MODULE 4: cellular machinery Flashcards
structural motifs
structural motifs are combinations of protein secondary structures
zinc-finger motif:
- both alpha and beta
- histidines and cytosines are cross braced by electrostatic interactions (e.g. zinc)
ring-finger motif:
- protein-protein interactions for substrate recognition
invaginations of the inner mitochondrial memrbrane are known as _________________
cristae
Ca2+/CaM switch
allosteric regulation (not chemically bound)
Ca2+ interactions changes calmodulin (CaM) tertiary structure to allow binding to a target protein
Ca2+/CaM binding opens up the catalytic site on this kinase to allow phosphorylation
increase in [Ca2+] acts as “on” switch
the GTPase switch
allosteric regulation (not chemically bound)
on = GTP off = hydrolysed to GDP
TO TURN OFF:
GAP activates GTPase activity, making GTPase hydrolyse GTP to GDP, inactivating switch
TO TURN ON:
GEF removes GDP and places GTP in place, activating switch
functions of cellular machines (5)
- transcription:
- transcription initiation complex ~ 30 subunits
- polymerases, linkers, adaptors, activators and DNA binding proteins - translation:
- machine to synthesize polypeptides from mRNA.
- yhe machine is made up of ribosomal RNA (rRNA) and proteins (ribonucleoprotein)
- protein synthesis requires ATP - protein folding:
- chaperonins –> barrel-shaped folding machine
- process driven by ATP hydrolysis - transport:
- kinesin: a microtubule motor required for cellular transport
- ATP binding and hydrolysis by the motor domains drives the “walking motion” - protein degradation:
- molecular machine for controlled proteolysis, 20S catalytic barrel core, 2 x 19S regulatory subunits
- ubiquitin (Ub) chains recognised by 19S regulatory subunit
- 19S-binding directs target protein into the core for proteolysis.
- polyubiquinitated proteins unfolded and degraded in 20S core in ATP-dependent manner
the nuclear membrane
surrounded by two membranes: nuclear lamina and nuclear envelope
- lamina supports nuclear envelope basally
- comprised of meshwork of intermediate filaments –> nuclear lamins
- this lattice interconnects with nuclear pores
inner nuclear membrane (INM) defines nucleus, whilst outer nuclear membrane (ONM) continuous with rough ER
INM and ONM are each phospholipid bilayers separated by perinuclear space
chromatin and regulation of chromatin structure
- DNA wraps around histone octomers
- DNA + histone octomer = nucleosome
- nucleosomes stack on top of each other
- chromatin structure is open/closed
- structure determines gene expression
structural regulation:
- post-translational modification of histones determines whether DNA is tightly packed or open
- N and C terminal tails come out of nucleosome
- these tails are sites for post-translational modification (acetylation etc)
- controls whether tails promote condensed or open DNA
Unacetylated: chromatin is highly condensed (transcriptionally inactive) – HETEROCHROMATIN
Acetylation – chromatin is less condensed, (transcriptionally active) – EUCHROMATIN
mechanism of transcription
- transcriptional regulators bind to DNA –> recruit chromatin remodelling structures –> chromatin opens
- a) also recruit a mediator (protein bridge) to recruit TFs to a promoter sequence
- b) mediator complex facilitates assembly of the pre-initiation complex that includes loading a RNA
polymerase (RNA pol II) on DNA - after initiation, transcription is paused by an elongation factor complex (NELF/DSIF).
- elongation pause is relieved by phosphorylation and remodelling of the elongation factors by a cdk/cyclin pair (P-TEFb)
nuclear pore complex (NPC)
sole gateway in/out of nucleus
spans both membranes
only small molecules can pass via passive diffusion
cytoplasmic NUPS form basket structure
NUPS form structure of pore (NPC) and anchor NPC to membrane
FG-NUPS = act as barrier to stop larger proteins passing through
FG-NUPS open up on signal to allow passage
** NPC has limit of ~40kDa
nuclear import mechanism
** diagram **
- importin and cargo move into nucleus via NPC
- in nucleus, ran-GTP binds importins with high affinity, causing the importins to release their cargo
- a) ran-GTP and the importin move back into cytoplasm (asymmetry arises).
- b) cytoplasmic GAP activity converts Ran-GTP to Ran-GDP lowering affinity —> release importins
- importins recycled to transport more cargo
- ran-GDP randomly diffuses back into nucleus to be converted into Ran-GTP by nuclear GEFs
(same ran-GTP/ran-GDP gradient drives both import and export)
nuclear export: ran-dependent mechanism
- ran-GTP binds exportins, promoting its association with cargo (opposite to import)
- hydrolysis of Ran-GTP to Ran- GTP in cytoplasm releases the exportin and cargo
- exportins and Ran-GDP move back through the NPC and mechanism reset by nuclear GEFs.
