Protein orientation affects the efficiency of functional protein transplantation into the xenopus oocyte membrane.
Xenopus oocytes incorporate into their plasma membrane nicotinic acetylcholine receptors (nAChRs) after intracellular injection of lipid vesicles bearing this protein. The advantage of this approach over the classical oocyte expression system lies in the transplantation of native, fully processed proteins, although the efficiency of functional incorporation of nAChRs is low. We have now studied the incorporation into the oocyte membrane of the Torpedo chloride channel (ClC-0), a minor contaminant protein in some nAChR preparations. nAChR-injected oocytes incorporated functional ClC-0: i) in a higher number than functional nAChRs; ii) retaining their original properties; and iii) with a right-side-out orientation in the oocyte membrane. In an attempt to elucidate the reasons for the low efficiency in the functional incorporation of nAChRs into the oocyte membrane, we combined electrophysiological and [125I]alpha-bungarotoxin-binding experiments. Up to 3% of injected nAChRs were present in the oocyte plasma membrane at a given time. Thus, fusion of lipoproteosome vesicles to the oocyte plasma membrane is not the limiting factor for an efficient functional transplantation of foreign proteins. Accounting for the low rate of functional transplantation of nAChRs is their backward orientation in the oocyte membrane, since about 80% of them adopted an out-side-in orientation. Other factors, including differences in the susceptibility of the transplanted proteins to intracellular damage should also be considered.
Ivorra
I
I
Dpto. de Fisiología, Genética y Microbiología, Univ. de Alicante, E-03080 Alicante, Spain.
Fernández
A
A
Gal
B
B
Aleu
J
J
González-Ros
J M
JM
Ferragut
J A
JA
Morales
A
A
D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D018118:Chloride Channels / Q000302:isolation & purification / Q000378:metabolism*; D004553:Electric Conductivity; D004557:Electric Organ / Q000166:cytology; D005260:Female; D007457:Iodine Radioisotopes / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D009865:Oocytes / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014101:Torpedo; D014981:Xenopus
D002038:Bungarotoxins; D018118:Chloride Channels; D007457:Iodine Radioisotopes; D011978:Receptors, Nicotinic