Hettema (Spring) Flashcards
Where are most proteins synthesised?
- cytoplasm
Where are proteins transported to?
- 50% delivered to specific membrane compartments
- many transported to lumen or inserted into membrane of organelles and further transported to other organelles
What is targeting and where does it start?
- starts in cytosol
- delivers newly synthesised proteins to particular membrane of organelle
What is translocation?
- transporting protein across membrane
What is protein sorting?
- transports protein from 1 membrane-bound compartment to another
How do proteins find the right place in cell?
- signal seq hypothesis
- proteins contain codes in AA seq that direct to specific membrane
What is a signal seq?
- relatively short AA seq that directs to specific location w/in cell
General model for protein targeting
- signal seq
- receptor
- docking at target membrane
- translocation
- recycling of receptor
- energy source
- protein folding
What did studying import of proteins into ER show?
- confirmed signal seq hypothesis
- provided 1st biochem insight into how proteins translocated into lumen of organelle
How was it shown that secretory proteins are localised to ER lumen shortly after/during synthesis?
- label cells w/ radioactive AAs
- homogenise
- protease prod assay
What is a microsome?
- ER vesicles formed by homogenisation
What did Milstein’s experiments provide evidence for?
- signal seq
- co-translational import
How does presence of microsomes affect cell-free protein synthesis?
- 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
Evidence for co-translational import
- 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
What is the signal peptide?
- 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
What does signal recognition particle (SRP) bind, block and deliver?
- binds signal seq
- binds specific receptor on endoplasmic membrane
- blocks translation temporarily
- delivers nascent protein complex to ER membrane translocation site
What is the series of events carried out by SRP?
- 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
Cotranslational translocation of secretory proteins across ER membrane
- specificity –> signal seq
- receptor –> SRP
- docking –> SRP receptor
- protein conducting channel –> translocon
- energy –> translation (or pulling by Hsp70 for posttranslational import
How many types of transmembrane protein are there?
- 5 types
How is type I transmembrane protein inserted into membrane?
- 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
How is type II single-pass transmembrane protein synthesised and inserted into ER membrane?
- 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
Post translational import
- completed secretory protein targeted to ER membrane by binding of signal seq to translocon
- unfolded polypeptide chain pulled in by Hsp70
- req ATP hydrolysis
What does GFP mito targeting signal (MTS) consist of?
- amphipathic helix of 20-50 AAs
What does post translational uptake of precursor proteins into isolated mito req?
- cytosol
- ATP
- respiring mito
What happens during post translational uptake of precursor proteins into isolated mito?
- 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
What are the steps of protein import into mito matrix?
- 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
What provides the energy for steps of protein import into mito matrix?
- Hsp70
Import into mito
- 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)
What do some proteins form and assemble in cyto?
- form oligomers
- assemble prosthetic groups
Are proteins folded for import?
- yes, unfolding not req
What targeting signal do most proteins have?
- C-terminal
How is energy provided for protein formation and assembly?
- recycling of receptors
What is hyperoxaluria type 1?
- hereditary kidney stone disease
- accum of CaC2O4
- 1st kidney and urinary tract affected
- later deposition in almost every organ and tissue
How is AGT different in hyperoxaluria type 1 patients?
- lack AGT (ala-glyoxylate aminotransferase)
- 70% no or reduced activity
- rest have normal amount and activity but still develop kidney stones
How can patients w/ normal amount and activity of AGT still develop kidney stones?
- AGT mistargeting
- double mutation = Gly170Arg slows dimerisation and folding of AGT
- Pro11Leu creates MTS
- so folded AGT imported into peroxisomes, not mito
Common principles in protein targeting and translocation
- 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
How are vesicles are transported?
- coat assembly and conc of cargo in bud
- bud off from donor membrane enriched for cargo
- uncoating
- docking and fuse specifically w/ target membrane
How are coat proteins involved in budding?
- drive budding by oligomerisation on membrane, bending it
- coat proteins bind sorting signals
Examples of diff coat proteins
- clathrin
- COPI
- COPII
How is vesicle formed at ER membrane?
- 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
What is coat assembly controlled by?
- GTPase
How does vesicle fusion occur?
- 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
Stages of anterograde transport (forward)
- coat assembly and budding
- coat released and tethered to donor membrane
- specificity factors of vesicle and donor membrane pair up –> fusion
How do ER resident proteins stay in ER and what evidence is there?
- escape prevented, those that escape transported back
- evidence is golgo mods found on ER proteins
Stages of retrograde transport (backward)
- 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)
How are vesicles trafficked from trans golgi network?
- specific glycosylation signals target some proteins from golgi to lysosomes
- N-linked protein glycosylation
- processing N-linked oligosaccharides in RER
N-linked protein glycosylation
- starts in ER
- added to nascent chain in RER
- linked to Asn residue
- complex oligosaccharide
Processing N-linked oligosaccharides in RER
- all glucose residues removed when protein properly folded in ER
- then will leave ER to be transported to golgi
What does transport to lysosomes depend upon?
- 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
Process of transport to lysosomes
- 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
What are the symptoms of I-cell disease?
- skeletal deformations
- psychomotor deformation
- mental retardation
- growth stops during 2nd year
- die from cardioresp complications at 5-8 yrs
Molecular basis of I-cell disease
- 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
