Dennis K. Stone

Vacuolar proton pumps

Recently a new class of proton-translocating ATPases has been localized to endomembrane compartments in plant, fungal, and mammalian cells. These proton pumps are large hetero-oligomers which have an ATP hydrolytic sector that is functionally and structurally distinct from a transmembranous proton pore. Enzymatic characteristics of these proton pumps are discussed as well as the current state of knowledge regarding subunit composition and function. In addition, recent primary sequence data are discussed which indicate that these proton pumps share a common ancestor with F1F0-type proton pumps of mitochondria

Molecular Characterization of the 50- and 57-kDa Subunits of the Bovine Vacuolar Proton Pump

The vacuolar type proton-translocating ATPase of clathrin-coated vesicles is composed of two large domains: an extramembranous catalytic sector and a transmembranous proton channel. In addition, two polypeptides of 50 and 57 kDa have been found to co-purify with the pump. These proteins, termed SFD (sub-fifty-eight-kDadimer) activate ATPase activity of the enzyme and couple ATPase activity to proton flow (Xie, X.-S., Crider, B.P., Ma, Y.-M., and Stone, D. K. (1994) J. Biol. Chem. 269, 28509–25815). It has also been reported that the clathrin-coated vesicle proton pump contains AP50, a 50-kDa component of the AP-2 complex responsible for the assembly of clathrin-coated pits, and that AP50 is essential for function of the proton pump (Liu, Q., Feng, Y., and Forgac, M. (1994) J. Biol. Chem. 269, 31592–31597). We demonstrate through the use of anti-AP50 antibody, identical to that of the latter study, that hydroxylapatite chromatography removes AP50 from impure proton pump preparations and that purified proton pump, devoid of AP50, is fully functional. To determine the true molecular identity of SFD, both the 50- and 57-kDa polypeptides were directly sequenced. A polymerase chain reaction-based strategy was used to screen a bovine brain cDNA library, yielding independent full-length clones (SFD-4A and SFD-21); these were identical in their open reading frames and encoded a protein with a predicted mass of 54,187 Da. The SFD-21 clone was then used in a reverse transcription-polymerase chain reaction-based strategy to isolate a related, but distinct, transcript present in bovine brain mRNA. The nucleotide and predicted amino acid sequences of this isolate are identical to SFD-21 except that the isolate contains a 54-base pair insert in the open reading frame, resulting in a protein with a predicted mass of 55,933 Da. Both clones had 16% identity toVMA13 of Saccharomyces cerevisiae. No sequence homology between the SFD clones and AP50 was detectable. Anti-peptide antibodies were generated against an epitope common to the two proteins and to the unique 18-amino acid insert of the larger protein. The former reacted with both components of native SFD, whereas the latter reacted only with the 57-kDa component. We term the 57- and 50-kDa polypeptides SFDα and SFDβ, respectively.

Recombinant SFD Isoforms Activate Vacuolar Proton Pumps

The vacuolar proton pump of clathrin-coated vesicles is composed of two general sectors, a cytosolic, ATP hydrolytic domain (V1) and an intramembranous proton channel, V0. V1 is comprised of 8–9 subunits including polypeptides of 50 and 57 kDa, termed SFD (SubFifty-eight-kDa Doublet). Although SFD is essential to the activation of ATPase and proton pumping activities catalyzed by holoenzyme, its constituent polypeptides have not been separated to determine their respective roles in ATPase functions. Recent molecular characterization of these subunits revealed that they are isoforms that arise through an alternative splicing mechanism (Zhou, Z., Peng, S.-B., Crider, B.P., Slaughter, C., Xie, X.S., and Stone, D.K. (1998) J. Biol. Chem. 273, 5878–5884).  To determine the functional characteristics of the 57-kDa (SFDα)1 and 50-kDa (SFDβ) isoforms, we expressed these proteins in Escherichia coli. We determined that purified recombinant proteins, rSFDα and rSFDβ, when reassembled with SFD-depleted holoenzyme, are functionally interchangeable in restoration of ATPase and proton pumping activities. In addition, we determined that the V-pump of chromaffin granules has only the SFDα isoform in its native state and that rSFDα and rSFDβ are equally effective in restoring ATPase and proton pumping activities to SFD-depleted enzyme. Finally, we found that SFDα and SFDβ structurally interact not only with V1, but also withV0, indicating that these activator subunits may play both structural and functional roles in coupling ATP hydrolysis to proton flow.

