Human voltage-dependent anion-selective channel expressed in the plasmalemma of Xenopus laevis oocytes.
Recent studies indicate a plasmalemmal localisation of eukaryotic porin, i.e. voltage-dependent anion-selective channel (VDAC), and there is evidence that the channel in this cell compartment is engaged in cell volume regulation. Until recently, others and we have used immuno-topochemical and biochemical methods to demonstrate the integration of the channel into the cell membrane and endoplasmic reticulum of vertebrate cells. In the present study, we used molecular biological methods to induce the heterologous expression of tagged human type-1 porin in oocytes of Xenopus laevis and to illustrate its appearance at the plasma membrane of these cells. Applying confocal fluorescent microscopy, green fluorescent protein attached to the C-terminus of porin could clearly be recorded at the cell surface. N-terminal green fluorescent protein-porin fusion proteins remained in the cytoplasm, indicating a strong influence of the porin N-terminus on protein trafficking to the plasma membrane. FLAG-tagged porin was also expressed in frog oocytes. Here, plasmalemmal expression was observed using anti-FLAG M2 monoclonal antibodies and gold-conjugated secondary antibodies, followed by silver enhancement through scanning electron microscopy. In contrast to the EGFP-porin fusion protein, the influence of the small FLAG-epitope (8 amino acids) did not prevent plasmalemmal expression of N-terminally tagged porin. These results indicate the definite expression of human type-1 porin in the plasma membrane of Xenopus oocytes. They thus corroborate our early data on the extra-mitochondrial expression of the eukaryotic porin channel and are essential for future electrophysiological studies on the channel.
Schwarzer
C
C
Max-Planck-Institut für Experimentelle Medizin, Abteilung Immunchemie, Hermann-Rein Strasse 3, 37075, Göttingen, Germany.
Becker
S
S
Awni
L A
LA
Cole
T
T
Merker
R
R
Barnikol-Watanabe
S
S
Thinnes
F P
FP
Hilschmann
N
N
D000818:Animals; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D006801:Humans; D008845:Microinjections; D008855:Microscopy, Electron, Scanning; D008856:Microscopy, Fluorescence; D009865:Oocytes / Q000166:cytology* / Q000378:metabolism* / Q000648:ultrastructure; D018272:Porins / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050994:Voltage-Dependent Anion Channels; D014982:Xenopus laevis
D018272:Porins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D050994:Voltage-Dependent Anion Channels