Journal of Experimental Botany, Vol. 50, No. 341, pp. 1715–1726, December 1999
REVIEW ARTICLE
Proteoliposomes and plant transport proteins
Guy Hanke, Caroline Bowsher, Malcolm N Jones, Ian Tetlow and Michael Emes1
School of Biological Sciences, 3.614 Stopford Building, University of Manchester, Manchester M13 9PT, UK
Received 6 July, 1999; Accepted 13 August 1999
Abstract
Artificial membrane systems are being used increasingly to study the function and role of plant membrane
proteins, particularly solute transporters which catalyse counter-exchange of metabolites. Performing
such studies requires (i) solubilization of the protein
with a suitable surfactant, (ii) functional reconstitution
of the protein in a well characterized liposome system.
Much of the technology has been derived from studies
on non-plant systems and there are many pitfalls of
which to be wary before applying it to a new protein.
This short review outlines the key parameters which
should be considered when attempting the study of
plant membrane proteins in artificial lipid bilayers,
including types of surfactants, lipid composition
of vesicles, membrane permeability, and protein
orientation.
Key words: Membrane protein reconstitution, liposomes,
surfactants, membrane solubilization.
Reconstitution of plant transport proteins
Model membrane systems
Phospholipid bilayers divide plant cells into cytosol,
plastids, mitochondria, endoplasmic reticulum, and other
subcellular compartments. Transport of solutes across
these membranes therefore dictates the relationship
between metabolism in different compartments, and in
some cases (for example, starch synthesis in potato tubers,
Tjaden et al., 1998) is fundamental in controlling metabolism. Considerable effort has therefore been invested in
the study of proteins that facilitate transport, and in
particular in their functional reconstitution. Such techniques are often adapted or adopted from other systems
(usually animal or prokaryotic). The purpose of this
review is not to provide a comprehensive discussion of
these techniques, which has been extensively reviewed
elsewhere (Jones and Chapman, 1995a, b), but more to
discuss the rationale of such approaches, including considerations and controls. It is hoped that the references
provided will supply the reader with any more detailed
information that is required.
Model bilayer systems are often used for studying
membrane transport as they negate many of the problems associated with the use of membrane vesicles
( Klingenberg, 1985; Hochstadt et al., 1975) and some
types of intact plant organelles ( Tetlow et al., 1996). The
model system approach involves removal of membrane
proteins, or specific protein(s), from the cell or purified
organelle, followed by functional reconstitution into a
closed bilayer system produced specifically for that purpose. Transport measurements can then be made. The
advantage of such a system is that conditions on either
side of the membrane can be manipulated in order to
characterize the function of a single transporter or transport complex in isolation from other cellular or membrane
processes. The disadvantage being that the activity may
not accurately reflect transport in vivo (Racker, 1979).
Two types of reconstitution system are routinely used,
planar lipid bilayers and liposomes, both represented in
Fig. 1. The planar lipid bilayer system consists of a bilayer
of phospholipid supported by a single pore of approximately 1 mm diameter in a hydrophobic ( Teflon) partition
separating two aqueous chambers of between 1 and 10 ml
volume (Miller, 1986). The liposome system consists of
spherical lipid bilayers separating aqueous compartments
(Szorka and Papahadjopoulos, 1980). Unilamellar liposomes (as illustrated) have a single bilayer membrane
and multilamellar liposomes have numerous concentric
bilayers. Both systems allow accurate measurement of
solute transport from one aqueous compartment to
another by reconstituted membrane proteins.
The type of transport being catalysed determines the
choice of system (as discussed by Miller, 1986). Surface
area to volume ratio varies by 12 orders of magnitude
1 To whom correspondence should be addressed. Fax: +44 161 275 3938. E-mail: [email protected]
© Oxford University Press 1999
1716 Hanke et al.
(A)
(B)
Fig. 1. Diagrams of model membrane systems. (A) Planar lipid bilayer.
(B) Cross-section of a proteoliposome.
between the two systems, being approximately 10−5 cm−1
for planar lipid bilayers and 107 cm−1 for liposomes.
Transport which rapidly achieves equilibrium, for
example, that catalysed by ion channels, is more accurately measured over a small surface area between two
large compartments in a system such as a planar lipid
bilayer. Planar bilayer lipid membranes are particularly
appropriate for the study of single ion channels, where
the existence of a channel can be directly monitored
electrically and the opening and closing of the channel
can be followed (Coronado, 1986). Planar lipid bilayers
have the advantage of well-defined geometry, and transport between the two aqueous compartments on either
side of the membrane is simple to follow. The major
disadvantage, however, is that it is difficult to control the
extent of penetration of the protein into the bilayer and
even more difficult to measure the amount that has
penetrated. Thus, in contrast to proteoliposomes that can
be separated by gel filtration and analysed in terms of
the lipid–protein ratio, planar lipid bilayers cannot be as
simply characterized. Planar lipid biliayers are also inherently unstable and very sensitive to the presence of free
surfactant.
Proteoliposomes lend themselves to the study of relatively slower carrier-mediated transport, which is better
measured over a large surface area into a small volume.
For instance, an active transport process, reconstituted in
liposomes, that reached 50% equilibrium in 2 min would
only achieve this state after 106 years in a planar lipid
bilayer system! (Miller, 1986). This review focuses on
methods and considerations for the reconstitution of
plant transport proteins in the liposome system.
