Regulation of V-ATPase Activity and Organelle pH by Phosphatidylinositol Phosphate Lipids

Abstract

Luminal pH and the distinctive distribution of phosphatidylinositol phosphate (PIP) lipids are central identifying features of organelles in all eukaryotic cells that are also critical for organelle function. V-ATPases are conserved proton pumps that populate and acidify multiple organelles of the secretory and the endocytic pathway. Complete loss of V-ATPase activity causes embryonic lethality in higher animals and conditional lethality in yeast, while partial loss of V-ATPase function is associated with multiple disease states. On the other hand, many cancer cells increase their virulence by upregulating V-ATPase expression and activity. The pH of individual organelles is tightly controlled and essential for function, but the mechanisms for compartment-specific pH regulation are not completely understood. There is substantial evidence indicating that the PIP content of membranes influences organelle pH. We present recent evidence that PIPs interact directly with subunit isoforms of the V-ATPase to dictate localization of V-ATPase subpopulations and participate in their regulation. In yeast cells, which have only one set of organelle-specific V-ATPase subunit isoforms, the Golgi-enriched lipid PI(4)P binds to the cytosolic domain of the Golgi-enriched a-subunit isoform Stv1, and loss of PI(4)P binding results in mislocalization of Stv1-containing V-ATPases from the Golgi to the vacuole/lysosome. In contrast, levels of the vacuole/lysosome-enriched signaling lipid PI(3,5)P₂ affect assembly and activity of V-ATPases containing the Vph1 a-subunit isoform. Mutations in the Vph1 isoform that disrupt the lipid interaction increase sensitivity to stress. These studies have decoded "zip codes" for PIP lipids in the cytosolic N-terminal domain of the a-subunit isoforms of the yeast V-ATPase, and similar interactions between PIP lipids and the V-ATPase subunit isoforms are emerging in higher eukaryotes. In addition to direct effects on the V-ATPase, PIP lipids are also likely to affect organelle pH indirectly, through interactions with other membrane transporters. We discuss direct and indirect effects of PIP lipids on organelle pH, and the functional consequences of the interplay between PIP lipid content and organelle pH.

Keywords: Golgi apparatus; PIKfyve; V-ATPase; acidification; endosome; lysosome; organelle; phosphatidylinositol phosphate.

FIGURE 1

Distribution of V-ATPase isoforms, PIP lipids and the pH of subcellular organelles. Subcellular localization of isoforms of the 100 kDa a-subunit of the V-ATPase, in yeast and mammals, are indicated by separately coloring the a-subunit isoforms. The key to the respective a-subunit isoforms are indicated on the right side of the figure. On the left bottom, a key to the different organelles in the figure is present. The distinct enrichment of PIP lipids on the membranes of subcellular organelles and compartments are indicated by different colors. The key to the color-coded PIP species is on the bottom right side of the figure. Enrichment of PI(4)P is indicated in the compartments of the Golgi network and the plasma membrane. In addition to PI(4)P, the plasma membrane maintains an enrichment of PI(4,5)P₂ and PI(3,4,5)P₃. Secretory vesicles are also indicated to be enriched in PI(4)P. The endosomal compartments, including the early-, late- and recycling-endosomes and lysosomes, are characteristically enriched in PI(3)P. The late endosomes and lysosomes, in addition, maintains the highly regulated species PI(3,5)P₂. pH or pH-ranges of all the different organelles are indicated by numbers inside or beside the respective organelles.

FIGURE 2

Structure of the yeast V-ATPase holoenzyme. (A) Cryo-electron micrograph structure of the assembled yeast V₁-V_o complex (Zhao et al., 2015). A combination of the electron density and a chain trace of the different subunits is used to demonstrate the structural features of the V-ATPase holoenzyme. The subunits and distinct domains are indicated. The three distinct EG heterodimers (green and purple) are indicated by numbers on the side of the respective heterodimers. Upper-case letters indicate the subunit of the V₁ sector and lower-cases represent the V_o-sector subunits. (B) A cartoon of the V-ATPase holoenzyme together with the functions performed by the V₁ and the V_o sectors is demonstrated. Precisely, the V₁ sector performs the ATP hydrolysis which is coupled to the proton transport function of the V_o sector. The direction of rotation of the rotor subunits (c8c′c″dDF) is indicated by a circular arrow on the hetero-decameric c-ring (cc′c″).

FIGURE 3

A unified model of proton pumps, transporters, and H⁺/ion exchangers that dynamically regulate the pH of subcellular compartments in yeast. A holistic regulation of pH of the cytosol, the vacuole, and the Golgi network is maintained by the coordinated transport of several ions. The plasma membrane Pma1 (green), vacuolar V-ATPase complex (chrome and black), and the Golgi V-ATPase complex (chrome and black) pump H⁺ out of the cytosol into the extracellular milieu, the vacuole, and the Golgi, respectively. Simultaneously, different ion transporters and H⁺/ion exchangers dynamically regulate the pH of the organelles and the cytosol (Li and Kane, 2009; Yenush, 2016). A key identifying the H⁺ pumps, ion channels, and H⁺/ion exchangers is provided at the bottom of the figure. BAA refers to basic amino acids. The arrowheads indicate the direction of ion transport.

FIGURE 4

Structural features of the a-subunit. (A) A cryo-EM based model indicating the dynamic structural re-orientation of the N-terminal domain of the a-subunit in an assembled and active holoenzyme-conformation, colored in red (Zhao et al., 2015), and a disassembled and autoinhibited conformation in the isolated V_o sector, colored in green (Stam and Wilkens, 2017). The chain traces of the c-ring comprized of c8c′c″ is colored in gray and the spherical atoms of the d-subunit is colored in blue. (B) A superimposition of the cryo-EM structure of the NT-domain of Vph1 in blue (Zhao et al., 2015) and a Phyre2 based structural model of the Stv1-NT in gray. Stv1NT was modeled to the available low-density structure of the Stv1-V-ATPase (Vasanthakumar et al., 2019). PI(4)P and PI(3,5)P₂ molecules are indicated with phosphates drawn in pink on the respective positions of a phosphatidylinositol lipid below the proximal and distal subdomains of the aNT domain, respectively. The PI(4)P binding site in Stv1, comprized of a W⁸³KY sequence in the proximal end of the Stv1NT domain is indicated using red ball-and-stick atomic side chains. A PI(3,5)P₂ recognition site in Vph1, comprized of a K²³¹TREYKHK sequence in the distal end of the Vph1NT domain is indicated using green ball-and-stick atomic side chains.