Methods for lateral movement
Fused human and mouse GFP
Heterokaryons
Fused by polyethylene glycol or Sendai virus
Rapid mixing of dyes
Rotational movement methods
Loss of fluorescence polarisation
Eosin maleimide reacts with AE1
Suggests a fast rotating and slow rotating population
Slow maybe linked to cytoskeleton
Flip flop movement methods
Membrane impermeable enzymatic probes
Side selective membrane bleaching
Freeze fracture of synapse and paranodes
RBCs show random distribution of IMPa
Other cells have adhesomes where IMPs near synapse
Overcomes free diffusion
Paranodes show high concentrations of ion channels
Mitochondrial IMPS
Subjected to electric field before freezing
Normally random, but seen at one end
Lateral diffusion after FRAP
Outer leaflet labelled
Bleach a small area
Lipid diffuse into bleached area restoring fluorescence
Diffusion coefficient can be determined
Integrins recovery is slow as bound to ECM
Single particle tracking
Used to measure lateral diffusion
Colloidal gold attached to Integrins, becomes stuck on matrix
Gold particles are corralled by the cytoskeleton
Gold-DOPE
Diffusion indexes
X10,000 difference between ACH and bacteriorhodopsin
Aggregation into purple patches stops diffusion
Hydrophobicity scales
Kyle Doolittle
Hopp-woods- potential antigenic sites of proteins. Rich in charged residues, so gives a hydrophilic index. Window size 6
False negatives and positives
Does not show the 7 TMs of GPCRs
More accurate is to use a hidden Markov model which is a statistical representation
Synthesis of class I proteins
N terminal signal sequence binds to a SRP
This SRP then binds to an SRP receptor before the protein is fed through the translocon
When both termini are on the same side it’s type I
N terminal signal sequence is cleaved by a signal peptidase
Will have the N in the lumen. Positive charge in near C
Synthesis of Class II proteins
Hydrophobic transmembrane sequence
Due to charges which flank the TM
Positive charges always face towards the cytoplasm not lumen
So if there is a positive next to the C, then type I
Positive next to N is type 2.
When the charges are swapped, this converts 2 to 1
But without swapping, the default is a type 2 with N into cytoplasm
Signal anchor will always insert in type 2 unless charges are switched
Type 2 will have the N terminus facing inwards
Mutations in the signal sequence don’t make a difference
Default in N in
With a signal sequence, N will always be in cytoplasm
Definition of channel
Approach free limits of diffusion
Not saturable
Direction by charge and gradient
Open and closing of tube
Definition of carriers
Stereo selective
Below free diffusion
Outward and inward facing
50 families
Aquaporin
6 TM helices and 2 half, 30 tilt with NPA loop
Cytoplasmic termini
Pore in tetrameric centre
His180 Arg195 make size and repel H3O
Water enters oxygen last but is switched by Positive dipoles of helix
Hg binds to C189
In tobacco plants transports CO2 shown by acidification
Potassium channel
8 TM helices, two from each tetrameric
Wide end of cone to extra cellular
C=O of pore loops coordinate to ion
4 sites but two at a time
Mutual repulsion drives through channel, no large barriers
1000/1 selective, selectivity loop removes water shell
Large vestibule rehydrates the ions
Movement of helices thought to restrict pore
Voltage gated postassium channel
4 TM helical extensions
4 beta subunits
S4 helix has Arg which is pulled to membrane
Depolarisation causes pull to extra cellular which opens channel
The M2 proton channel
Acidification of endosome release virus from receptor and detach RNA
Inhibited by amantadine
Homotetramer
4 Trps block channel, coordinate with His
As pH increases, Trp points downwards instead of across and opens channel
Two possible mechanisms-
His passes protons to water
Proton hopping of water
NA/k ATPase
3na out, 2 k in
Needed to maintain conc gradient and action of secondary transporters
A, b, y subunits
Beta is a chaperone and type 2
Gamma regulated by PKA and PKC by adrenaline , type 1
Alpha has Asp phosphorylation. Binds cholesterol to activate only at membrane
3 domains: nucleotide, actuator, phosphorylation
Drugs to affect NA/K ATPase
Ouabain- glycosides which inhibits, mimics cholesterol. Blocks NA leaving.