Transport mediated by proteins reconstituted into liposomes (giving proteoliposomes) is most commonly measured as movement of radiolabelled substrate into vesicles.
A population of proteoliposomes is incubated with a
known concentration of radiolabelled substrate and after
a time period the vesicles are removed from the surrounding medium and the amount of substrate inside the
proteoliposomes may be quantified by liquid scintillation
counting. The same principle lies behind measuring movement of substrate out of vesicles, except in this case
liposomes are preloaded with radiolabelled substrate.
After a period of incubation, vesicles are removed from
the reaction medium and the amount of radiolabel encapsulated or released into solution can then be quantified.
In the case of counter-exchange mechanisms the same
approach is used except that the liposomes are first preloaded with counter-exchangeable substrate.
The flexibility of the proteoliposome system has led to
its successful application in many cases to characterize
transport by populations of membrane proteins from
isolated plant organelles. For example Mg2+/2H+ antiport by Hevea brasiliensis lutoid tonoplast membrane
proteins (Amalou et al., 1994) and ATP/ADP transport
by pea root plastid membrane proteins (Schünemann
et al., 1993). Similar methods have also been used to
characterize transport by specific isolated plant proteins,
for example, the malate carrier of barley mesophyll vacuoles (Martinoia et al., 1991), the sucrose carrier of sugar
beet leaf plasmalemma (Li et al., 1992) and the chloroplast phosphate translocator (Flügge and Heldt, 1981).
Isolation of functional membrane proteins for
reconstitution into liposomes
The preparation of membrane proteins from plant tissues
is often a time-consuming task in itself. Large amounts
of tissue (many grammes) are often required to obtain
relatively small amounts (micrograms) of even ‘crude’
membrane proteins from a particular cell or organelle
(Amalou et al., 1994; Li et al., 1992; Tetlow et al., 1996).
It may be necessary to purify a particular membrane
fraction from other cellular compartments and lysed
organelle membranes, which often involves centrifugation
through, for example, sucrose density gradients. In general, complete purification of a membrane protein often
yields an active preparation when reconstituted (Racker,
1979), although the reconstitution of certain metabolite
transport processes in various plant tissues is possible
with crude homogenates ( Flügge and Weber, 1994). In
certain cases, where distinct cellular compartments may
transport common intermediates (eg adenylate transport
which can occur in mitochondria, various plastid types,
and the endoplasmic reticulum) purification of the transport protein, or at least the membranes from the particular cellular compartment/organelle is essential. The
Proteoliposomes and plant transport proteins
anchoring of membrane proteins and glycoproteins to the
bilayer of cell/organelle membranes depends largely either
on the hydrophobic interaction between bilayer lipids and
the transmembrane regions of the integral membrane
proteins; or on hydrophobic and/or ionic interactions in
the case of extrinsic membrane proteins. Indeed in the
case of intrinsic membrane proteins, which include transporters, ion channels and many receptors, a major proportion of the protein is in intimate contact with the lipid of
the bilayer. Thus, the successful isolation and reconstitution of such proteins depends initially on the breakdown
of some or all of these interactions in a manner that
causes as little change as possible in the conformation of
the membrane protein and hence with the retention and
preservation of function. To this end, the extraction and
initial solubilization should be as short as possible. In
some cases, inclusion of substrate in the solubilization
medium has been reported to protect the transport protein
along with the inclusion of glycerol (up to 20%, v/v) and
lipid (1–2 mg ml−1) to protect the hydrophobic regions
of the protein (Ambudkar et al., 1986; Tetlow et al.,
1996). In common with all protein purification strategies,
temperature should be kept low (2–4 °C ) and protease
inhibitors and reductants, for example, dithiothreitol, are
included at all stages of extraction, purification and
reconstitution to minimize the considerable loss of protein
and transport activity which inevitably occurs in such
experiments. All aspects of the extraction and reconstitution procedure should be carefully optimized for each
protein under study, not least the lipid environment in
which the protein is solubilized and into which it is
eventually reconstituted. Studies with ‘model’ membrane
reconstitution systems indicate that the lipid environment
is crucial to the biological activity measured ( Tefft
et al., 1986).
Solubilization
In order that membrane proteins may be reconstituted in
a model system they must be removed from their original
lipid environment. The surrounding lipid membrane is
removed from proteins using surfactants (commonly
known as detergents, although strictly detergents are a
formulation containing a surfactant as the active species)
in a process known as solubilization. Solubilization has
been discussed in general terms (Lichtenberg et al., 1983;
Jones and Chapman, 1995a; Jones, 1999) and also using
specific surfactant examples ( Kragh-Hansen et al., 1993;
Ollivon et al., 1988; de la Maza and Parra, 1994;
Paternostre et al., 1988). Membrane solubilization is often
a necessary prerequisite to the functional reconstitution
of a membrane protein. It is therefore important to be
able to assess whether or not the protein under study has
been solubilized at a given surfactant concentration. This
typically involves centrifugation of the surfactant/mem-
1717
brane-sample mixture following relatively short periods
of solubilization (solubilization times should also be
optimized for each protein under study). The centrifugation (typically 100–150 000 g for 1 h) separates solubilized and insoluble material; the former being recovered
in the supernatant and assayed for activity ( Tetlow
et al., 1996).
