Biomembranes,Subcellular Organization, and Membrane Trafficking

Biomembranes

Fundamental structure and function of all cell membranes depends on lipids (phospholipids, steroid derivatives)

Specific function of each membrane depends on Lipid compositionMembrane proteins, present in that specific membrane

Membrane lipids and proteins may be glycosylated

Phospholipid structureExamples of the most frequent types

FA chains can be long, short, and various degrees of branched. Branching makes the sidechains more bulky.

Phospholipid structure

Due to the amphipathic nature of phospholipids, these molecules spontaneously assemble to form closed bilayers

Bilayer structure of biomembranes

In aquous solution phospholipids spontaneously form organized polar structures driven by hydrophobic effect and van der Waals interactions between the FA tails

Electron micrograph

Membrane lipids - sterols

Eucaryotic membranes also contain sterols.

Sterols align with and stabilize the FA sidechains giving membranes ehanced thermo-stability

Cholesterols are precursors for bile acids, steroid hormones, vitamin D, and function as signaling molecules

Fluid mosaic model of biomembranes

3 classes of membrane interacting proteins:

Integral lipid-anchored peripheral

Measuring the dynamics of membranes

Flourescence recovery after photo-bleaching (FRAP) can quantify the lateral movement of proteins and lipids within the plasma membrane

Mobility (diffusion) of a given membrane component depends on:

the size of the moleculeits interactions with other moleculestemperaturelipid composition (tails, cholesterol)

Each closed compartment has two faces

Some organelles are enclosed by double bilayers (=2 membranes)

The two faces of a membrane are asymmetric in terms of lipid and protein composition

Cytosolic=cytoplasmic

Animal cell structure

Functions of the plasma membrane

Regulate transport of nutrients into the cellRegulate transport of waste out of the cellMaintain “proper” chemical conditions in the cell eg pHProvide a site for chemical reactions not likely to occur in an aqueous environmentDetect signals in the extracellular environment Interact with other cells or the extracellular matrix (in multicellular organisms)

Organelles of the eukaryotic cell

LysosomesPeroxisomesMitochondriaChloroplasts (plants)the Endoplasmic Reticulum (ER) the Golgi complexthe Nucleusthe Cytosol

Lysosomes-acidic organelles containing a battery of degradative enzymes

Responsible for degrading certain cell components material internalized from the extracellular environment

Key Featuressingle membranepH of lumen ≅ 5acid hydrolases carry out degradation reactions

Several hundred lysosomes in a single cell

Location of lysosomes and mitochondria in living cell

Tay-sachs disease is caused by a defect in a lysosomal enzyme -> ganglioside glycolipids can not be broken down -> nerve cell dysfunction -> dementia + blindness (infant death)Confocal deconvoluted image of mitochondria (green flourescence stain) and lysosomes (red flourescence stain)

Peroxisomes

Responsible for degrading fatty acids toxic compounds (alcohol ☺)

Key Featuressingle membrane contain oxidases and catalase

FA or ETOH + O2 acetyl groups + H2O2 (ΔG = negative)

2H2O2 2H2O + O2

catalase

Mitochondria

Site of ATP production via aerobic metabolism

Key Featuresouter membrane (very porous)intermembrane spaceinner membrane MatrixHas its own genome!!

The endoplasmic reticulum (ER)

Responsible formost lipid synthesismost membrane protein synthesisCa++ ion storageDetoxification

Key Featuresnetwork of interconnected closed membrane tubules and vesicles composed of smooth and rough regions

The Golgi complex

Modifies and sorts most ER products

Key Featuresseries of flattened compartments & vesiclescomposed of 3 regions: cis (entry), medial, trans (exit)each region contains different set of modifying enzymes

Secretory proteins are synthesized in the ER and pass through the Golgi on the way to the extracellular

environment

mRNA coding secreted proteins contain a signal that target it to the outer ER membrane. Ribosomes then translate the protein directly into the ER

The nucleus

Separates DNA from cytosol transcription from translation

Key Featuresouter membrane inner membranenuclear poresNucleolusCarries the main genome of cells

The cytosol

The portion of the cell enclosed by the plasma membrane but not part of any organelle Key Features

the cytoskeleton polyribosomesmetabolic enzymes

Transmembrane Transport of Ions and Small Molecules

Where are my zebra‐stripes ?

Sorry Mate, you seem to have a 

transport problem... 