Digoxin- increases cytoplasmic NA, less NCX activity which increase Ca conc to give stronger contraction
Secondary transporters
Sodium always flows out to in, so after first pumping it out the reentry can be paired with another transporter
Symporters Glucose (creates internal x30,000), nucleosides
Antiporters
Extrusion of acid (NHE) or calcium (NCX)
Action potential opens Ca in membrane and SR
Moves troponin allowing contraction
Ca pumped back to SR using ATP or exchanged for NA entry
Secondary active transporters in kidney
SLC, KCC4, NKCC2 (NaKcl)
KCC3 in the PT, NCC and KCC4 in cortex and NKCC2 in outer medulla
Transporters of SLC12A are electro chemically neutral and regulate ion balance. Diuretic targets
SLC21 transporters in kidney
NKCC2 brings in NA, 2Cl, K
ROMK pumps K back out
NA is pumped back into blood by exchange for K
Cl into blood by CLCK
NCC brings in NA and Cl
TRPV5 brings in Ca
NA/K exchanger
NCX1 brings NA from blood exchanging for Ca
Ca can also be returned to blood by the H/Ca exchanger (PMCA1b)
Actions of diuretics
Sodium has a hydration she’ll
Acts on NA transporters to prevent water reabsroption
Furosemide- NA/KCl
Thiazides- NaCl
Transporters in homeostasis
Glycolysis makes pyruvate and H before kerbs making 6H MCT removes lactate and H AE removes HCO3- NHE removes H for NA NA is exchanged for K by NKA
NHE1 and metastasis
Large ATP at leading edge
Extrusion of acid
NHE1 and lysosomes are redistributed to the pseudopod tip to extrude acid and protease
Focalised proteolysis of ECM and cell attachments
Deteriorating environment gives positive feedback to NHE1
Mesenchymal to ameboid transition, moves through gaps in ECM
NHE1 regulated by cofillin
NHE1 removes 2H+ from the asp122,cofillin,his133 complex
At a low Ph His binds to PIP2
NHE1 makes alkali so loses PIP2 interaction
Complex is activated for barbed end formation
PIP2 -> IP3 and DAG by PLC
Tertiary transporter
NA/K ATPase
NA flows back in with AA1
AA1 then exchanged for AA2
Could be used to remove high amounts of AA1 to create useful exchange
GLUT1 structure
12 TM Lys mod by n-Hydroxy-succinimide esters -> 90% inhibition in Cys mutations Neutral bilayer interaction Few hydrophilic show complexes? Large N and O and cleft bottom shows C=O binding to sugars
In bacteria, transport H too to overcome energy, by mutating GLUT1 can create Glu/H symporter
Eukaryotes lost ability as constant high glucose
Beta barrel proteins
Humans have a low H gradient but high NA
Gram negative have peri plasm and peptidoglycan
Beta barrels excreted in peri plasm as hydrophilic before outer mem
Only needs 7 AA to cross
Tilted from 20-45 degrees
Porins for transport e.g. Maltose
Surface loops are antigenic sites
Variations in Eyelet in antibiotic resistant bacteria
Present in short-lived to inactive state delcour 1997
Squalene hopeless cyclase
Each half is enzyme
Substrate in ER membrane
Long chain to sterol ring
Two non polar plateaux
Prostaglandin H2 synthase 1
Targets head group of A acid
Channel links cavity to membrane binding surface
Mobile loops release
Myristoylstion
2 examples
Myristic acid 14:0 to n terminal glycine Donor of myristoyl-CoA Proteolytic event of N terminal signal Specific 7 residues Regulated membrane association Permanent and cotranslational
MARKS- regulates actin in neurons. PKC phosphorylates Ser which repels membrane
Recoverin- Ca sensor, ca exposes myristoyl group
Isoprenylation
2 examples
Strong with bilayer- targeting
CAAX box c terminus
Transfers from polyisoprene pyrophosphate precursors
AAX removed, often methylated
Precursor mevalonic acid, based around farnesyl and geranylgeranyl (X is leu/Phe)
Lamin B
Cell cycle dependent
Rab/Ral A and B
RabGDP is soluble and binds to GDI
Recycled back to membrane where GEF activates
Palmitoylation
Palmitate to Cys via palmitoyl-CoA by palmitoyltransferase
Can be C, internal or N
Reversed by hydrolysis protein acylthioesterases
Membrane targeting
Ras family
TM proteins e,g, b adrenergic at internal
Ankyrin
Membrane fusion SNAP-25
Hypothesis for palmitoylation
FarnesylTed protein samples membranes
The PAT is present in target membrane which then fixes it
Vehicle mediated transport moves it to other membranes where there are protein acylthioesterases
Palmitoylation only in Golgi
Makes sure that it is only present in PM
Example of palmitoylation
Targetin regulation and kinetic trapping of eNOS
Palmitoylation for eNOS in caveolae
Down regulate by endocytosis
Phosphorylation of myristoyl removes from PM
De palmitoylation removes from caveolae
Disease linked to PAT
ZdHHc9 is a PAT, found in complex with GCP16 in the Golgi
Modifies NRAS and HRAS
Upstream of MAPK which regulate transcription for division
By changing 181C and 184C HRAS remains in Golgi
Both lead to non specifically localised on all endomembranes
Development of nervous system needs synapse maturation