Surfactants, in common with phospholipids, are amphipathic. However, the hydrophobic moieties of surfactant
molecules occupy far less volume than those of phospholipids. This confers solubility in an aqueous environment
by enabling ready formation of micelles (aggregates of
amphiphiles internalizing their hydrophobic regions) by
all surfactant molecules above a critical micelle concentration (CMC ). During solubilization the surfactant is added
to biological membranes and, being amphipathic, it is
partitioned into the bilayer causing swelling and leakiness
of the membrane. With increased surfactant concentration
the membrane is lysed and the contents of the cell/
organelle are released as unsealed lipid–protein–surfactant bilayers. Above a certain surfactant concentration
(the critical mixed micelle concentration) the bilayer is
disrupted and mixed micelles are formed from any combination of lipid, surfactant and membrane proteins. It is
unlikely that solubilized membrane proteins will be totally
free of phospholipid molecules and this will depend on
the affinity constants of the surfactant and phospholipids
for the particular protein.
Surfactants
Choice of surfactant for use in reconstitution studies is
dependent on several factors. Primarily it is essential that,
in stripping the lipid surrounding membrane proteins, no
damage is caused which may disrupt functionality in the
reconstituted state. The properties of various surfactants
have been reviewed (Jones and Chapman, 1995a). As a
rule the anionic polar groups of ionic surfactants such as
sodium n-dodecylsulphate (SDS) and n-alkylsulphates
bind to cationic sites on the surface of proteins, causing
denaturing and permanent loss of function. By contrast
some anionic surfactants such as cholates and the nonionic surfactants Triton X-100 and n-octylglucoside (OG),
bind to the tertiary structure of proteins, but do not
unfold them, making these ideal candidates for use in
reconstitution studies, particularly in many of the plant
membrane protein reconstitution studies to date. This is
aptly demonstrated (Doige et al., 1993) in work on the
ATPase active P-glycoprotein from mammalian cell lines.
When the surfactants, digitonin and SDS, were used for
solubilization, activity was eliminated in the reconstituted
system, whereas when non-ionic surfactants such as Triton
X-100 and OG were used, reconstituted enzyme activity
was detected. This study also serves to demonstrate that,
depending on the protein being studied, there may be
1718 Hanke et al.
exceptions to the general rule. In this case the zwitterionic derivative of cholic acid 3-[(3-cholamidopropyl )dimethylammonio]-1-propane-sulphonate (CHAPS ) preserves more activity than any other surfactant. Table 1
describes the properties of some of the most commonly
used surfactants in reconstitution studies; where possible,
examples have been taken from functional reconstitution
studies with plant membrane proteins.
Critical micelle concentration
The critical micelle concentration (CMC ) is another
important factor in selecting a surfactant. The CMC gives
an indication of the surfactant concentration required to
incorporate all phospholipid molecules into mixed lipid–
surfactant micelles (critical mixed micelle concentration),
therefore achieving solubilization. The critical mixed
micelle concentration tends to be between the CMC
values of the surfactant and that of the phospholipids in
the membrane (Ollivon et al., 1988). The more hydrophobic a surfactant, the lower its CMC and the lower
the concentration necessary to solubilize a bilayer.
Surfactants with low CMCs include Triton X-100 and
other non-ionic polyglycol surfactants such as Lubrol PX
and Tween 80. It is hypothesized (Mimms et al., 1981)
that some membrane proteins might require detergents
with long alkyl chains (and therefore lower CMCs) to
maintain structural integrity.
In some methods surfactant must be removed or diluted
below the CMC after solubilization in order that it does
not interfere with model systems into which proteins will
be reconstituted. Surfactants with high CMCs such as
n-octyl b--glucopyranoside (OBG) or n-octyl b--thiopyranoside are more easily dialysed away from protein
fractions or diluted to concentrations where effects on
membranes are negligible. The detergent Triton X-100
has a low CMC but may be removed by rapid adsorption
onto hydrophobic Bio-Beads SM , as described (Lévy
2
et al., 1990). Therefore, depending on the method of
reconstitution and the proteins under investigation either
a high or low CMC may be preferable. The CMC of a
surfactant may also change according to temperature and
salt concentration to the extent that values taken for a
single surfactant may vary considerably between studies
( Table 1). For example, the critical mixed micelle concentration of OBG is dependent upon experimental conditions and ranges from 16.6 mM to 25 mM (Almog et al.,
1990; Jackson et al., 1982; Jones and Chapman, 1995a).
Biological effects of surfactants
Surfactants that interfere with experimental procedures
should be avoided. All Triton-based surfactants contain
a phenyl group that absorbs light at 280 nm and so may
interfere with assays that measure optical density close to
this wavelength, for example, where the aromatic sidechains of proteins/glycoproteins also absorb (Jones and
Chapman, 1995b). This problem can be avoided by using
reduced Tritons in which the phenyl group is reduced to
cyclohexyl. The CMC of reduced Triton X-100 is approximately 12% higher than that of the dehydrogenated form,
conferring an added advantage for reconstitution procedures involving surfactant dilution or dialysis ( Tiller et al.,
1984). Triton X-100 has been reported to activate several
enzymes, including alkaline pyrophosphatase ( Tetlow
et al., 1993), glucose 6-phosphatase (Behyl, 1986) and
ATPase-active P-glycoprotein (Doige et al., 1993).