Outline

Overview of membrane transportersClassificationExamples:

Uniport Transport (example: glucose)ATP-powered pumps & intracellular ionic environment (V-class, P-class, and ABC family)Nongated ion channels and the resting membrane potentialCotransport by symporters and antiporters

Multiple transport proteins cooperate

Pure phospholipid bilayers have only limited permeability

Requirements for facilitated flow (transport)

Diffusion rate of a molecule across a lipid bilayer is determined by the molecule’s:

concentration gradient across the lipid bilayerhydrophobicitySizeRelation to the electrical potential across the bilayer (only for charged molecules)

If low diffusion rate transporters required (= facilitated flow)

ΔGFlow < 0 transport will proceed with facilitated bilayer crossing

ΔGFlow > 0 transport must be coupled to energetically favorable process:cotransport with another molecule (the sum of the ΔGFlow for all transported molecules must be

negative)hydrolysis of the energy-rich terminal phosphoanhydride bond of ATP

Overview of membrane transport proteinsTransport proteins are a unique sub-class of integral membrane proteins capable of assisting flow of molecules to which the membrane is non-permissive

Primary active transportPassive transport

Secondary active transport

The 3 main classes of membrane transport proteins!

Transporters, Uniporters

Glucose uniporters; a simple glucose transport mechanism

There are 12 known mammalian glucose transporters with different glucose affinities and tissue specific expressions transporting glucose into mammalian cells

Transporters, UniportersGlucose uniporters; a simple glucose transport mechanism

ATP-powered pumps

The four classes of ATP-powered transport proteins

ATP-powered pumps

Active transport of ions against their concentration gradients and electric potential, driven by specific ATP-powered pumps

Ionic gradients and an electric potential is maintained across the plasma membrane

Represents stored energy that can be used for transmitting electrical signals (nerve cells), facilitating other transports or membrane located molecule synthesis

ATP-powered pumps

P-type: the Calcium ATPase in the Sarcoplasmatic reticulum of muscle cells

A high concentration of Ca2+ is stored in the muscle ER

Ca2+ influx in muscle cell cytosol induces muscle contraction. Muscle relaxation requires an active pump

ATP-powered pumps

P-type: the Na+/K+ ATPase maintains the intracellular Na+ and K+ concentrations in animal cells

1997 jens Skou recieved the Nobel price for discovering the function of this transporter, thereby opening for an understanding and investigation of membrane potentials

Special importance: nerve cells and cardiac electrical impulse (heart beat initiation)

ATP-powered pumps

V-type: the H+ ATPase maintains the acidity of lysosomes and vacuoles

To obtain acidity the electrogenic effect of the H+ transport must be overcome.

This is achieved by concurrent import of negative ions, or export of another positive ion.

ATP-powered pumps

ABC family: Bacterial permeases import nutrients from the environment against concentration gradients.

ATP-powered pumps

ABC family: Transport of a wide variety of substances in eucaryotic cells.

Approximately 50 different mammalian ABC transporters are known

ATP-powered pumps

ABC family: Certain ABC proteins “flip” lipid soluble substrates from one half of the bilayer to the other

Flippase model of transport by the multidrug resistance protein (MDR1)

Ion channels

Can be gated (regulated) or non-gated (always “relaxed”)

Mammalian plasma membranes contain many K+ and a few Na+ channels the membrane has capacitor function with the inside being negative.

Na+/K+ pumps maintain a conc gradient that gives a flow of K+ back through the ion channels resulting in an equilibrium net membrane potential (resting membrane potential = 70mV))

In a few cell types (nerve, muscle) charge fluctuations (impulse) occur when gated ion channels are suddenly opened

Ion channels

Ion flux through individual channels can be calculated from patch clamp tracings

Two patches of a muscle plasma membrane were clamped

The patch electrode contained NaCl

When the Na+ channel was open a flux of 10 million Na+ ions per second was measured.

The electrical depolarization “travelled” the surface of the membrane, reaching the second clamp after 10ms

Ion channels

Novel ion channels can be investigated by a combination of oocyte expression and patch clamping

Transporters, Symporters

Na+ linked symporters import amino acids and glucose into animalcells against high concentration gradients

The favorable negative free energy of Na+ transport down its

concentration gradient “compensates” the symport of glucose

Transporters, Symporters

The 2-Na+/one-glucose symporter mediates glucose uptake from the intestine

Transporters, AntiportersPutative Na+/Ca+ antiporter determines skin color!

When the gene SLC24A5 is mutated the zebrafish looses its stripes and becomes “golden”!

This gene appears to encode a Na+/Ca+ antiporter....

The human parallel is very prevalent in africans, and mutated in europeans...

The antiporter influences the accumulation of melanin in the skin

Further investigation pending!

The human genomes contains hundreds of putative transporters, the function of most of these is unknown.....