lipids, membranes and transport outline

7/29/2019 Lipids, Membranes and Transport Outline

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Lipids, Membranes and Transport Outline

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Biological Membraneso Framework of the membrane is the phospholipid bilayero Phospholipids are amphipathic (forms bilayer) molecules

Hydrophobic (water-fearing) faces in Hydrophilic (water-loving) faces out

o Membranes contain Proteins and carbohydrates Fluid Mosaic Model

o The membrane is considered a mosaic of lipid, protein and carbohydrate moleculeso Membrane resembles a fluid- lipids and proteins move around relative to each other

Lateral movement across membrane Membrane Proteins

o Integral or Intrinsic membrane proteins Transmembrane Proteins

Region physically embedded in the hydrophobic region of bilayer Lipid anchored Protein

Noncovalent attachment of a lipid to amino acid side chain within a proteino Peripheral membrane (extrinsic proteins)

Noncovalently bound to regions of integral membrane proteins that project out frommembrane or bound to polar head groups of phospholipid

o Hydrophilic Head- loves watero Hydrophobic Tails- hates water

Hydrophobic

Amino acids


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Approximately 25% of all genes Encode membrane proteinso Membranes are extremely important- biologically and medically

Membranes are Semifluido Fluidity

Individual molecules remain in close association but have ability to move within themembrane

o Semifluid Most lipids can rotate freely around their long axes and move laterally within the

membrane leaflet

Flipflop of lipids from one leaflet to the opposite leaflet does not occur spontaneously Flippase requires ATP to transport lipids from one leaflet to another

2. Multiple

alpha helices

1.Alpha-helix

3. series of

alpha helices4. 1 layer

of bilayer

5. Lipid

anchored


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Factors affecting Fluidityo Length of fatty acyl tails

Hydrophobic tails- the shorter the tail, the more fluid it iso Presence of double bonds in the acyl tails

Big factor in fluidity Double bond creates a kink in the tail, making it more difficult to interact with

nearby tails and making the bilayer more fluido Presence of Cholesterol

Tends to stabilize membranes Depends on temperature

Cold-rigid Warm-fluid

Saturationo Saturated is when there are no C=C double bondso Unsaturated is when there are C=C double bonds

Cholesterol Stabilizes Membraneso High Temperatures

Lipid becomes fluid Cholesterol becomes more rigid

o Low Temperatures Lipid becomes more rigid Cholesterol becomes more fluid

Loves water

Hates water

Head

Tails

Head

2 C=C

bonds

1 C=C

bonds

1 C=C

bonds

0 C=C bo

Saturated


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Lateral TransportExperimento Mouse and Human cells fused togethero Added a florescent label antibody and sawo 0 C- Cold temperature

Protein unable to move laterally and remains on one side of the cello 37 C- Higher temp

Movement observed, the protein is on both sides of the cell Some Integral Membrane Proteins have restricted movement

o 10%-70% of membrane proteins may be restricted in their movement Anchored, attached to cytoskeleton or extracellular matrix

o Membrane proteins may be attached to molecules outside of the cell, so the network of proteinsthat forms the extracellular matrix

How to study Membraneso TEM (Transmission Electron Microscopy)

Thin section sample stained with heavy metal dyes, gives electron dense, binds topolar bits since metal has a charge

Train track looking membrane bilayer (polar heads dyed)

Transmembrane

Protein


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o FFEM (Freeze Fracture Electron Microscopy Specialized form of TEM, can be used to analyze the interiors of phospholipid bilayers Sample is frozen in liquid Nitrogen and fractured with a knife Leaflets separate into P face (protoplasmic) and E face (extracellular) Provide 3D detail about membrane protein form and shape Using FFEM- Branton in 1966 was able to conclude that membranes are formed from

phospholipid bilayers, with proteins intercalated within

Synthesis of Membrane Componentso In Eukaryotes, Cytosol and endomembrane system work together to synthesize most lipidso Process occurs at cytosolic leaflet of smooth ERo Fatty acid building blocks made via enzymes in cytosol or taken into cells from food


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All phospholipids are synthesized in the Smooth ER Transfer of Lipids to other Membranes

o Lipids in ER membrane can diffuse laterally to nuclear envelopeo

Transported via vesicles to Golgi, lysosomes, vacuoles, or plasma membrane (blebbing)o Lipid exchange proteins- extract lipid from one membrane for insertion in anothero 2 Phospholipids into 1

Membrane Protein Synthesiso Most transmembrane proteins are directed to ER membraneo From the ER, membrane proteins can be transferred via vesicles to other regions of the cello Trans-membrane (expand layer) proteins are translated directly into the phospholipid bilayer

of the rough ER

Stretches of 20 hydrophobic amino acids (universal for transmembrane proteins) Tends to form alpha-helical structure Length of region is 20 amino acids long

