The structure and function of the fungal V-ATPase.

The fungal vacuole is a prominent organelle that functions as a storage site for amino acids, Ca, storage carbohydrates, inorganic phosphate and numerous hydrolases (Fig. 1) (Klionsky et al. 1990). A hallmark of fungal vacuoles is that they are acidic compartments involved in the turnover of cellular macromolecules. In the yeast Saccharomyces cerevisiae, the hydrolase activities in the vacuole are required for diploid cells to progress normally through sporulation and meiosis. The goal of this chapter is to present an overview of what is known about the enzyme complex responsible for acidifying the fungal vacuole and to summarize the current view of the function of acidification of the vacuolar network. The vacuolar H-ATPase (V-ATPase) is responsible for acidification of the fungal vacuolar network (Klionsky et al. 1990; Raymond et al. 1992). This enzyme functions by coupling the hydrolysis of cytoplasmic ATP to the translocation of protons across the vacuolar membrane. The V-ATPase has been isolated from several fungi, and the enzymes isolated from Neurospora crassa and the yeast Saccharomyces cerevisiae have been extensively characterized. In all cases, the enzyme is a multisubunit complex of at least 500 XlO relative molecular mass (Mr) (Hirata et al. 1989) composed of both membrane-bound polypeptides and peripherally associated subunits. Assigning functions to each of the polypeptides associated with the purified, enzymatically active V-ATPase complex remains a challenge for the future. The molecular cloning of the genes encoding the V-ATPase subunits has provided an avenue for the detailed characterization of the structure and function of the various polypeptides. Subunit-encoding genes were first isolated and characterized for the Neurospora crassa V-ATPase 69 and 59X 10Mr polypeptides (Bowman etal. 1988a,&). These investigations were followed by the isolation of the genes for the corresponding yeast subunits (Shih et al. 1988; Nelson et al. 1989; Hirata et al. 1990; Yamashiro et al. 1990). The availability of the yeast genes provided an opportunity for the complete genetic dissection of the fungal V-ATPase subunit composition and function through the use of yeast mutants lacking individual subunits as a result of gene disruptions. These genetic analyses have allowed each biochemically defined subunit to be individually scrutinized for its in vivo requirement for function and assembly of the V-ATPase complex. These topics are considered in detail in this paper and the other papers of this fungal V-ATPase chapter.