Molecules to cells Flashcards
Signal hypothesis
Blobel - proteins have intrinsic signals that govern their transport + localisation in the cell intracellular postcodes
-> signal sequences made up of specific types of a. acids (can be removed after), often hydrophobic side chains.
70% proteins remain in cytosol
Features of nucleus
Envelope - composed of 2 membranes w/ underlying lamina (protein network), continuous w/ ER
Pores - 30+ proteins act ac gateways, small water sol mols diffuse freely through, larger components (RNA, proteins) actively transported across pore complex
Nuclear localisation signal (NLS)
Target proteins to nucleus, common feature is many +ve a. acids (Arg, Lys), short + can be located anywhere in protein, not removed after.
Protein import into nucleus
- Receptor binds proteins w/ NLS in cytosol
- cytosolic fibrils direct receptor to pore + binds pore proteins, cargo proteins moved into nucleus through gel-like meshwork of fibrils
- pores large so folded proteins can be imported
GTP hydrolysis by RAN
Drives nuclear import
Small GTPase RAN is GTP or GDP bound w/ different localisations
- RAN-GAP (reg1) triggers GTP hydrolysis
- GEF (reg2) promotes exchange of GDP for GTP
High Ran GTP in nucleus displaces cargo protein from receptor - receptor recycled back to cytosol
GTP hydrolysed in cytosol so Ran-GDP dissociates
How is NLS exposed?
Nuclear factor of activated T-cells (NFAT) - family of TFs - stimulated to enter nucleus by calcium (changes their conformation).
-> exposes NLS
Cut and paste experiments used to conclude NLS required + sufficient on its own for nuclear import.
Mitochondria features for protein import
Inner membrane impermeable BUT outer membrane permeable to all mols < 5000 Da.
Targeting sequences normally have high Arg(+) & Ser/Thr (nonpolar)
- located N terminus, 20-80 a.acids long
- cleaved after import
-> can form ampiphilic a-helix w/ 2 different sides
Mechanism of import into mitochondria
- Targeting signal recognised by receptor on outer mem.
- Translocator (TOM) channel moves protein into inter-membrane space.
- Signal binds TIM in inner mem.
- Signal sequence cleaved (protein must be open/unfolded to enter matrix)
-> chaperone protein (Hsp70s) pulls protein into matrix + helps refold it.
Protein targeting to ER
Pancreatic cells have extensive rER, hepatocytes have extensive sER.
ER entry point for proteins destine to other organelles/cells, delivered by vesicular transport
Targeting sequence has 8+ hydrophobic a. acids (leu, val, isoleucine) residue near N terminus.
Proteins enter whilst still being synthesised (co-translational)
Mechanism of import into ER
- SRP binds ER signal sequence as it emerges from ribosome (translation paused)
- SRP binds SRP receptor adjacent to translocator protein (Sec61) on ER mem.
- SRP displaced + released for reuse
- ribosome passes through translocator (translation resumed)
Binding of signal sequence by Sec61 opens channel.
Polypeptide threaded though channel as loop.
Signal sequence cleaved by signal peptidase.
Membrane protein insertion
- ER signal sequence binds Sec61 opening channel
- Hydrophobic stop-transfer seq stops polypeptide movement through channel
- Stop-transfer seq released into bilayer forming transmembrane domain
- Protein inserted into bilayer w/ fixed orientation, N terminus in lumen.
- Signal sequence cleaved
Alternating start/stop transfer sequences generate complex multi pass proteins
Post translational modification in ER
Folding assisted by molecular chaperones.
e.g. BiP is an ATPase that binds exposed hydrophobic residues, Calnexin binds N-glycosylated proteins
Disulphide bonds formation: oxidation of cysteine residue -> increases tertiary structure stability
N-linked glycosylation (N=asparagine): lipid donor dolichol donates oligosaccharide to protein, catalysed by OST, only done in specific consensus sequence
Functions of post-translational modifications
- Assist protein folding
- Creation of manmose-6-phosphate tags act as lysosome sorting signal
- Act as ligand for specific cell-cell recognition events.
Glycocalyx (protective layer) made at ER + golgi - used to coat eukaryotic cells
Quality control in ER
Chaperones bind misfolded proteins + stop them leaving ER.
Unfolded protein response (UPR) occurs when build up of misfolded proteins in ER lumen.
- activates ER sensor protein which activates chaperone genes
SDS-polyacrylamide gel electrophoresis
Sodium dodecyl sulphate (SDS) binds protein to give them negative charge, acts as ionic detergent so will unfold proteins
Polyacrylamide gel is mesh like gel that separates charged proteins by size.
They move towards positive anode faster if they’re smaller
Can use coomassie blue, silver stain, radiolabel or antibodies to visualise proteins.
Membrane lipid synthesis
- Catalysed by enzymes on cytosolic face of ER mem.
- Scramblase transporters transfer phospholipids between leaflets non selectively until equilibrium reached
*far more phosphatidyl serine in inner leaflet
-> flippase transporters in golgi flip specific phospholipids from outer -> inner mem
Membranes + proteins retain orientation during vesicular transport -> lumen domain joins extracellular surface
Vesicle formation
- Protein coat deforms membrane into bud captures cargo.
- Dynamin (GTPase) helps bud pinch off.
- Coat proteins (clathrin in endocytic pathway of plasma mem + golgi) removed.
How is cargo selected and vesicle separated?
Adaptins help clathrin attach to mem forming clathrin-coated pit on cytosolic face.
They also bind cargo receptors which recognise specific sorting signals on cargo proteins, recruiting them into vesicle.
-> clathrin cage causes membrane to invaginate, dynamin assembles ring around neck of bud - GTP hydrolysis changes dynamin conformation contricting neck of bud
How does vesicle uncoating occur?
Coat proteins (e.g. clathrin) removed - requires molecular chaperones + ATP
Mechanism of vesicle docking + fusing
- Rab protein binds vesicle, v-SNARE acts as marker
- specific tethering protein on target organelle binds Rab
- v-SNARE & t-SNARE interact + wrap tightly to allow vesicle docking
- bilayers must be close (1.5nm) to fuse, water must be displaced (energetically unfavourable)
Retention & sorting in ER & golgi
Sorting/trafficking signals vital
ER retention signal: KDEL sequence at C terminus of soluble proteins recognised by KDEL receptor in golgi
-> recruited in COPI vesicles + returned to ER
(BiP & PDI contain KDEL sequences)
- short TM domain (18a. acids) retains proteins in golgi
- addition of Mannose-6-phosphate to N linked glycans of some glycoproteins sends them to lysosome.
Golgi apparatus
cis network - entry, carrying vesicles from ER
trans network - exit, carrying proteins onwards
Vesicular transport model in Golgi
Cisternae static components containing specific enzymes. Vesicles bud + fuse through each cisternae .
- cargo mols present in small transport vesicles (100nm)
Cisternal maturation model
Cisternae matures as it migrates outward through the stack (cis -> trans), resident enzymes carried forward are returned to earlier compartment
- transport large proteins like collagen (300nm), too big for typical vesicles