NMDA (Glu) -> HRAS and NRAS -> AMPA recruitment for long term signalling (exocytosis of AMPA receptors)
Also migration of AMPA on dendrite surface
Experiments for determining greasy feet
4
PLC or PLD cleave at phosphate
Nitrous acid cleaves between glucosamine and inositol ring releasing protein-glycol and phosphatidyinositol
C terminal peptide hydrophobic in tryptic digest, will be in detergent or hydrophobic phase
Acid hydrolysis reveals ethanol amine
Biosynthesis of GPI anchors
Preformed anchor in ER membrane
N of ethanol amine is joined to sugar
C-terminal TM of protein is cleaved and added to anchor
Two peaks on a hydrophobicity plot- one for signal sequence and one for c-terminal TM. Type 1 protein
GPI anchor disease
Paroxysmal nocturnal haemoglobinuria Mutation in single haemopoetic stem cell Haemolytic anaemia Improper expression of CD55 and CD59 Mutation in PIG-A, X linked, which transfers GlcNAc to PI Higher freq due to X inactivation
Types of singer proteins
1- N is intracellular
2
Signal anchor and c terminal anchor
Poly topic
The 3 classes of peripheral proteins
Steps of association
I and H penetrate the hydrocarbon core
S is only interfacial region
Initial membrane adsorption due to electrostatic interactions and diffusion
Could be triggered by Ca switch
Membrane penetration or binding to lipid
For many phosphoinositide binding proteins, both occurs
Classes of lipids
Tricyglycerols
Phospholipids- glyceroPL and sphingoPL
Glycolipids- Sphingolipids and galactolipids
Ester linkage or amide linkage
Phospholipids- phosphodiester
Glycolipids- glycosidic linkage
Glycerophospholipids
Derivatives of phosphatidic acid Polar alcohol can be: P-4,5BP (negative) Phosphoserine (neutral) P-choline or P-ethanol amine (positive)
Phospholipase a
A1- first fatty acid carbon
A2- second fatty acid carbon.
C- cleaves between the oxygen and the phosphate
D- cleaves between phosphate and head group
Sphingolipids
Amino groups at C2 has amide linkage to saturated fa
Ceramide is parent compound
Phosphocholine -> sphingomyelin
Gangliosides have very complex oligosaccharides
Carrying antigens as well as barriers
Melting points of lipids by DSC
Input of constant energy raises temperature
At 30 degrees more temp is needed to induce a change
Lipids absorb energy as they undergo structural changes
Large input is the energy needed for lipid tails to melt
Bending of membrane in the ripple phase
Asymmetry in the membrane
Unsaturated more inward facing
In apoptosis, phosphatidyserine goes to extra cellular face
Sugars face outwards and create the glycolax layer
Outer layer- phosphatidycholine, sphingomyelin
Inner- Pserine, all PI lipids
X-Ray diffraction of the bilayers
High densities of head groups
Can determine where the cholesterol is by comparing to cholesterol lacking membrane
In myelin produced by oligodendrocytes, cholesterol is not symmetrical
The raft hypothesis
Different ratios of components makes Ld, Lo and So regions
Phase separation of phosphatidycholine and sphingomyelin upon cholesterol addition
Calveolin
Peripheral
Monomers has 3x acyl groups by palmitoylation and myristoylation
Polymerises when contacts cholesterol
Central hydrophobic domain
Raft function in T cell activation
CD45 dephosphorylates the tyrosine kinase LCK
Can also act on LCK substrates such as ITAMs
Ligation of TCRs pulls rafts together, by excluding CD45 this activates LCK
Allows signalling
Detergent resistant membranes
GPI anchors became insoluble
Associated via acyl chains with phospholipid acyl chains such as SM and PI.
Cholesterol also stabilises
Tightly packed bilayer
Low density
Cyclodextrans which remove cholesterol disrupts rafts
But detergent can cause artifacts
Proteins to change lipid bilayers
Flippases - change aminophospholipids from outer to inner
Need ATP, p atpases
Floppases- from inner to outer need ATP , ABC transporters
Scramblases- no ATP, activated by calcium. During cell death Ca increases, so phosphatidylserine exposed on extra cellular
Synthesis of dolichol phosphate
Addition of GlacNAc by UDP Mannose addition Translocation (flipase) Transfer to Asn residue Protein in ER lumen
Lipid linked diseases
Tay-Sachs- underdeveloped, paralysis, blind. Abnormal ganglioside deposits in the lysosomes
Affects the enzyme hexoseminidase A which removes GalNac
Niemann-pick- defect in sphingomyelinase. Prevents the breakdown of sphingomyelin into Ceramide and phosphcholine
Lipids as signalling molecules
PIP2 -> DAG -> CALDAG-GEFI -> Rap1 -> RIAM
PIP3 is a marker for recycling endosomes in epithelial cells
PIP2 ->PIP3 actives PKB which inhibits GSK
This allows glycogen synthase to be activated
Dynamin PH domain binds to PIP2 whereas a GED domain is involved in the oligomers satin and regulation of GTPase activity
Tetraspanins
Large family with 4 TM segments
10^4 per cell except from red cells
Aggregate with other proteins in membrane
CD151 is a glycoprotein which controls neurite outgrowth.
