SUBCELLULAR FRACTIONATION

Reading for the interested student:Mathias Dreger (2003). Proteome analysis at the levelof subcellular structures. Eur. J. Biochem. 270:589-599.

Lukas A. Huber, Kristian Pfaller and Ilja Vietor(2003). Organelle proteomics. Implications for subcellular fractionation in proteomics. CirculationRes. 92:962-968.

Both available from PubMed.

SUBCELLULAR FRACTIONATIONSubcellular fractionation techniques were developed in the1950-ies, as a result of the development of ultracentrifuges. Although fractionation of subcellular structures in some cases may be carried out by other procedures, the ultracentrifuge is involved in an overwhelming majority.

Subcellular fractionation techniques were instrumental to thediscovery of several organelles, particularly the lysosomes and the peroxisomes. These, and related discoveries resulted in Nobel prizes to: Palade, Porter and De Duve in 1974.

The ability to correlate the fact that certain enzymaticactivities sedimented with similar kinetics with observablestructures in the electron microscope formed the basis for thediscovery of new organelles.

George Palade Albert Claude Keith Porter Christian de Duve1974 1974 xxxxx 1974

The founders of modern cell biology

An aim within biochemical research is not only to unravel themechanisms of biochemical reactions, but to find out in whatcellular structures these activities take place, and how differentbiochemical processes are co-ordinated in the different regions of the cell.

The cellular fluid is called the cytoplasm, enclosed by theplasma membrane. Here you may list the names of organellesyou have heard of:

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At the end of the 1980-ies and 1990-ies, subcellularfractionation became less fashionable, because it wasproposed that biological questions could be solvedwithout destroying the cell, or to limit the desrtuctionto permeabilisation of the cell membrane.

However, with the emerging fields of organelle-proteomics, controlled subcellular fractionationbecomes increasingly important.

The initial phase of in vitro assays may also be classified as subcellular fractionation techniques.

Animal cell

Plant cell

Further fractionation includesseparation of the inner and outermembrane of mitochondria.

THE AIMS OF SUBCELLULAR FRACTIONATION

*Preparation of organelle fractions-Localisation of enzymes and other proteins -Distribution of endocytosed ligands -reconstitution of cellular processes in vitro-starting point for proteomics

The plasma membrane is a barrier to introduction of most water solublesubstances to the cell interior. Access to organelles may be achieved by homogenisation of the cells, or otherpermeabilisation techniques.

+ the nucleus

THE STEPS IN SUBCELLULAR FRACTIONATION EXPERIMENTS

1. ISOLATION OF CELLS OR TISSUEInitially subcellular fractionation studies were only carried out withtissue, preferentially liver, but also kidney. One clearly has to remember that tissues consist of several different kinds of cells. Theliver is built of hepatocytes, endothelial cells, Kupffer cells, and stellate cells. To be sure about the origin of the organelles, one wouldhave to separate the cells first.

2. HOMOGENISATION

3. SEPARATION OF ORGANELLES INTO FRACTIONS

4. BIOCHEMICAL ANALYSIS

THE STEPS IN SUBCELLULAR FRACTIONATION EXPERIMENTS

1. ISOLATION OF CELLS OR TISSUE

2. HOMOGENISATION

Homogenisation is the most critical step in everyprocedure. The aim is to open most of the cells, butkeeping the organelles as intact as possible. The”best compromise” will be the final conditions.

3. SEPARATION OF ORGANELLES INTO FRACTIONS

4. BIOCHEMICAL ANALYSIS

THE STEPS IN SUBCELLULAR FRACTIONATION EXPERIMENTS

1. ISOLATION OF CELLS OR TISSUE

2. HOMOGENISATION

3. SEPARATION OF ORGANELLES INTO FRACTIONS

Separation of organelles into fractions is usuallycarried out by centrifugation, although othertechniques have been used (2-phase extraction, electrophoresis, immunoisolation for instance).

4. BIOCHEMICAL ANALYSIS

THE STEPS IN SUBCELLULAR FRACTIONATION EXPERIMENTS

1. ISOLATION OF CELLS OR TISSUE

2. HOMOGENISATION

3. SEPARATION OF ORGANELLES INTO FRACTIONS

4. BIOCHEMICAL ANALYSIS

Analysis of marker enzymes specific for certainorganelles, or markers introduced to organelles, for instance by endocytosis. This is to characterise theresults of the applied procedure.

Homogenisation: The human touch

Cellular dimensions:Animal and plant cells are typically 5 to 100 µm in diameter, while many bacteria are only 1 to 2 µm long. Mycoplasma, sometimes classified as bacteria are 300 nm in diameter, while a single bacterial ribosome is about 20 nm in its longest dimension.

The general rule is: the larger the cell, the easier to open – less force is needed.

The homogenisation is usually carried out in an isotonic buffer withsucrose and neutral pH. The isotonisity avoids osmotic effects to influence the integrity of the organelles.

Hypotonic medium is used in some cases to aid the rupture of the cellmembrane, because it induces swelling of the cells that reduces theforce needed.

