QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online November 3, 2003
doi: 10.1242/10.1242/jcs.00791

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Morel, N. Articles by Philippe, J.-M. Search for Related Content PubMed PubMed Citation Articles by Morel, N. Articles by Philippe, J.-M.

Specific sorting of the a1 isoform of the V-H⁺ATPase a subunit to nerve terminals where it associates with both synaptic vesicles and the presynaptic plasma membrane

Nicolas Morel^1,*, Jean-Claude Dedieu² and Jean-Marc Philippe^1,

¹ Laboratoire de Neurobiologie Cellulaire et Moléculaire, CNRS, 91198 Gif sur Yvette, France
² Centre de Génétique Moléculaire, CNRS, 91198 Gif sur Yvette, France

View larger version (92K): [in a new window]   Fig. 1. Amino acid sequence alignment of the T. marmorata and mouse a1 isoforms of the V-ATPase a subunit. The T. marmorata sequence obtained in the present report was compared with the sequences of two splice variants (a1-I and a1-II) of the mouse a1 isoform of the V-ATPase a subunit (Nishi and Forgac, 2000). Identical residues are shaded in grey. The nine putative transmembrane domains assigned in the yeast homologue Vph1 (Leng et al., 1999) are shown with a dark bar (I to IX). Conserved residues whose mutation affects the activity or assembly of yeast V-ATPase are in black (Leng et al., 1998).

View larger version (74K): [in a new window]   Fig. 2. The a1 isoform of the V-ATPase a subunit does not co-localize with the c subunit in neuron cell bodies. A rabbit antiserum was generated by immunization with a synthetic peptide (13 C-terminal amino acids of the a1 isoform of T. marmorata V-ATPase subunit a) linked to BSA. Specific anti-a1-isoform antibodies were affinity purified on beads coated with the immunopeptide (purified antibody), whereas the serum fraction that did not bound to the beads corresponds to the adsorbed antiserum. (A) Binding of the anti-a1 antiserum (left), adsorbed antiserum (middle) and purified antibodies (right) on T. marmorata cerebellum frozen sections, indirectly visualized with FITC-conjugated anti-rabbit IgG antibodies. Scale bar, 200 µm. (B) Binding of the anti-a1 antiserum (1), adsorbed antiserum (2) and purified antibodies (3) on an immunoblot of synaptosomal proteins (40 µg protein per slot). (C) Distribution of the SV2 synaptic vesicle antigen in a parasagital section of T. marmorata brain (indirect immunolabelling by an anti-SV2 antibody, a peroxidase-conjugated anti-mouse Ig antibody and di-amino-benzidine staining). Scale bar, 5 mm. (D) Double staining of the c subunit (green) and a1 isoform of the a subunit (red) of V-ATPase in T. marmorata electric lobe frozen sections. Binding of mouse anti-subunit-c and rabbit anti-a1-isoform antibodies was indirectly visualized by anti-mouse or anti-rabbit Ig antibodies conjugated to FITC or Alexa 633, respectively. Right-hand panels (merge) correspond to the superimposed stainings. Scale bars, 400 µm (top row) and 200 µm (bottom row). EL, electric lobe; Rh, rhombencephalum.

View larger version (58K): [in a new window]   Fig. 3. The a1 isoform of the V-ATPase a subunit is concentrated in nerve terminals. The a1-subunit distribution in different tissues (liver, kidney, brain and electric lobe) was studied by western blotting of tissue aliquots containing similar amounts of the V-ATPase c subunit (c-V-ATPase) and compared with the distribution in the same samples of the nerve terminal SNARE proteins VAMP-2 and syntaxin-1. The distribution of these antigens in the somatodendritic (electric lobe) and nerve terminal (synaptosome) compartments of T. marmorata electromotoneurons was compared. The protein amount in each sample is indicated (µg protein). SV, purified synaptic vesicles.

View larger version (108K): [in a new window]   Fig. 4. Immunogold detection of the V-ATPase subunit c in a freeze-fractured nerve ending. A typical nerve terminal, frozen in situ in the electric organ, was fractured and replicated. The fracture plane crosses the nerve terminal cytoplasm (right), which contains many synaptic vesicles (arrowheads). It splits the plasma membrane (left), exhibiting a large surface area of the presynaptic membrane E face. Binding of mAb 15K1 to the VATPase c subunit embedded in the replica was indirectly visualized using anti-mouse Ig antibodies conjugated to 15 nm gold particles (arrows).

View larger version (92K): [in a new window]   Fig. 5. V-ATPase c-subunit distribution in freeze-fractured synaptosomes. Typical freeze-fractured synaptosomal profiles that illustrate the preferential detection of the V-ATPase c subunit in the external leaflet (E) of the synaptosomal membrane. Synaptic vesicles in the synaptosome cytoplasm (right) are also labelled by gold particles (arrowheads). P, cytoplasmic leaflet of the synaptosomal membrane.

View larger version (174K): [in a new window]   Fig. 6. V-ATPase c subunit is a constituent of large protein complexes. A flat surface of a freeze-fractured E face of a nerve terminal plasma membrane was labelled by anti-VATPase-c-subunit antibodies. Gold particles (15 nm, arrows) are preferentially associated with large intramembrane particles that correspond to large intramembrane proteins (arrowheads).

View larger version (31K): [in a new window]   Fig. 7. Comparison of V-ATPase in the synaptosomal plasma membrane and synaptic vesicles. After biotinylation of intact synaptosomes, the biotinylated plasma membrane (B) was isolated on streptavidin-coated beads. Its antigenic content was compared with that of purified synaptic vesicles (sv). Samples were adjusted to contain similar VAMP-2 amounts. C, background binding of unbiotinylated synaptosomal membranes to streptavidin beads.

View larger version (62K): [in a new window]   Fig. 8. Association of the V-ATPase membrane domain to SNARE complexes. (A) Solubilized synaptosomal proteins were incubated in parallel with different immunobeads, coated with mAbs to VAMP-2 (19K2), syntaxin-1 (HPC-1) and SNARE complexes (1567). The SNARE protein contents of immunoprecipitated (ip) and unbound (nr) samples deriving from 10 µg synaptosomal protein are compared. (B) Samples depleted of VAMP-2 (19K2 nr in A) or of syntaxin-1 (HPC-1 nr in A) were submitted to a second incubation with the immunobeads (as in A). The syntaxin-1, SNAP-25 and VAMP-2 contents of the resulting immunoprecipitated and unbound samples are compared (deriving from 20 µg synaptosomal protein). (C) Detection of the a1 and c subunits of V-ATPase in the immunoprecipitated samples deriving from the first incubation (left) or the second incubation after VAMP-2 or syntaxin-1 depletion (as indicated). Samples derive from 200 µg synaptosomal protein. (D) Parallel immunoprecipitations of VAMP-2 (19K2) or the SNARE complexes (1567) from solubilized synaptosomes (syn; 200 µg protein) or electric lobe homogenates (el; 2000 µg protein) as in (A).