How transmembrane proteins are translated

Protein traffikingo Proteins destined for any part of the endomembrane system or secretion from the cell are

translated directly into the ER

Anything membrane bound is directly translated into the ER membrane itself- evensome non-membrane proteins also

Protein finished

being translated


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o How proteins are sorted once in the endomembrane system is reliant on the physical propertiesof lipid vesicles

- Like a lava lamp- things can bud off and go back and forth, not unidirectional

-Most things can go back and forth with vesicles, not unidirectional


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Protein Sorting to the ER

Protein Sorting to the Golgio From ER to Golgio Uses membrane proteins V-Snare and T-Snare to merge protein-containing vesicles to the correct

target

o Certain proteins are classified as V-Snare or T-Snare

Signal

recognition

protein

(SRP)

Docks with

protein in

Rough ERto translate

Translated

into ER

lumen

Transmembrane

Protein- 20 amino

acids, forms alpha

helice

Polypeptide

chain- inside

ER lumen

Proteins that

form matrix

(cage)

Pinches off

(Blebs off)

Attaches to

T-Snare

Gol i

Rough ER

Inside ER Lumen


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Protein Sorting in the Golgi (continued)o Cargo protein, targeted to the golgi, binds to a cargo receptor protein in the ER membrane. Coat

proteins on the outside aid in the formation of vesicle buds, which will contain the appropriate

cargo. In the vesicle membranes are also V-snares.proteins embedded in the membrane which

target the vesicle to the correct site. Upon approaching the target (Golgi), T-snare proteins

within the golgi membrane bind with the v-snares, causing the membranes to fuse, and the cargo

protein to be released into the golgi.

Coat Proteins cause the membrane to Bleb Off

SARE Proteins allow vesicles to join together- (Direct mechanism)o 2 Nerves

- Transmembrane Proteins


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Glycosylationo Process of covalently attaching a carbohydrate to a protein or lipid

Glycolipid- Carbohydrate to lipid (sugar stuck to fats) Glycoprotein- Carbohydrate to protein (sugar stuck to protein)

o Proteins ER to Golgio Can serve as recognition signals for other cellular proteinso Plays a role in cell surface recognitiono Protective effects

Cell coat or glycocalyx- carbohydrate rich zone on the cell surface shielding cell

Clicker Questiono The bond that attaches Carbohydrates to Lipids and Proteins in the Plasma Membrane

Covalent Bond Because were transferring to O or N

Spontaneously Happens

-Forms one larger unit

-T-Snare attaching to Golgi


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Protein Glycosylationo N-Linked

In ER & Golgi Attachment of Carbohydrate (sugars) to Nitrogen (N) atom of asparagine side chain

o O-Linked Only in Golgi Addition of sugars to Oxygen (O) atom of Serine or Threonine side chains

Both have hydroxyl groups (OH), so sugars get added on there N-Linked Glycosylation

o Carbohydrate (sugars) to N atom of asparagine side chaino In ER & Golgio Dolichols are previously made in the ERo Transfer to target Asp sequenceo Commonly found in membrane proteins


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O-Linked Glycosylationo Occurs only in Golgio Links sugar chains to the O in Ser or Thr side chainso Important for production of proteoglycans

Mucous Extracellular matrix

o Transfers sugar group onto OH of Ser and Thr Endocytosis and Exocytosis

o Transport larger molecules such as proteins and polysaccharides, and even very large particleso Usually larger things gets endocytosed or exocytosed

Ex. Load of Insulin excreted from Pancreatic cello Exocytosis

From inside cell to outside cell Material inside the cell packed into vesicles and excreted into the extracellular matrix

o Endocytosis Plasma protein invaginates or folds inward to form a vesicle that brings substances into

the cell

Receptor-mediated endocytosis Pinocytosis- Small molecules in extracellular environment are brought into the cell

Used for absorption of extracellular fluids Phagocytosis-Eat Endocytose bacteria

Engulfing solid particles by the cell membrane Defense mechanism- to remove cellular debris- immune response

Exocytosiso Inside cell to Outside the cell


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Endocytosiso Into the Cell, receptor-mediatedo Blebs off into the cello Guided by cytoskeleton (carries cargo)

Phospholipid Bilayer Barriero Serves as a barrier to hydrophilic molecules and ions due to hydrophobic interior and hydrophilic

outside

o Rate of diffusion depends on chemistry of solute and its concentrationo Gasses and a few uncharged molecules can passively diffuse acrosso Ions and large polar molecules diffuse slowly

- Things that can get thru freely- Hydrophobic

molecules. Gasses, small molecules

-This is how cells breathe Diffusion

-Diffuse thru membrane freely

Small polar molecules diffuse thru

somewhat slow, uncharged.