Differential centrifugation

Particles of different sizesand shapes sediment withdifferent speed in a centrifugation field.

The centrifugal force:

Φ= mw2x

m:mass of particle

x:distance from rotational centre

w:angular speed

N

M

L

P

S

E

100

% protein0 50 100

N

M

L

P

S

The L fraction is themost heterogeneous and is most frequently thesubject of furtherseparation.

The P fraction alsocontains severaldifferent kinds ofmembrane.

Density gradient centrifugation

Sample: Fractions after differentialcentrifugation of tissue or a post-nuclear supernatant produced from a

cell line.

The organelles move in thegradient medium uponcentrifugation until they reachtheir isopycnic (their own) density. The density is influenced by the protein to

lipid ratio.

What kind of gradient media?

•Sucrose. Membrane permeable substance. Will result in thesame density of the solution within the organelle as outside in the gradient tube.

•Nycodenz / Optiprep. Often used in iso-osmotic gradients where the heavy portion of the gradient is composed ofNycodenz/Optiprep. And the light portion of sucrose.

•Percoll is a high-molecular weight medium based on silicaparticles. The gradient medium does not penetrate theorganelles, which thus do not reach the density. This is a self-generating gradient. Because of the particle size, the gradient generates during the centrifugal run and must not be made in advance as for the other gradients.

Linear gradients can be made withdifferent kinds ofgradient mixers.

The sample maybe applied at thetop of the gradient as shown, but alsoat the bottom aftermixing with heavysolution.

PERCOLL

A non-continousstep-gradient is usually made by laying layers ofsucrose withdecreasing density ontop of each other.

This can be used to concentrateorganelles withdensities between thedensities of thesucrose layers.

ROTORSThere are rotors of different types and sizes, depending on your sample volume and the application.

Fixed-angle rotors: Reorientation of gradients

The gradient reorients within thecentrifugation tube when the rotor starts to rotate.

SWING – OUT ROTORS

No reorientation of the gradients, longer distance travelled

Isopycnic centrifugation versus rate zonal

Isopycnic centrifugation is carried out until all organelles reach their bouyant density.

Rate zonal centrifugation takes advantage of thefact that some organelles reach their densitybefore others during centrifugation. This is thesame principle as that for differentialcentrifugation.

Some sedimentation coefficientsin Svedberg units

The sedimentation coefficient (s) says something about theability of a particle to sediment in a centrifugation field1S (Svedberg) tilsvarer 10-13sekunder.

Serum albumin: 4,6 S

The viscosity of the gradient medium, the temperature and the rotor type influences the sedimentation coefficient.

After centrifugation, the gradients are separated into fractions, either with an appropriate apparatus or with a pipette. Thefractions are subject to biochemical and other analysis.

The density profile of the gradient may be monitored by measurement of the refractive index in the gradient fractions

Several organelle fractions may be further fractionatedMitochondrial sub-fractions:

*Mitoplasts: Inner mitochondrial membranes, ”right side out” or ”inside out” by sonication or digitonin treatment.

*Outer membrane fractions, identified by monamine oxidase.

Peroxisomal sub-fractions: Peroxisomes do only have one limitingmembrane, but peroxisomes from some tissues do contain a core or matrix rich in certain enzymes, for instance urate oxidase.

Peroxisomes, like other organelles may be stripped of peripherallyassociated membrane proteins by bicarbonate wash at pH 11 (seepicture). Thus only trans-membrane proteins remain in the membranepellet fraction.

Peroxisomes (including ghost) and peroxisomal membranes treated withsodium carbonate.

How well do your subcellular fractionation conditions allow the recoveryof processes involving several proteins embedded in a undamagedmembrane?

Recovery of activity is a major concern in subcellular fractionation.

PITFALLS:

1. An organ consists of several cell types. When the whole tissue is homogenized, a certain activity may origin from different celltypes, without any possibility of controlling this. Alternatively, thedifferent cell types must be separated before homogenisation.

2. It is possible to overlook the localisation of an activity to a certainorganelle, because there is a preoccupied wiev of where theactivity is localised.

3. In studies of a sequence of reactions that might take place in a certain organelle, it is possible to overlook that on or more of thesteps might take place in a different organelle.

Remember: You do not isolate an organelle, you isolate a fraction thatno matter how pure it is will contain contaminating components.

4. An activity seems to be localised to the cytoplasm, because an organelle has been destroyed during homogenisation => leakage.

ER ER

Peroksisomer

ER

Cytoplasma

ER/mitokondrier

The conversion of cholesterolto bile acids depends onenzymes that are localised to different compartments of thehepatocyte.

SUBCELLULAR/ORGANELLE PROTEOMICS

*Subcellular fractionation and proteomics – the ideal partnership for analysis of intracellular organelles. The main limitations are theimpurity of subcellular fractions and the handling of massive amountsof data.

TOP

ActivityAdressing theproblem ofcontamination

Digesting the proteins of the organelle for further analysis