Associates with a3b1 Integrins
X linked mental retardation
Huntingdons, fragile X and myotonic dystrophy
The CO2 metabolon
AQP1 RhAG CD47 Glycophorin B 4.2 AE1 Ankyrin Carbonic anhydrase II
Decrease in pH causes oxygen release
Hereditary spherocytosis
1/5000 Small rounder and more fragile Trapped in the spleen High levels of production in bone marrow Aplastic crisis Increased numbers of reticulocytes Often treated by removing spleen to prevent breakdown Young kids at risk of sepsis with pneumococcal bacteria
Dominant: b Spectrin W202R, ank V4631, ae1 P327R,
Also rh null and 4.2 null
Recessive- heterozygosity
Hereditary elliptocytosis
Pyropoikilocytosis
Causes elliptical shaped cells
Either 4.1 null or inability for Spectrin tetrameric
Severity upon dimer:tetramer
B Spectrin L2025R, a Spectrin K48R V31A, protein 4.1 M1R
Sensitivity to to heat
Does spectrin bind to the membrane?
Radioactive labelled
Bind inside out vesicle
Binds to Ankyrin (palmitoylated)
Ankyrin binds AE1
Regulation of spectrin binding
Spectrin and actin coextract
4.1 and adducin also present
Adducin binds to + end
The AE1 complex
Attached through Ankyrin to Spectrin Tetrameric Protein 4.2 GPA, rh and rhag bind to AE1 CD47 and LW associate
Protein 4.1 complex
Membrane skeletal junctions Spectrin, f actin, 4.1 Actin binding proteins Ternary interaction with p55 and GPC Rh, kell and XK Dimer ae1
Sickle cell and haemoglobin
Deoxy HbS forms contacts between molecules and polymerises
Instead of a charged residue, can dock into a hydrophobic pocket (val6)
When water levels, there is more aggregation
Sickle cells and oxidation
Haeme and free iron leak leading to oxidation of GSH
This means that GSH cannot be oxidised to GSSG to protect
Oxidation activates the psickle channel
This allows calcium entry
KCl is lost via the gardos channel
Prevents colloidoosmotic lysis
In normal cells, de oxygenation deactivates the gardos channel
Senicapoc inhibits the Gardos channel and stops dehydration
C284 and C373 in actin
Disulphide bond between C284 and C373 Because of glutathionation of Cys residues Cytoskeleton locks Even when oxygenated still appear sickle Prenyl-actin used as a probe
N-acetyl cysteine
Potential to reduce oxidative stress
It is the rate limiting substance for production of GSH production
It enters cell and is converted to cysteine
Increasing GSH prevents oxidative damage
Beta Spectrin mutations in c.elegans
SR lost
Muscles tear
In heart cells, the SR is lined with Spectrin. If it can’t resist mechanical stress this leads to heart malfunction
Ankyrin B knock out mice
Loss of function mutation E1425G
Disrupts organisation of the NA pump, NA/Ca exchanger which reduces the targeting of these proteins to the tranverse tubules
This causes altering of Ca signalling
What is the effect on Ankyrin B loss in cardiomyocytes?
Gradient cannot be created by NA/k ATPase
Gradient is coupled to extrusion of Ca by NCX
Ankyrin stops reinternalisstion
Cardiomyocytes cannot reset after calcium release
Irregular heartbeat and sudden death
Spectrin and evolution
Binding partners integrated cells into tissues
Two rounds of whole gene duplication
4 beta spectrin said, but one alpha
In mammals alpha was duplicated and specialised for red cells (a1)
Isoprenylation and heart disease
Statins increase lifespan
Decreases specific protein prenylstion
Explains the non cholesterol related affects of statins
2012 clinical trial to inhibit prenylstion to treat Hutchinson Gilford progeria syndrome which causes accelerated atherosclerosis
Lipid linked diseases 2
B-galactosidase: generalised gangliosidosis
Hexosaminidase A: Tay Sachs, GlcNAc removal
Gauchers: glucocerebrosidase, removes Glc from Glc-Ceramide
Niemann pick: sphingomyelinase
Fabrys: a-galactosidase A
Sandhoffs: hexosaminidase A and B