Surfactants of the alkyl saccharide group contain sugar
moieties, which might interfere with some transport
experiments (Lichtenberg et al., 1983). If the solubilization procedure is time-consuming (in the region of several
days) then stability of the detergent to oxidation and
hydrolysis should also be considered, for instance n-octyl
b--thioglucopyranoside is more stable than the otherwise
similar OBG (Jones and Chapman, 1995b).
Reconstitution
Reconstitution is the opposite of solubilization, in that
the aim of the procedure is to return the protein to its
native state, inserted in a membrane. Surfactants, preventing formation of lipid bilayers, must be removed or
diluted below their CMCs. Depending on the procedure
and sensitivity of the protein to the aqueous environment,
detergent may be removed prior to or during reconstitution. Most membrane proteins have at least one
membrane-spanning domain, in which there is a high
proportion of hydrophobic amino acids. It is energetically
unfavourable for such hydrophobic sequences to be in
contact with an aqueous environment and this property
can be exploited to reconstitute solubilized proteins in a
model membrane system such as liposomes. Where membrane proteins have been incorporated into liposome
bilayers, it is known as a proteoliposome system.
The substrates for reconstitution are invariably solubilized protein–surfactant mixed micelles, and a lipid component present either in the form of vesicles or in lipid–
surfactant mixed micelles. The lipid to protein ratio is
typically in the region of 2000–10 000 (Jones et al., 1990).
At each stage of a reconstitution protocol it is important
to be able to monitor the stability of the proteoliposome
system, particularly with respect to the surfactant used.
Surfactant titration curves are often used to monitor
bilayer integrity of proteoliposomes as part of a preliminary study when designing a reconstitution protocol. The
stability of proteoliposomes can be assessed in a number
of ways; the simplest and most convenient of these are
illustrated in Fig. 2 using turbidity measurements of the
sample in a spectrophotometer (measuring absorbance of
the sample between 500–560 nm) and measurement of
Table 1. Surfactants most commonly used in the isolation and reconstitution of functional membrane proteins
Name
Surfactant type
Molecular mass
(monomer)
Triton X-100 (polyethylglycol
p-t-octylphenol, n=9–10)
Non-ionic
650
CMC
(mM )a
0.24–0.3
Reference
Chloroplast phosphate translocator
Metabolite transporters from green and non-green
plant tissues
Adenylate transporter from maize endosperm
amyloplasts
Auxin-binding protein from zucchini (Zea)
plasma-membrane
H+-ATPase from spinach chloroplasts (used as a
mixture with 30 mM n-octylglucoside)
Integral membrane proteins from human
erythrocyte plasma membrane
Cl−/HCO− exchanger from human erythrocytes
3
Flügge (1992)
Flügge and Weber (1994)
Möhlmann et al. (1997)
Triton X-114 (polyethylglycol
p-t-octylphenol, n=7–8)
Sodium cholate (3a, 12adihydroxy-5b-cholan-24-oate)
Sodium dodecyl sulphate (SDS )
Non-ionic
537
Anionic
430.6
13.0
Anionic
288.4
8.0
C E (octaoxyethylene mono-n12 8
dodecyl ether)
Nonyl glucosde (n-nonyl-b-glucopyranoside
Lubrol-PX (polyethylglycol (910) alcohol )
CHAPS (3-[(3chloroamidopropyl )dimethylammonio]-1-propanesulphonate
n-Octyl-b--glucopyranoside
(n-octylglucoside, OBG)
Non-ionic
538.8
0.056–0.087
Non-ionic
306.4
6.5
Ca2+-ATPase (CF ) from spinach chloroplasts
1
Patrie and McCarty (1984)
Non-ionic
582.0
0.02–0.1
Guanylate cyclase from sea urchin spermatozoa
Radany et al. (1985)
Zwitterionic
614.9
1.4
Acid lipase from Ricinus endosperm
Fuchs et al. (1996);
Altaf et al. (1997)
Li et al. (1992)
292.4
23.0
n-Octyl-b--thiopyranoside
Non-ionic
308.4
9.0
n-Dodecyl-b--maltoside
(dodecylmaltoside; lauryl
maltoside)
Non-ionic
510.6
0.16
MEGA-9 (nonanoyl-N-methylglucamide)
Non-ionic
335.5
19–25
Sucrose transporter from plasma membranes of
sugarbeet leaves
Hexose-phosphate/phosphate antiporter from
wheat endosperm amyloplasts
Mg2+/2H+ antiporter from lutoid tonoplasts of
Hevea brasiliensis latex
-glucose transporter from the plasma membrane
of Trypanosoma brucei
H+-ATPase, H+-PPase from Ricinus cotyledons
Glucose 6-phosphate/phosphate antiporter from
maize and cauliflower bud amyloplasts
Photosystem II from spinach chloroplasts (used
with n-octylglucoside)
High conductance solute channel from pea
chloroplasts
aThe CMCs for ionic surfactants decrease with increasing ionic strength: the values shown are those for water at 25 °C.
