Class H ATPases Pump Protons Across Lysosomal and Vacuolar Membranes

All V-class ATPases transport only H+ ions. These proton pumps, present in the membranes of lysosomes, endosomes, and plant vacuoles, function to acidify the lumen of these organelles. The pH of the lysosomal lumen can be measured precisely in living cells by use of particles labeled with a pH-sensitive fluorescent dye. After these particles are phagocy-tosed by cells and transferred to lysosomes, the lysosomal pH can be calculated from the spectrum of the fluorescence emitted. Maintenance of the 100-fold or more proton gradient between the lysosomal lumen (pH =4.5-5.0) and the cytosol (pH =7.0) depends on ATP production by the cell.

The ATP-powered proton pumps in lysosomal and vacuolar membranes have been isolated, purified, and incor porated into liposomes. As illustrated in Figure 7-6 (center), these V-class proton pumps contain two discrete domains: a cytosolic hydrophilic domain (Vj) and a transmembrane domain (V0) with multiple subunits in each domain. Binding and hydrolysis of ATP by the B sub-units in Vj provide the energy for pumping of H+ ions through the proton-conducting channel formed by the c and a subunits in V0. Unlike P-class ion pumps, V-class proton pumps are not phosphorylated and dephosphory-lated during proton transport. The structurally similar F-class proton pumps, which we describe in the next chapter, normally operate in the "reverse" direction to generate ATP rather than pump protons and their mechanism of action is understood in great detail.

Pumping of relatively few protons is required to acidify an intracellular vesicle. To understand why, recall that a solution of pH 4 has a H+ ion concentration of 10~4 moles per liter, or 10-7 moles of H+ ions per milliliter. Since there are 6.02 X 1023 molecules per mole (Avogadro's number), then a milliliter of a pH 4 solution contains 6.02 X 1016 H+ ions. Thus at pH 4, a primary spherical lysosome with a volume of 4.18 X 10~15 ml (diameter of 0.2 ^m) will contain just 252 protons.

By themselves ATP-powered proton pumps cannot acidify the lumen of an organelle (or the extracellular space) because these pumps are electrogenic; that is, a net movement of electric charge occurs during transport. Pumping of just a few protons causes a buildup of positively charged H+ ions on the exoplasmic (inside) face of the organelle membrane. For each H+ pumped across, a negative ion (e.g., OH~ or Cl_) will be "left behind" on the cytosolic face, causing a buildup of negatively charged ions there. These oppositely charged ions attract each other on opposite faces of the membrane, generating a charge separation, or electric potential, across the membrane. As more and more protons are pumped, the excess of positive charges on the exoplasmic face repels other H+ ions, soon preventing pumping of additional protons long before a significant transmembrane H+ concentration gradient had been established (Figure 7-10a). In fact, this is the way that P-class H+ pumps generate a cytosol-negative potential across plant and yeast plasma membranes.

In order for an organelle lumen or an extracellular space (e.g., the lumen of the stomach) to become acidic, movement of protons must be accompanied either by (1) movement of an equal number of anions (e.g., Cl_) in the same direction or by (2) movement of equal numbers of a different cation in the opposite direction. The first process occurs in lyso-somes and plant vacuoles whose membranes contain V-class H+ ATPases and anion channels through which accompanying CP ions move (Figure 7-10b). The second process occurs in the lining of the stomach, which contains a P-class H+/K+ ATPase that is not electrogenic and pumps one H + outward and one K+ inward. Operation of this pump is discussed later in the chapter.

▲ FIGURE 7-10 Effect of proton pumping by V-class ion pumps on H+ concentration gradients and electric potential gradients across cellular membranes. (a) If an Intracellular organelle contains only V-class pumps, proton pumping generates an electric potential across the membrane, luminal-side positive, but no significant change in the intraluminal pH. (b) If the organelle membrane also contains CP channels, anions passively follow the pumped protons, resulting in an accumulation of H+ ions (low luminal pH) but no electric potential across the membrane.

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