MODULE 4: cellular machinery

Question 1

structural motifs

Answer 1

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

Question 2

invaginations of the inner mitochondrial memrbrane are known as _________________

Answer 2

cristae

Question 3

Ca2+/CaM switch

Answer 3

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

Question 4

the GTPase switch

Answer 4

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

Question 5

functions of cellular machines (5)

Answer 5

1. transcription:
   - transcription initiation complex ~ 30 subunits
   - polymerases, linkers, adaptors, activators and DNA binding proteins
2. translation:
   - machine to synthesize polypeptides from mRNA.
   - yhe machine is made up of ribosomal RNA (rRNA) and proteins (ribonucleoprotein)
   - protein synthesis requires ATP
3. protein folding:
   - chaperonins –> barrel-shaped folding machine
   - process driven by ATP hydrolysis
4. transport:
   - kinesin: a microtubule motor required for cellular transport
   - ATP binding and hydrolysis by the motor domains drives the “walking motion”
5. 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

Question 6

the nuclear membrane

Answer 6

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

Question 7

chromatin and regulation of chromatin structure

Answer 7

• 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

Question 8

mechanism of transcription

Answer 8

1. transcriptional regulators bind to DNA –> recruit chromatin remodelling structures –> chromatin opens
2. a) also recruit a mediator (protein bridge) to recruit TFs to a promoter sequence
3. b) mediator complex facilitates assembly of the pre-initiation complex that includes loading a RNA
   polymerase (RNA pol II) on DNA
4. after initiation, transcription is paused by an elongation factor complex (NELF/DSIF).
5. elongation pause is relieved by phosphorylation and remodelling of the elongation factors by a cdk/cyclin pair (P-TEFb)

Question 9

nuclear pore complex (NPC)

Answer 9

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

Question 10

nuclear import mechanism

Answer 10

** diagram **

1. importin and cargo move into nucleus via NPC
2. in nucleus, ran-GTP binds importins with high affinity, causing the importins to release their cargo
3. a) ran-GTP and the importin move back into cytoplasm (asymmetry arises).
4. b) cytoplasmic GAP activity converts Ran-GTP to Ran-GDP lowering affinity —> release importins
5. importins recycled to transport more cargo
6. 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)

Question 11

nuclear export: ran-dependent mechanism

Answer 11

1. ran-GTP binds exportins, promoting its association with cargo (opposite to import)
2. hydrolysis of Ran-GTP to Ran- GTP in cytoplasm releases the exportin and cargo
3. 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)

Question 12

laminopathies

Answer 12

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

Question 13

shape of ER

Answer 13

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

Question 14

ER branches: membrane fusion

Answer 14

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

Question 15

cotranslational translocation

Answer 15

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

Question 16

insertion of type I membrane proteins into ER

Answer 16

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

Question 17

insertion of type II membrane proteins into ER

Answer 17

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

Question 18

insertion of type III membrane proteins into ER

Answer 18

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

Question 19

golgi function: glycosylation

congenital defects of glycosylation

Answer 19

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

Question 20

disease associated with ER architecture: hereditary spastic paraplegia

Answer 20

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

Question 21

golgi organisation

Answer 21

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:

1. ribbon structure
2. 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

Question 22

golgi machinery: golgins and GRASPS

Answer 22

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

Question 23

golgi machinery: formation of golgi ribbon

Answer 23

• 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

Question 24

golgi transport: ER to golgi

Answer 24

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)

Question 25

golgi function: cisternal maturation

Answer 25

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

Question 26

mitochondrial fission/fusion mechanism

Answer 26

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

Question 27

mitochondrial transport: protein import

Answer 27

• 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)

1. mitochondrial proteins synthesised in cytoplasm before targeted to mitochondria (unlike ER)
2. targeting signal recognized by import receptor and directs cargo to the TOM complex
3. unfolded cargo passes through both outer (TOMs) and inner membrane (TIMs) translocons simultaneously
4. during translocation cargo is bound by matrix chaparone (Hsc70) which hydrolyses ATP to “pull” the cargo into the mitochondria
5. targeting sequence at N-terminus processed (cleaved by protease, like ER) and cargo folded by chaperonins into mature tertiary state

Question 28

steps in generating ATP

Answer 28

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

Question 29

diseases associated with mitochondria

Answer 29

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

Question 30

the release of cargo proteins by importins in the nucleus is triggered by …. ?

Answer 30

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