Hicks et al. (1993)
Pick and Racker (1979)
Lundahl et al. (1992)
Maneri and Low (1988)
Tetlow et al. (1996)
Amalou et al. (1994)
Seyfang and Duszenko (1993)
Long et al. (1997)
Kammerer et al. (1998)
Hankamer et al. (1997)
Pohlmeyer et al. (1998)
Proteoliposomes and plant transport proteins
Non-ionic
0.21
Membrane protein reconstituted
and tissue source
1719
1720 Hanke et al.
Fig. 2. Stability of liposomes in n-octyl-b-D-glucopyranoside (OBG) at
25 °C measured as changes in turbidity at 500 nm (2) and vesicle
diameter ($) using photon correlation spectroscopy. Liposomes
composed of 10 mM asolectin were mixed with increasing 2 ml additions
of surfactant (1M OBG stock solution) in a well-stirred cuvette,
incubated for 5 minutes, and measurements taken. The various
structural transitions of the liposomes from lipid bilayers ( lamellar) to
mixed micelles (micellar) are indicated by RSAT, which is the effective
molar ratio of surfactant to phospholipid at the onset of the lamellar/
micellar transitions, and RSOL, the molar ratio of surfactant to
phospholipid at complete solubilization, when the sample is optically
clear. The surfactant CMC (23 mM ) is indicated by an arrow.
Surfactant titration curves serve as useful indicators of the range of
surfactant concentrations that can be used in a reconstitution protocol
to maintain structurally intact lipid vesicles.
liposome/proteoliposome diameters by photon correlation spectroscopy. The light-scattering data show the
solubilization curve of liposome suspensions made of
asolectin arising from the addition of increasing amounts
of OBG (Fig. 2). The different structural transitions from
lipid bilayers ( lamellar) to mixed micelles are typical of
solubilization curves for liposomes with other non-ionic
surfactants commonly used for functional membrane
reconstitution, such as Triton X-100 and n-dodecyl-b-maltoside ( Kragh-Hansen et al., 1993; Ollivon et al.,
1988; Paternostre et al., 1988). Such studies show the
range of surfactant concentrations that can be used in
the reconstitution process to maintain structurally stable
proteoliposomes. Other methods have been employed to
determine liposome stability, in conjunction with turbidity
measurements, these include fluorescence anisotropy and
electron microscopy (Jackson et al., 1982; Ruiz et al.,
1988). Various methods for reconstitution are discussed
below.
Lipid composition of vesicles
In the production of proteoliposomes consideration
should be given to the lipid component of the vesicles, as
this can influence efficiency of incorporation and the
functionality of the proteins incorporated. Variation in
the chain length of the non-polar fatty acyl chains, the
polar group, and presence or absence of cholesterol have
all been shown to have an effect. Much success in the
reconstitution of plant membrane proteins has been
achieved using the crude soybean phospholipid preparation known as asolectin. Due to its blend of a number of
lipid classes, asolectin is often useful for a first attempt
at reconstitution, particularly if the lipid requirements for
a transport protein are unknown. The phosphatidylcholine (PC ) in asolectin is often partially purified by
washing the commercially available preparations of asolectin with acetone (New, 1990). The lipid composition
of vesicles used in a reconstitution may also affect the
solubilization efficiency of the surfactants employed
( Urbaneja et al., 1987); this should be taken into account
by running pilot solubilization experiments (see above).
It has been reported that functionality of the bovine
heart mitochondrial ATPase complex is dependent on the
length of the fatty acyl chain of phospholipids used in
reconstitution (Bruni et al., 1975). It has been hypothesized that activation of different proteins by lipids with
different acyl chain lengths may be due to the width of
transmembrane sequences, known to vary from protein
to protein (Jones and Chapman, 1995b). Saturation of
fatty acyl chains causes the lengthening of hydrophobic
regions, and leads to a wider membrane. Proteins with
long transmembrane domains may, therefore, be better
incorporated (and hence function with greater efficiency)
in a more saturated lipid environment and vice versa.
This hypothesis may also explain the effects of cholesterol in inhibiting the functionality of some membrane
proteins in a reconstituted system. A high cholesterol to
lipid ratio (between 151 and 151.5 in various experiments)
during reconstitution caused an inhibition of rabbit
kidney (Na++K+)-ATPase activity (Papahadjopoulos
et al., 1973). These authors hypothesize that such inhibition is consistent with the role of cholesterol as an
amphipathic sterol which is incorporated in lipid bilayers.
When cholesterol interdigitates between phospholipid
molecules it causes widening of the membrane, decreasing
hydrophobic complimentarity between the bilayer and
proteins with short transmembrane domains. Cholesterol
also causes greater rigidity in phospholipid bilayers at
temperatures above phospholipid chain melting temperatures, and at high concentrations may inhibit proteins
catalysing active transport processes that require structural fluctuations in the surrounding lipid environment.
