins have?

A
  • C-terminal
32
Q

How is energy provided for protein formation and assembly?

A
  • recycling of receptors
33
Q

What is hyperoxaluria type 1?

A
  • hereditary kidney stone disease
  • accum of CaC2O4
  • 1st kidney and urinary tract affected
  • later deposition in almost every organ and tissue
34
Q

How is AGT different in hyperoxaluria type 1 patients?

A
  • lack AGT (ala-glyoxylate aminotransferase)
  • 70% no or reduced activity
  • rest have normal amount and activity but still develop kidney stones
35
Q

How can patients w/ normal amount and activity of AGT still develop kidney stones?

A
  • AGT mistargeting
  • double mutation = Gly170Arg slows dimerisation and folding of AGT
  • Pro11Leu creates MTS
  • so folded AGT imported into peroxisomes, not mito
36
Q

Common principles in protein targeting and translocation

A
  • targeting signal picked up by receptor in cytosol or on organelle membrane
  • complex binds to target membrane and protein passed on to translocation
  • receptor recycles
  • protein translocated
  • transport req energy
37
Q

How are vesicles are transported?

A
  • coat assembly and conc of cargo in bud
  • bud off from donor membrane enriched for cargo
  • uncoating
  • docking and fuse specifically w/ target membrane
38
Q

How are coat proteins involved in budding?

A
  • drive budding by oligomerisation on membrane, bending it

- coat proteins bind sorting signals

39
Q

Examples of diff coat proteins

A
  • clathrin
  • COPI
  • COPII
40
Q

How is vesicle formed at ER membrane?

A
  • Sar1-GDP binds Sec12 on ER
  • GDP exchange for GTP, anchors Sar1 into ER membrane
  • Sar1-GTP drives polymerisation of soluble coat factors
  • leading to budding
  • sorting signals in cargo receptors recognised by coat protein
  • GTP hydrolysis initiates uncoating, poss to isolate coated vesicles using non-hydrolysable GTP
41
Q

What is coat assembly controlled by?

A
  • GTPase
42
Q

How does vesicle fusion occur?

A
  • vSNARE on vesicle and tSNARE on target membrane form v tight complex, bringing membranes in close proximity and fusion occurs
  • Rab-GTP on vesicle binds complex on plasma membrane
  • SNARE proteins form complex, 4 helix bundle
  • ATP-dependent disassembly of SNARE complex by NSF and α- SNAP
  • GTp of Rab hydrolysed resulting in Rab dissociation
43
Q

Stages of anterograde transport (forward)

A
  • coat assembly and budding
  • coat released and tethered to donor membrane
  • specificity factors of vesicle and donor membrane pair up –> fusion
44
Q

How do ER resident proteins stay in ER and what evidence is there?

A
  • escape prevented, those that escape transported back

- evidence is golgo mods found on ER proteins

45
Q

Stages of retrograde transport (backward)

A
  • KDEL containing protein binds to specific receptor in golgi stack
  • complex incorp into retrograde vesicle
  • delivered to ER
  • contributes to keeping identity of ER
  • same principles for SNARE (not KDEL)
46
Q

How are vesicles trafficked from trans golgi network?

A
  • specific glycosylation signals target some proteins from golgi to lysosomes
  • N-linked protein glycosylation
  • processing N-linked oligosaccharides in RER
47
Q

N-linked protein glycosylation

A
  • starts in ER
  • added to nascent chain in RER
  • linked to Asn residue
  • complex oligosaccharide
48
Q

Processing N-linked oligosaccharides in RER

A
  • all glucose residues removed when protein properly folded in ER
  • then will leave ER to be transported to golgi
49
Q

What does transport to lysosomes depend upon?

A
  • depends on protein mod
  • transfer of phosphorylated glcNAC to C 6 atom of more than 1 mannose residue
  • after release from enzyme, a phosphodiesterase removes glcNAC, leaving phosphate
50
Q

Process of transport to lysosomes

A
  • Man-6-P added in cis golgi
  • Man-6-P protein binding to receptor in trans golgi and incorporation into clathrin coated vesicle
  • coat disassembles after budding
  • uncoated vesicle fuses w/ late endosome
  • release from receptor plus dephosphorylation and fusion of late endosome w/ lysosomes, receptor and coat proteins recyle
  • some receptors end up on PM
  • phosphorylated lysosomal protein occasionally secreted, can be picked up by PM sorted receptor via endocytosis
51
Q

What are the symptoms of I-cell disease?

A
  • skeletal deformations
  • psychomotor deformation
  • mental retardation
  • growth stops during 2nd year
  • die from cardioresp complications at 5-8 yrs
52
Q

Molecular basis of I-cell disease

A
  • lysosomes contain large inclusions of glycolipids and glycosaminoglycans
  • soluble lysosomal enzymes elevated in blood and urine
  • lysosomes lack at least 8 diff acid hydrolases
  • formation of Mannose-6-phosphate affected

MBB164 (7 decks)

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