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  • In addition to pH regulation

    2024-05-15

    In addition to pH regulation by ion (counter-) fluxes, acidification is regulated by the abundance of V-ATPase on organelles, by the pumping activity of the complex and by proton leakage. In yeast, the V-ATPase VO subunit a is the only subunit expressed in two isoforms termed Stv1p and Vph1p. V-ATPase complexes containing Stv1p are targeted to the Golgi apparatus, Vph1p-containing V-ATPases to the vacuole (Kawasaki-Nishi et al., 2001a). These two isoforms additionally regulate the efficiency of the proton pump, and therefore luminal pH, by tight (vacuole) or less tight (Golgi) coupling of ATP-hydrolysis on sector V1 with proton pumping through the VO sector (Kawasaki-Nishi et al., 2001b). Likewise, mammalian V-ATPases can contain either of four different VO a-subunits that are specifically localized to certain tissues and organelles (Nishi and Forgac, 2000, Oka et al., 2001). This is exemplified by VO a-subunit isoform 3 which is highly expressed on the ruffled border in osteoclasts (Toyomura et al., 2000) and it is also the major a-isoform in macrophages (Sun-Wada et al., 2009). However, it is not known whether, like in yeast, mammalian a-subunits can affect coupling efficiency of the proton pump. Temporary regulation of V-ATPase activity can be further achieved by increased association of the proton pump with the target membrane or its disassembly into V1 and VO sectors which occurs in baker’s yeast and in mammals in response to Cisatracurium Besylate receptor starvation (Kane, 1995, Parra and Kane, 1998, Sautin et al., 2005). Finally, the extent of acidification is limited by leakage of protons from the organelle, for example by spontaneous flux directly through the hydrophobic membrane core. In studies using macrolid inhibitors of V‐ATPase, the proton leakage was followed as a gradual alkalinization of the lumen and calculated to be in the order of 0.1 ΔpH/min for lysosomes and phagosomes (Lukacs et al., 1991, Steinberg et al., 2007). Attempts to knockout essential subunits of the proton-pumping ATPase (e.g., VOc) result in early lethality in higher eukaryotes (Dow et al., 1997, Oka and Futai, 2000, Sun-Wada et al., 2000) yet depletion of some subunit isoforms yields viable organisms likely because it does not lead to complete loss of the complex. Such knockout systems helped to understand subunit functions and roles of the V-ATPase that exceed those of a mere proton pump.
    Tools to quantify phagosome pH The most frequently used method to determine V-ATPase-driven phagosome acidification is based on ratiometric measurements of fluorescence emission (Fig. 2): Particles to be phagocytosed are surface labeled with a pH-sensitive fluorophore, often fluorescein, whose emission at 520nm quantifies the intracellular pH due to its exquisite sensitivity towards the surrounding pH when excited at 490nm (Lukacs et al., 1990, Lukacs et al., 1991). Fluorescein is most useful in the pH range from 5.0 to 7.5 but pH-sensitive fluorophores covering different pH ranges are also available, e.g., Oregon Green for the more acidic range, (Schneider et al., 2000). To control and correct for different particle sizes and numbers ingested by phagocytes a second, pH-insensitive fluorophore is co-coupled to the particle surface. Often rhodamine derivatives are used for this purpose. Ratiometric analysis of pH-dependent and pH-independent signals in connection with extensive calibration of the system yields the average phagosome pH over a large cohort of cells (Yates and Russell, 2008). As an alternative choice, fluorescence emission of fluorescein and its derivatives can be determined at their isosbestic point at which fluorescence is not pH-dependent. This kind of measurement alleviates the need for a second fluorophore and can be applied to a growing range of fluorescent groups. For example pH-sensitive variants of the green fluorescent protein (GFP) such as pHluorin (Miesenbock et al., 1998), pHluorin 2 (Mahon, 2011) and dual emission GFP (Hanson et al., 2002) are available that show pH-dependent spectral shifts. Using modified protocols of ratiometric approaches calibration and pH determination can be done microscopically on single phagosomes (Oh and Straubinger, 1996, Schramm et al., 1996) or on single cells using flow cytometry (Simons, 2010).