(same ran-GTP/ran-GDP gradient drives both import and export)
laminopathies
premature aging
protease cleaves precursor lamin into mature form
mature lamins control vital nuclear functions
mutations knocks out protease or mutate protease sites on lamins
results in defective nuclear architecture and premature ageing
shape of ER
rough = sheet-like structure or "cisternae" smooth = highly branched, "tubular"
without energy, phospholipid bilayers would be flat
reticulons have wedge-like structure with certain angle
- can bend tightly to make two layer flat disc
- can bend to make circle
ER branches: membrane fusion
smooth ER contains many 3-way branches
3 way branching comes about from fusion of an extending tube with side of another tubule
fusion facilitated by small G protein - atlastin
atlastin is a reticulon that contains GTP binding domain
two atlastin proteins on opposing membranes will bind and dimerise
GTP hydrolysis pulls membranes together for fusion
cotranslational translocation
secretory proteins: live in vesicles then secreted via exocytosis
1) targeting signal on N-terminus is detected
2) polypeptides direct protein to ER membrane
4) SRP (signal recognising particle) binds to both signal sequence of peptide and large ribosomal subunit
5) SRP facilitates docking onto ER membrane and stops signal sequence
5) receptor on membrane recognises SRP and polypeptide docks to translocon (pore)
- this is facilitated by GTP
- hydrolyses of GTP = SRP released from receptor
6) polypeptide enters ER and signal peptidase cleaves signal sequence
7) polypeptide folds within lumen of rough ER
insertion of type I membrane proteins into ER
TYPE 1: N-terminal in lumen and C-terminal in cytoplasm
SRP recognises N-terminal targeting sequence and halts translation
SRP docks polypeptide to ER membrane
docking opens translocon and translation continues
signal cleaved as protein passed through pore
stop transfer anchor (STA) signal
- membrane stops passing through and anchors itself to membrane
- protein translation completed
results with N-terminus in cytoplasm and C-terminus in lumen
insertion of type II membrane proteins into ER
TYPE 2: N-terminal in lumen and C-terminal in cytoplasm
type II membrane proteins DO NOT have a cleavable N-terminal signal sequence —> instead translation occurs in cytoplasm
internal “signal anchor” sequence recognised by SRP and protein is directed to ER translocon
once SA sequence is embedded, it is moved laterally along the bilayer and ribosome continues co- translation into ER lumen
results with C-terminus in cytoplasm and N-terminus in lumen
insertion of type III membrane proteins into ER
TYPE 3: very short N-terminal in lumen and C-terminal in cytoplasm
N-terminus us very short –> cannot be cleaved
translation occurs in cytoplasm
internal “signal anchor” sequence recognised by SRP and protein is directed to ER translocon –> SA sequence located very close to N-terminus
once SA sequence is embedded, it is moved laterally along the bilayer and ribosome continues co- translation into ER lumen
results with N-terminus in lumen and C-terminus in cytoplasm
golgi function: glycosylation
congenital defects of glycosylation
many secretory proteins are glycoproteins that have been glycosylated
these proteins are transported from ER then remodified in the Golgi
each compartment of golgi has different function, as each contains different sugar modifying enzymes
substrates in medial compartment come from cis, substrates in trans compartment come from medial = assembly line
glycosidases –> remove sugars
glycosyltransferases –> add individual sugars
congenital defects of glycosylation (CDG):
- deficiency in glycan-modifying enzymes = lose architecture of sugar branches
- muscular dystrophy, psychomotor retardation, cutis laxa (wrinkled, inelastic skin), autoimmune disease
disease associated with ER architecture: hereditary spastic paraplegia
progressive stiffness, contraction (spasticity), loss of co-ordination and weakness of lower limbs
~60% of mutations impact reticulons and atlastin –> defect in ER membrane shape
ER membrane shape and complexity is required to maintain axon function —> loss of peripheral neurons that innervate sensory and motor nerves
golgi organisation
golgi consists of stacked cisternae i.e. flat membrane discs stacked on top of each other
stacks connected laterally to one another
cisternae organised into cis, medial and trans compartments
this is further flanked on two sides by fenestrated tubular networks: the cis- golgi network (CGN) and trans golgi network (TGN) —> GOLGI IS POLARISED
CGN receives vesicles (protein + lipids) from the ER —> processed/modified/tagged as they move through the stack —> secretory cargo is packaged and leaves the golgi from the TGN
microtubules required to maintain:
- ribbon structure
- perinuclear localisation
during mitosis, reorganization of microtubules into spindles is accompanied by restructuring of the golgi.
golgi microtubules —> temporarily fragmented into mini-stacks and individual cisternae
golgi machinery: golgins and GRASPS
2 types of golgi proteins for formation:
1) GRASPs = golgin reassembly and stacking proteins
- hold cis, median and trans compartments in stack formation
- proteins on membrane dimerise and hold stacks
- do not facicilate membrane fusion
2) golgin tethers
- long coiled cord structure
- interacts/dimeriss with other golgins
- stick out in cytoplasm to tether two compartments together
- brings two golgi stacks together = membrane fusion
golgi machinery: formation of golgi ribbon
- Microtubule network and microtubule dependent transport clusters golgi mini- stacks at the perinuclear region
- Tethering protein (golgins) then draw mini-stacks into close proximity to allow membrane fusion.