Large molecules, more charged andharder to diffuse, cant go thru bilayer


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Membrane Transporto Selectively permeable plasma membrane

Outside environment cant dictate inside environmento Structure ensures

Essential molecules enter Metabolic intermediates remain Waste products exist

Movement Across Membraneo Passive transportdoesnt require an input of energy- down or with gradient

Passive Transport Diffusion of a solute through a membrane without transport protein

Facilitated Diffusion Diffusion of a solute through a membrane with the aid of a transport protein

o Active transport requires energy- up or against gradient

-Simple diffusion

-No transport protein

-Down/with gradient, no

energy required

Ex.) O2, CO2

-Diffusion with Aid oftransport protein

-Down/with gradient, no

energy required

Spontaneous, favorable

G = -

-Up/Against gradient

-Requires energy

Non-spontaneous,

unfavorable

G = (+)


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Active Transporto Movement of a solute across a membrane against its gradient from a region of low to higher

concentration (Low Concentration High Concentration)

o Energetically unfavorable, non-spontaneouso Requires input of Energy to drive movemento Primary Active Transport

Uses a pump- directly uses energy to transport soluteo Secondary Active Transport

Very common Uses pre-existing gradient to drive transport of solute Relying on concentration gradient- ATP is not always used

Cells Maintain Gradientso Living cells maintain a relatively constant internal environment different than outside the cello Transmembrane gradient

Concentration of a solute is higher on one side of a membrane than the othero Ion electrochemical gradient

Both an electrical gradient and chemical gradient

Low Co

High Co

Glucose

Transmembrane Gradient

Conc. Of glucose is higher

outside than it is inside the

cell


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Tonicityo Comparison of solute concentrations across a barriero In relation to the cell

Isotonic- Equal amounts of solute inside and out of the membrane (and equal amounts ofwater)

Hypertonic- Solute concentration is higher on one side of the membrane (and waterconcentration lower)

Hypotonic- Solute concentration is lower on one side of the membrane (and waterconcentration is higher)

Ion Electrochemical

Gradient

-Electrical and Chemical

gradient

-More Na+ outside the cell

and higher Positive charge

outside the cell

-Solute concentration outside the cell is Isotonic to the

inside of the cell

-Solute Concentration outside the cell is Hypertonic to th

inside of the cell

-Solute concentration outside the cell is Hypotonic to the

inside of the cell


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Osmosiso The movement of water towards a soluteo Water diffuses through a membrane from an area with more water to an area with less watero If solutes cannot move, water movement can make the cell shrink or swell as water leaves or

enters the cell

o Osmotic Pressure- The tendency for water to move into any cello Animal cells must maintain a balance between extracellular and intracellular solute

concentrations to maintain their size and shape

o Crenation-shrinking in a hypertonic solution- (can kill cells this way) Aquaporins

o Channel proteins that move water across membraneo Discovery of Aquaporins

CHIP28 Water passively diffuses across plasma membrane Certain cell types allow water to move across the plasma membrane at a much

faster rate than would be predicted by passive diffusion

Peter Agre and colleagues first identified a protein that was abundant in RBCsand kidney cells, but not found in many other cell types

Striking difference was observed between frog oocytes that expressed CHIP28versus the control

CHIP28- renamed Aquaporins Transport Proteins

o Transmembrane proteins that provide a passageway for the movement of ions and hydrophilicmolecules across membranes

o 2 classes bases on movement type Channels- forms a pore to shuttle thru Transporters- Carrier proteins, transforms and changes shape to pass through


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Transporterso Carrierso Conformational change transports soluteo Principal pathway for the uptake of organic molecules

Sugars, amino acids, nucleotides

o Types of Transporterso Uniporter- Single molecule/ion moves in and out of the cell o Symporter/Cotransporter- 2 or more ions or molecules transported in same

direction

o Antiporter- 2 or more ions or molecules transported in opposite directions Glucose Transporters

o Some glucose transporters are uniportero Ex.) Blood brain barrier (BBB), glucose is transported across endothelial cells of small

blood vessels into astrocytes

o Uniporters that transport Glucose down its concentration gradiento Glucose transporters undergo conformational changes that result in a reorientation of

their substrate binding sites across membranes


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Channelso Form an open passageway for the direct diffusion of ions or molecules across the

membrane

o Most are gatedo Ion Channels

Hydration of Ionso Ions in aqueous solutiono Influences their flux through Transmembrane poreso Salts dissolved in water form hydrated ionso The hydrophobicity of lipid bilayers is a barrier to movement of hydrated ions across cell

membranes

Ion channels Dehydrate Ionso All ion channels dehydrate ionso Hydration shell must be removed for ions to be selectively shuttled through a channelo Allow for the rapid selective transport of ions across membraneso Dehydration of Ions costs energy, whereas hydration of ions frees energyo Almost every ion channel is gated