Examples of phospholipid polar group-dependent functionality include the mitochondrial ATP/ADP transporter, the glucose transporter of the human erythrocyte
membrane and the ATPase complex from bovine heart
mitochondria. The ATP/ADP transporter is entirely inactive in proteoliposomes composed of pure phosphatidylcholine (PC ), and functionality is restored by the addition
of acidic phospholipids or cardiolipin to the liposome
components ( Klingenberg, 1985). By contrast, the
ATPase complex is activated in a negatively charged
phospholipid environment, independent of the fatty acyl
chain length (Bruni et al., 1975). Activation energy of
Proteoliposomes and plant transport proteins
the glucose transporter varies according to the polar
groups of the phospholipids comprising the surrounding
bilayer, possibly due to electrostatic effects (Jones and
Chapman, 1995b).
When measuring activity of membrane transporters in
a reconstituted system it is essential that the phospholipid
membrane be as impermeable as possible to the substrate
in question. Permeability of a membrane to a solute
depends on the phospholipid composition of the membrane and the hydrophobicity and size of the solute (New,
1990). It is known that the inclusion of phospholipids
with charged head groups in the bilayer greatly decreases
leakage, possibly due to electrostatic repulsion between
liposomes preventing aggregation and fusion ( Kinsky,
1974). When measuring leakage or transport of a charged
compound, phospholipids of the same charge should be
used to prevent association of substrate with the bilayer.
Reconstitution of membrane proteins into bilayers
has been shown to increase the permeability of the
membrane to large polar molecules in several cases
(Papahadjopoulos et al., 1973). This may be reduced by
the inclusion of low concentrations of cholesterol in the
bilayer. Cholesterol is a much smaller molecule than most
phospholipids and it is considered that it may interdigitate
at disrupted points in the membrane, thus reducing permeability. Cholesterol reduces permeability of liposomes
into which no proteins have been inserted (New, 1990;
Kinsky, 1974). For instance, it has been found that a
phospholipid5cholesterol ratio of approximately 3651
sufficient to reduce leakage of glucose from vesicles dramatically ( Tetlow et al., 1996). Cholesterol has also been
shown to catalyse spontaneous protein incorporation into
vesicles at concentrations as low as 0.1 mol% (Scotto and
Zakim, 1986), possibly by causing packing defects in the
bilayer (see later). It is also considered that unsaturated
lipids and mixtures of lipids reduce leakage of solute from
proteoliposomes by reducing mismatch between protein
transmembrane domains and neighbouring fatty acyl
chains (Jain and Zakim, 1987).
The charge and species of the phospholipid head group
and the length of fatty acyl chains that effect functionality
vary considerably from protein to protein. Such effects
can only be determined by careful experimentation with
isolated proteins. A non-specialized lipid environment,
such as asolectin is often used in a system where a varied
population of integral membrane proteins is reconstituted,
with an unknown number of proteins mediating the
transport under investigation. For example, in the reconstitution of the entire membrane protein fraction from
the endoplasmic reticulum, egg yolk PC was used (Guillén
and Hirschberg, 1995), or in the reconstitution of the
entire plastidic membrane protein fraction from cauliflower bud amyloplasts where asolectin was used
(Möhlmann et al., 1995).
1721
Reconstitution by surfactant removal
Adaptations of two basic methods are commonly used in
the reconstitution of membrane transport proteins, either
proteoliposome formation by surfactant removal or insertion into preformed vesicles. Both methods are diagrammatically represented in Fig. 3 and have been discussed
in detail (Jones et al., 1990; Jones and Chapman, 1995b).
To reconstitute membrane proteins by surfactant
removal the lipid that will form the vesicle bilayer must
first be solubilized using surfactant. A solution of mixed
micelles containing surfactant, lipid and membrane proteins is produced. Surfactant concentration is then
reduced below the CMC, facilitating formation of vesicles
that include the transmembrane domains of proteins.
Alternative procedures differ mainly in the method of
surfactant removal, which may be achieved by dialysis,
gel filtration or adsorption of the surfactant onto hydrophobic resin beads such as Bio-Beads SM (Holloway,
2
1973) or Amberlite XAD-2 (Cheetham, 1979) which can
be employed using columns or a simple batch procedure.
A comparison of the activity of the myometrial oxytocin
receptor from guinea pig uterus, reconstituted using
each of these techniques, has been made ( Klein and
Fahrenholz, 1994). Gel filtration on Sephadex G-25 columns was found to diminish activity in contrast to dialysis
Fig. 3. (A) Reconstitution by surfactant removal. (B) Reconstitution
by insertion into preformed vesicles.
1722 Hanke et al.
or adsorption onto Bio-Beads. The advantage of BioBeads over dialysis was the time taken for surfactant
removal, as dialysis took place over a 24 h period. By
contrast detergent removal with hydrophobic media such
as Bio-Beads SM can be achieved in less than 1 h,
2
depending on temperature (Lévy et al., 1990).
An alternative method of surfactant removal is one
developed earlier (Racker et al., 1979) in which mixed
micelles of lipid, surfactant and protein are rapidly
injected into a large volume of aqueous medium, causing
dilution of the surfactant below its CMC and formation
of proteoliposomes. The vesicles are collected by highspeed centrifugation and so are removed from any residual surfactant in the supernatant. This method has been
successfully used to reconstitute diverse membrane transporters including the sucrose carrier of sugar beet leaf
plasmalemma (Li et al., 1992), the Mg2+/2H+ antiporter
of the lutoid tonoplast from Rubber tree latex (Amalou
et al., 1994) and the lactose transport system from
Escherichia coli (Newman and Wilson, 1980).
