Heterologous expression of plant membrane proteins in Xenopus oocytes

• Introduction
• Injection of oocytes
• Assaying the activity of a foreign protein
• Two-electrode voltage clamping
• References

The best way to characterise the function of a transport protein is to study the activity of the protein in a membrane. Plant membrane transporters can be expressed in immature eggs or oocytes from the South African clawed frog, Xenopus laevis.

This figure shows a diagrammatic summary of the steps involved in obtaining expression in an oocyte. The most commonly used route for expression is to prepare mRNA from the cDNA and then to inject this into the cytoplasm of the cell. Direct injections of DNA into the nucleus are technically more difficult because the nucleus must be located and can be damaged by the injection.

The foreign plant protein can be synthesised, glycosylated, phosphorylated and targeted to the oocyte plasma membrane. The final result is an oocyte expressing moderate amounts of a foreign transporter protein, and this cell can then be used to characterise a single gene product in isolation from other interacting proteins.

Injection of oocytes


Healthy (A) and damaged (B) oocytes. Note the well-defined interface between the animal and vegetative poles in A which is not present in B. Healthy oocytes are essential for good expression of foreign DNA.

As shown in the previous section, there are two routes for introducing the foreign genetic information into the oocyte and both require injection of the cell. In an earlier review we have discussed some ways of checking why expression may have failed, but it is very important to inject good quality RNA which is not degraded and this can be easily checked on a gel.

The chief problem for cytoplasmic injections is the instability of the mRNA transcripts, this can be improved by adding a poly(A) tail. We have found improvements in the expression of a plant sucrose carrier expressed in oocytes when a 75 poly(A) tail was added to the expression vector.

Most RNA molecules have a 5'7-methyl guanosine residue, the cap structure, which functions in the protein synthesis initiation process, protects the mRNA from degradation and is essential for oocyte expression. The relative importance of both capping and the poly(A) tail of the mRNA for expression in oocytes changes during oocyte development, but the two processes work synergistically stimulating translation as the oocyte matures. This is one reason for checking that the oocytes chosen for injection are at the correct developmental stage to optimise expression, other reasons will be described later.

A different approach is to directly inject the nucleus with double-stranded cDNA. The biosynthetic machinery of the oocyte nucleus then does the transcription, capping, polyadenylation and exporting to the cytoplasm of the processed mRNAs. However, the cDNA must be cloned into a suitable expression vector before nuclear injections can be performed. This involves inserting the cDNAs, in the correct orientation, into a vector which contains a eukaryotic promoter, examples are viral promoters but even plant regulatory elements have been used to drive expression in oocytes.
microinjection of mRNA into the vegetative pole of an oocyte
Assaying the activity of a foreign protein

The activity of a foreign membrane protein expressed in oocytes can be studied by using conventional techniques such as uptake of a radiolabelled substrate or depletion from the external solution.

If the transport process included the net movement of current across the membrane then electrophysiological techniques provide powerful tools for the characterisation of plant transport proteins.

The large cell size (1 mm diameter) of an oocyte favours using the two-electrode voltage clamp technique (see next section) to assay the protein's activity. The application of this technique to oocytes permits the transporter-mediated currents to be assayed as a function of membrane potential. This offers an extra dimension to analysis of the kinetic behaviour of the protein because for transporters which are electrophoretic, the membrane potential is a component of the driving force and is normally uncontrolled in transport assays. This characterisation of the carrier can include a complete kinetic analysis for each of the transported species.

Two-electrode voltage clamping

For the electrophysiological characterisation of a membrane protein in single cells, including Xenopus oocytes, the two-electrode voltage clamp technique can be used. The technique can be used in other cell types, for example root hairs but then it may be necessary to correct for loss of current into adjoining cells.

The technique requires the insertion of two electrodes (or a single double-barrelled electrode) into a cell, each of which is then used to measure voltage and current. One electrode (or barrel) reports the membrane potential while the other is used to pass current to maintain the voltage at a predetermined value. Current-voltage (I-V) relationships are obtained with a pulsed protocol generated by a computer linked to an A/D interface. The cell membrane potential is clamped at a particular voltage, the holding potential, from which the membrane is pulsed in a series of steps to a range of predetermined voltages. Usually the membrane potential is returned to the holding voltage between each of these steps.

Typically, a Xenopus oocyte is voltage clamped over the range +20 to -150 mV, and to maintain the membrane voltage during each of these pulses it is necessary to pass a recorded amount of current through the second electrode.

For an oocyte expressing a foreign membrane carrier protein, the transported substrate is then added to the external bathing solution and the oocyte's membrane potential is again voltage clamped through the range of values and the required current is measured. The oocyte is usually treated with the transported substrate for less than a minute to minimise the accumulation within the cell. This is done to avoid possible negative feedback on the activity of the transporter caused by accumulation of the substrate. Substrate-dependent currents are obtained by subtracting the currents measured before from those obtained after the addition of the transported substrate.

The substrate-elicited currents are then plotted against each of the predetermined membrane voltages and an I-V profile is obtained. For a cotransporter expressed in an oocyte at any particular voltage, the steady-state substrate-dependent currents are measured as a function of external ligand concentration (either substrate or driver ion, usually H+ for a plant carrier). These currents can be fitted to Michaelis-Menten kinetics and Km and imax values determined and based on these measurements a kinetic model for the cotransporter can be built.

This method enables many different possible substrates for a transporter to be tested quickly and cheaply without the need for radiolabelled substrates.

References

For more details of this methodology see the following publications:

Miller AJ, Smith SJ & Theodoulou FL (1994) The heterologous expression of H+-coupled transporters in Xenopus oocytes. In: Membrane Transport in Plants and Fungi: Molecular Mechanisms and Control. Blatt M.R., Leigh R.A. & Sanders D. (eds.) The Company of Biologists Ltd. pp 167-178.

Theodoulou FL & Miller AJ (1995) Xenopus oocytes as a heterologous expression system. In: Plant Gene Transfer and Expression Protocols. Jones H. (ed.) Humana Press pp 317-340.

Theodoulou FL & Miller AJ (1995) Xenopus oocytes as a heterologous expression system for plant proteins. Molecular Biotechnology 3: pp 101-115.

Miller AJ & Zhou J-J (2000) Xenopus oocytes as an expression system for plant membrane proteins. Biochim. Biophys. Acta - Biomemb. 1465: pp 343-358.