Electrochemical gradientso Across the cell membrane generate the Membrane Potentialo Nernst Equation- used to calculate the membrane potential as a function of ion concentrationso Cells maintain a negative resting membrane potential with the inside of the cell slightly more

negative than the outside

o Membrane potential is a prerequisite for electrical signals and for directed ion movement acrosscellular membranes


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K+ Potassium Channelso Catalyze selective and rapid ion permeationo Function as water-filled pores that catalyze the selective and rapid transport of K+ ionso Complex of 4 identical subunits, each of which contributes to the pore (tetramer)

o Selectivity filter- evolutionary conserved structureo K+ channel selectivity filter catalyzes dehydration of ions, which confers specificity and speeds

up ion permeation

o How it works:


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P5 is where the K+ hydrated ion enters and in here, it dehydrates the K+ ion and allows it to fit specifically into

P4 to P0

Gating of K+ Channelso Gating means being able to shut if off (plug it up)o Different gating mechanisms define functional classes of K+ channelso Distinct from the selectivity filtero Regulated by the membrane potential

Packets

0,P1,P2,P3,P4,P5

Configurations

1,3 or

2,4

Keeps moving K+

up one rung-

every other, so it

pushes up, itsstaggered every

other one.

-Because positive

charges repel,

repulsion moves

cells up the

channel

Pushes up by repulsion

Opens when K+ is

resent

Pinch closed at bottom

Moves paddles, inverts

Able to close around

inside of channel


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Aquaporin channelso Selective water transport occurs through Aquaporin Channelso Have to dehydrate ions to move them througho Allow rapid and selective water transport across cell membraneso Tetramers of four identical subunits, with each subunit forming a pore

Aquaporin Selectivity Filtero Has 3 major features that confer a high degree of selectivity for water

Size restriction Electrostatic repulsion Water dipole orientation

o Water molecules have to line up single file based on dipole momentso Tetomer- forms like an hourglass, thin in the middle

Action Potentialso Electrical Signalso Depend on several typs of ion channelso Enable rapid communication between cells

Nerve cells and muscle cells use theseo Na+, K+, Ca2+ currents are key elements of action potentials, but not the only oneso One way that cells communicate with each other (rapidly)o Uses electrochemical gradientso Resting Potential- The electrical potential that exists due to a difference in charge across a

membrane. The charge thats maintained so the signal can be transmitted

Resting Potential- forces K+

against its gradient

+ charge outside, - inside

Voltage gated channels-

-Triggers gated ion channels to

open, triggers others to open up

as well

Action potentials are mediated

by ion currents- depends on ion

concentrations


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Action Potentialso Membrane depolarization is mediated by the flow of Na+ ions into cells through voltage

dependent Na+ channels

o Repolarization is shaped by transport of K+ ions through several different types of K+ channelso Electrical activity of organs can be measured as the sum of action potential vectors

Na+ Gradiento Used for getting lots of stuff into the cell

If we want to get something into the cell we can use Na+ ion to couple it, to get it inside to Transmembrane Na+ gradient is essential for the function of many transporterso Plasma membrane Na+ gradient is maintained by the action of the Na+/K+-ATPaseo Used for Glucose and other ionso Na+/K+-ATPase is an ion driven pump

ATP-driven Ion Pumps Generate Ion Electrochemical Gradientso Na+/K+-ATPase

Actively transport Na+ and K+ against their gradients by using the energy from ATPhydrolysis (to ADP)

3 Na+ exported for 2 K+ imported into the cell Antiporter- different directions Electrogenic pump- export 1 net positive charge (3 ions out, 2 ions in)


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Reaction cycle is described by the Post-albers scheme, which proposes that the enzymecycles between two fundamental conformations

Model for transport by t

Na+/K+-ATPase

1. 3 Na+ ions bind

a. ATP to ADP, and th

protein now has a

phosphate

2. Conformational chang

of molecule (protein)

3. Release 3 Na+ ions

outside the cell and its

opened up to allow K+

4. 2 K+ binds into pocke

protein gets

dephosphorylated

5. Protein closes outside

cell portion, protein gets

phosphorylated with AT

6. Releases K+ ions into

the cell

Starts all over again


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Na+ gradient formed by Na+/K+-ATPase powers the transport of:o Examples

Na+/H+ and Na+/HCO3-cotransporters Regulates cytostolic and extracellular pH

Na+/Glucose transporter Intestinal cells absorb glucose

Na+/K+/Cl- cotransporter Regulates intracellular Cl- concentrations

Na+/Mg2+ exchanger Transport Mg2+ outside of the cell

Na+/Ca2+ exchanger Major transport mechanism for removal of Ca2+ from the cytosol of excitable

cells

lipids, membranes and transport outline

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