Reconstitution by insertion into preformed vesicles
When inserting membrane proteins into preformed phospholipid vesicles it is important that the liposome population is of as uniform size and composition as possible
and is predominantly of a unilamellar nature. Vesicles for
use in this procedure may be formed by surfactant
removal using similar techniques to those described in
the previous section. Alternatively, a dry lipid film is
vortexed to produce multilamellar vesicles which are
sonicated under an inert gas (e.g. argon or nitrogen) to
form unilamellar vesicles. This technique has been
described in detail (Jones et al., 1990; New, 1990). It is
possible that vesicles may be formed around other vesicles,
giving a multilamellar structure. This is highly undesirable
as it leads to an underestimation of transport activities.
It has been reported that sonication (violent agitation)
reduces the size of phospholipid vesicles and therefore
the probability of further vesicles being included within
them (Bangham et al., 1974). Sonication may be performed using a probe or preferably (to avoid contamination) in a sealed container lowered into a sonication bath.
In order that proteins may be inserted in the membrane
of preformed vesicles the organization of the bilayer must
be disrupted. This can be achieved through the use of
low surfactant concentrations; disrupting amphipaths
(fusogens); or a freeze–thaw procedure.
Low concentrations (below the CMC ) of surfactant
may be insufficient to destroy the bilayer structure of
unilamellar vesicles, whilst causing sufficient disruption
to catalyse spontaneous insertion of some proteins (Jain
and Zakim, 1987). The membrane can then be stabilized
by surfactant removal using any of the methods mentioned in the previous section. The authors describe
reconstitution of several membrane proteins into preformed vesicles using this technique, including UDP
glucuronosyltransferase and cytochrome c. These
examples demonstrate an advantage in using preformed
vesicles, that proteins tend to be inserted in an asymmetric
manner, the benefits of this are discussed in the section
on protein orientation. The authors report that
cytochrome c is incorporated symmetrically when using
surfactant removal to produce proteoliposomes, but has
a unidirectional orientation when inserted into preformed
vesicles. The orientation of bacteriorhodopsin from
Halobacteria halobium, when reconstituted by surfactant
removal or by insertion into preformed vesicles, was
compared (Rigaud et al., 1988). It was found that proteins
incorporated into preformed vesicles orientated unidirectionally (80–95% facing externally depending on surfactant used) whereas proteins in vesicles formed by
surfactant removal were orientated more randomly
(60–75% facing externally depending on surfactant used).
Methods used to cause minor disruption in vesicle
membranes without using surfactant, by including fusogens in the vesicle lipid have been described (Jain
and Zakim, 1987). Fusogens are amphipaths such as
cholesterol and myristate, which interdigitate in the
bilayer, but disrupt overall structure, increasing the
ground state energy of the bilayer by causing organizational defects. The exact mechanism of insertion is
unclear. Organizational defects are thought to be localized, reducing the activation energy necessary for fusion
of vesicles (hence the name) or insertion of membrane
proteins at these points. Cholesterol and myristate have
been used as fusogens in the reconstitution of bacteriorhodopsin from Halobacteria halobium, UDPglucuronyltransferase from pig liver microsomes and cytochrome
oxidase from bovine heart mitochondria and pig heart
mitochondria (Scotto and Zakim, 1985, 1986).
A freeze–thaw–sonication method for protein reconstitution was first used in 1977 to functionally reconstitute
the -glucose transporter from human erythrocytes
( Kasahara and Hinkle, 1977). In this technique the
solubilized membrane proteins (usually devoid of surfactant) are added to a population of unilamellar vesicles,
produced by sonication, and the mixture is rapidly frozen
in liquid nitrogen. A slow thaw step on ice is followed by
brief sonication for 10–20 s. Freeze–thawing results in the
formation of larger vesicles by fusion of smaller ones.
The exact method of protein insertion is not fully understood, but it is thought that ice crystallizes on charged
phospholipids, forming planes between separate vesicles
and so rupturing membranes, and that during thawing
the proteins insert in the membranes as the vesicles fuse
(Jones and Chapman, 1995b; Pick, 1981). The second
sonication step is thought to accelerate the rate at which
packing defects (produced by the thaw procedure) are
dissipated (New, 1990). A wide range of transport pro-
Proteoliposomes and plant transport proteins
teins have been functionally reconstituted using adaptations of this method, including a glucose transporter from
bovine heart mitochondria ( Wheeler and Hauck, 1985),
a 2-oxoglutarate carrier from bovine heart mitochondria
(Indiveri et al., 1987), a malate carrier of barley mesophyll
vacuoles (Martinoia et al., 1991), adenylate transporters
from pea root plastids, spinach chloroplasts, and pea leaf
mitochondria (Schünnemann et al., 1993), and a hexose
phosphate translocator from wheat endosperm amyloplasts (Tetlow et al., 1996).
Disadvantages of this technique are that: depending on
the CMC, surfactant removal resulting in protein denaturation may be necessary prior to reconstitution;
sonication for longer than a few seconds will disrupt the
structure and function of many proteins (Jones et al.,
1990); and that charged vesicles must be used (New
1990). Advantages are that the technique is quick, reproducible and well characterized for reconstitution of membrane transporters.
