Differential targeting to the plasma membrane of the Torpedo 15-kDa proteolipid expressed in oocytes.
Xenopus laevis oocytes were injected with poly(A)+ RNAs extracted from the electric lobes of Torpedo marmorata, which contain a homogeneous population of cholinergic neurons. These primed oocytes were able to synthesize acetylcholine and to release the neurotransmitter in a calcium-dependent manner. Fractionation of oocyte membranes as well as immunofluorescence experiments showed that the 15-kDa proteolipid, a common subunit of the vacuolar H(+)-ATPase and of a presynaptic membrane protein capable of calcium-dependent acetylcholine translocation called the mediatophore, was located at the oocyte plasma membrane. In contrast, oocytes injected with separate transcripts encoding the 15-kDa proteolipid and choline acetyltransferase were unable to release acetylcholine in spite of an equivalent acetylcholine content and a higher level of 15-kDa proteolipid expression. We observed by immunofluorescence that under these conditions, the 15-kDa proteolipid was expressed in granular cytoplasmic membranes, which were then identified as being Golgi vesicles by cell fractionation. The striking difference in the distribution of the 15-kDa proteolipid expressed in oocytes primed with Torpedo electric lobe mRNA as compared with that seen in oocytes injected with the cRNA alone suggests that another protein endogenous to the electric lobe may be implicated in the localization of the 15-kDa proteolipid at the plasma membrane. Moreover, such a targeting mechanism could contribute to the capacity of electric lobe mRNA-injected oocytes to release acetylcholine.
Leroy
C
C
Département de Neurotransmission, CNRS, Gif sur Yvette, France.
Meunier
F M
FM
D000109:Acetylcholine / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000502:physiology; D002462:Cell Membrane / Q000378:metabolism; D003594:Cytoplasmic Granules / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D007267:Injections; D009865:Oocytes / Q000378:metabolism*; D011510:Proteolipids / Q000235:genetics / Q000378:metabolism*; D018075:RNA, Complementary; D012333:RNA, Messenger; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution; D014101:Torpedo / Q000378:metabolism*; D014982:Xenopus laevis
D011510:Proteolipids; D018075:RNA, Complementary; D012333:RNA, Messenger; D000109:Acetylcholine; D002118:Calcium