Identification of a 14-kDa Subunit Associated with the Catalytic Sector of Clathrin-coated Vesicle H-ATPase

The clathrin-coated vesicle H-ATPase is composed of a peripheral catalytic sector (V) and an integral membrane proton channel (V), both of which are multiple subunit complexes. This study was conducted to determine if subunit F, previously identified in vacuolar proton pumps of tobacco hornworm and yeast, was present in mammalian pumps. Using a polymerase chain reaction-based strategy, we have isolated and sequenced cDNA clones from bovine and rat brain cDNA libraries. A full-length clone from rat brain encodes a 119-amino acid polypeptide with a predicted molecular mass of 13,370 Da and with approximately 72 and 49% identity to subunit F of tobacco hornworm and yeast, respectively. Southern and Northern blot analyses indicate that the protein is encoded by a single gene. An anti-peptide antibody, directed against deduced protein sequence, was affinity-purified and shown to react with a 14-kDa polypeptide that is present in a highly purified pump prepared from clathrin-coated vesicles and also isolated V. When stripped clathrin-coated vacuolars and purified chromaffin granule membranes were treated with KI in the presence of ATP, the 14-kDa subunit was released from both membranes, further indicating that it is part of the peripheral catalytic sector. In addition, direct sequencing of this 14-kDa component of the coated vacuolar proton pump confirmed its identity as a subunit F homologue.

Proton pump of clathrin-coated vesicles.

Publisher Summary This chapter describes solubilization, isolation, and reconstitution of the clathrin-coated vesicle proton-translocating complex. By sucrose density gradient centrifugation determination, the molecular weight of the proton pump is 530K. In addition, the purification of a 116K ATPase from clathrin-coated vesicles is described. An ATP-driven proton pump activity is present in clathrin-coated vesicles, which are distinguished from the well-characterized proton pump of mitochondria by its insensitivity to oligomycin, azide, and efrapeptin. The activity of the pump is expressed in arbitrary optical density units, either in terms of the initial slope or the extent of ATP-induced decrease in absorbance, under the assay conditions described. Coated vesicles contain a transporter through which chloride or bromide pass—thereby, balancing the electrogenic proton translocation. Chloride transport can be functionally dissociated from the proton pump. It is facilitated either by ATP-driven proton movement, or by an inside positive membrane potential generated by K + moving with valinomycin from the outside to the inside of the vesicles.

[3] Purification of calelectrins

Publisher Summary This chapter describes purification methods for calelectrin from the electric organ of Torpedo marmorata and from bovine tissues. Although the different calelectrins are differentially expressed in a cell-type specific manner as determined by immunocytochemistry, they can be purified from several different tissues. Calelectrins have the following characteristics: (1) Binding of Ca 2+ at low Ca 2+ concentrations; (2) immunologic cross-reactivity with Torpedo calelectrin, a 34-kDa calcium-binding protein isolated from the electric organ of Torpedo marmorata ; (3) retention on hydrophobic affinity columns such as phenyl-Sepharose or phenothiazine-agarose in the presence of calcium and elution with EGTA; (4) binding to biomembranes in a calcium-dependent and specific manner. Electric organ from Torpedo marmorata is frozen in liquid nitrogen, crushed to a fine powder, and extracted with 0.4 M NaCl, 1 mM EGTA, 1 mM PMSF, and 10 mM Tris, pH 7.4, with 2–3 vol of buffer per volume of tissue. The supernatant from this step is concentrated by NH 4 SO 4 precipitation (95%, 700 g/liter) and further purified from low–molecular-weight contaminants by gel filtration. From this gel column, calelectrin elutes as a single peak at a position corresponding to its monomeric molecular weight and is homogeneous by SDS polyacrylamide gel electrophoresis.