Vesicle characterization
Before measuring transport with proteoliposomes several
physical properties of the vesicles must be established in
order that the system be fully characterized. The most
important criteria that should be considered are lamellarity of vesicles, protein orientation, and membrane
permeability.
Vesicle lamellarity
When measuring transport it is desirable that solutes are
moving across a single bilayer. If proteoliposomes are
multilamellar then transport activity may be masked,
as solutes traverse membranes within other vesicles.
Characterization of the proportion of multilamellar vesicles within a proteoliposome population is therefore
important. The most accurate methods rely on visualizing
a representative population of vesicles using electron
microscopy ( EM ). Freeze-fracturing EM and negative
staining EM methods have been extensively used for this
purpose (Jones et al., 1990), but are hampered by possible
artefacts and the technique of cryofixation (as described
by Schubert et al., 1991) is acknowledged to yield the
best results. Cryofixation requires the rapid freezing of
vesicles in liquid ethane and the use of an EM stage at
liquid nitrogen temperatures to maintain liposomes in an
appropriate ionic environment.
When vesicles are reduced to a minimum average
diameter by prolonged sonication (as described by Huang
and Thompson, 1974; New, 1990), they are predominantly too small to encompass other vesicles, and so are
considered unilamellar. In this instance proteoliposome
size is often taken as an indication of lamellarity, as any
increase in size from the original liposome population
1723
could indicate vesicles large enough to encompass others.
Commonly used methods for determination of vesicle size
include negative staining or freeze–fracture electron
microscopy and gel filtration (Jones et al., 1990), nuclear
magnetic resonance (NMR), and analytical centrifugation
(Szorka and Papahadjopoulos, 1980), and photon correlation spectroscopy (Payne and New, 1990).
Protein orientation
In vivo membrane proteins are orientated asymmetrically,
with all equivalent active sites on the same side of the
membrane. When incorporated into proteoliposomes
randomization may occur, with equivalent active sites
facing both the internal compartment and the external
medium. The protein to lipid ratio may affect the degree
of randomization and type of detergent used (Jones et al.,
1990) and varies from protein to protein. For instance
on
insertion
into
dimyristoylphosphatidylcholine
(DMPC ) vesicles M13 coat proteins orientated with the
N-terminus consistently on the external surface whereas
bacteriorhodopsin orientates randomly (Jones et al.,
1990). Reconstitution methods can also influence the
degree of randomization that occurs, as discussed in the
section above on reconstitution into preformed vesicles.
The importance of orientation in transporter protein
reconstitution depends upon the type of transport being
measured. If transport is unidirectional it is of paramount
importance to characterize the proportion of active sites
facing internally and externally. When a system of counter
exchange is being investigated orientation is of lesser
importance. However, many counter-exchanging transporters possess two separate active sites with different
substrate affinities, for example, the mitochondrial
ATP/ADP transporter ( Klingenberg, 1985), so a high
degree of homogeneity in orientation is desirable to ensure
accurate characterization.
Characterization of protein orientation in a vesicle
system requires some knowledge of the structure of the
proteins in question. Several methods for quantifying
orientation of proteins in a proteoliposome system have
been described (Jones et al., 1990). The most reliable of
these involve comparing the effects, on solubilized and
intact proteoliposomes, of non-membrane permeable factors. For example, radiolabelled ligands/toxins that bind
to only one exposed side of a specific membrane protein
and monoclonal antibodies to one exposed side of a
specific membrane protein, allowing immunoprecipitation
of previously radiolabelled proteoliposomes. When vesicles are solubilized all proteins will be labelled, whilst
labelling of the intact proteoliposomes will depend on the
orientation of proteins within the bilayer, as binding sites
facing the internal compartment will be inaccessible to
non-permeable factors.
1724 Hanke et al.
Vesicle permeability
In the characterization of protein-mediated transport
across liposome membranes it is important to establish
the degree of membrane permeability to the substrate
being transported. If the membrane is permeable, preloaded substrate, and substrate that has been imported,
will leak out of liposomes and measurements of transport
will be inaccurate. All methods of measuring permeability,
or leakage, follow similar principles (as outlined by Jones
et al., 1990; Kinsky, 1974). Vesicles are prepared in the
presence of the relevant substrate, any unincorporated
substrate being removed by gel filtration, ion exchange
chromatography or dialysis. Leakage of metabolites from
liposomes is then followed by spectrophotometric assay
( Kinsky, 1974) or release of radiolabelled substrate (Jones
et al., 1990).
Conclusion
In many cases the most practical and flexible approach
to characterizing transport by plant membrane proteins
is through the use of a reconstituted proteoliposome
system. Increasingly, such techniques are being used,
many of which are adaptations of methods developed for
use in mammalian or prokaryotic systems. It is hoped
that the discussion, in this article, of parameters that
should be investigated and optimized before measuring
transport by reconstituted proteins, will be of use to those
applying this valuable technique to plant membrane
proteins.
Acknowledgements
GTH gratefully acknowledges the financial support of the
BBSRC. CGB is in receipt of the Royal Society Pickering
Research Fellowship, IJT holds a Leverhulme Special Research
Fellowship; both acknowledge the financial support of the
Royal Society and the Leverhulme Trust, respectively.
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