- Golgi membrane fusion likely by SNARE mediated docking mechanism as with vesicle membrane fusion
golgi transport: ER to golgi
ANTEROGADE TRANSPORT:
- rough ER —-> cis golgi
- facilitated by COPII (coat protein)
- at rough ER, sorting signal on cytoplasmic face of membrane cargo is recognised by coat proteins
- coat proteins select cargo with signal for inclusion in budding vesicle
- coat proteins pinch off vesicle and it travels to cis side of Golgi and enters via endocytosis
RETROGADE TRANSPORT:
- cis golgi —-> rough ER
- facilitated by COPI (coat protein) and KDEL sequence (sorting signal)
- lower pH in golgi is recognised and triggers binding of KDEL to membrane receptor
- cargo is retrieved back to rough ER by COPI
- retrograde transport maintains cis-, medial and trans-golgi compartment residency (cisternal maturation)
golgi function: cisternal maturation
cargo doesn’t move, COMPARTMENTS MOVE
vesicles from ER form cis —> matures into cis cisternae —> matures into medial —> matures to trans —> vesicles disasemble trans
think standing wave = no net movement but compartments moving through stack
mitochondrial fission/fusion mechanism
mitochondrial morphology (elongated network) related to balance of fusion and fission events and depends on stage in cell cycle
fission:
- fission controlled by mitochondrial fission factors = MFF
- recruit small G-proteins called DRP1
- DRP1s hydrolyse GTP to pinch membranes (endocytosis/exocytosis)
fusion:
- fusion controlled by mitofusins = MFN
- hydrolyse GTP to fuse membranes
- different MFNs for outer and inner membranes
- outer membrane fusion followed by inner membrane fusion
- dimerise to bind membranes then hydrolyse GTP to fuse
mitochondrial transport: protein import
- proteins have to make it through narrow pores in mitochondria —> fully folded proteins cannot pass —> use chaperones to keep proteins unfolded —> needs ATP
- inner and outer membrane proteins require separate translocons (TIM and TOM)
- mitochondrial proteins synthesised in cytoplasm before targeted to mitochondria (unlike ER)
- targeting signal recognized by import receptor and directs cargo to the TOM complex
- unfolded cargo passes through both outer (TOMs) and inner membrane (TIMs) translocons simultaneously
- during translocation cargo is bound by matrix chaparone (Hsc70) which hydrolyses ATP to “pull” the cargo into the mitochondria
- targeting sequence at N-terminus processed (cleaved by protease, like ER) and cargo folded by chaperonins into mature tertiary state
steps in generating ATP
STEP 1: SUGAR + FAT METABOLISM
- sugars/lipids broken down into intermediate molecules which are transported into mitochondrial matrix
- glucose oxidised to pyruvate via glycolysis —> generates ATP and NADH in the process (high-energy electron carriers)
- while sugars/lipids broken down, carbon molecules funnelled into common intermediate —> energy released in C-H bonds —> stored by reducing NAD to NADH
STEP 2: GIVING UP ELECTRONS
- fatty acids and sugars broken down into acetyl CoA
- acetyl CoA then taken through citric acid cycle —> oxidatively broken down into CO2, whilst transferring energy to NAD and FAD —> NADH + FADH2
- sugars/lipids eventually oxidised to CO2
- proteins broken down into aa’s - different process
STEP 3: PUMPING PROTONS
- NADH and FADH2 donate their electrons
- electrons shuttled between membrane complexes by electron carriers (products are H2O + CO2)
- this energy is harnessed to pump protons into inter-membrane space —> creates charge across inner membrane
STEP 4: ATP SYNTHASE
- charge gradient harnessed by ATP synthase (want to return to equilibrium)
- ATP-synthase has pore which allows protons from inner membrane to matrix
- components in ATP synthase harness proton motor force into kinetic energy to turn base
- smallest known rotary electric motor spins to generate ~400 ATP molecules per second
diseases associated with mitochondria
mitochondria undergo fission/fusion for:
- growth/replication
- segregation during cell division
- cope with energy demand
- ** repair or remove damaged mitochondria **
parkinson’s disease
- tremors, bradykinesia, loss of movement and speech control
- mutations on genes encoding PINK (a kinase) and Parkin (a ubiquitin ligase)
- PINK accumulates in damaged organelles
- PINK recruits Parkin which ubiquitinates mitofusins (no fusion)
- unable to fuse, damaged organelle targeted for degradation
- in Parkinson’s damaged organelle not effectively removed
the release of cargo proteins by importins in the nucleus is triggered by …. ?
an excess of Ran-GTP in the nucleus
the hydrolysis of Ran-GTP to Ran-GDP by nuclear GAPs DOES NOT TRIGGER RELEASE, facilitates it
