Crystal structure of the regulatory subunit H of the V-type ATPase of Saccharomyces cerevisiae

Abstract

In contrast to the F-type ATPases, which use a proton gradient to generate ATP, the V-type enzymes use ATP to actively transport protons into organelles and extracellular compartments. We describe here the structure of the H-subunit (also called Vma13p) of the yeast enzyme. This is the first structure of any component of a V-type ATPase. The H-subunit is not required for assembly but plays an essential regulatory role. Despite the lack of any apparent sequence homology the structure contains five motifs similar to the so-called HEAT or armadillo repeats seen in the importins. A groove, which is occupied in the importins by the peptide that targets proteins for import into the nucleus, is occupied here by the 10 amino-terminal residues of subunit H itself. The structural similarity suggests how subunit H may interact with the ATPase itself or with other proteins. A cleft between the amino-and carboxyl-terminal domains also suggests another possible site of interaction with other factors.T he vacuolar proton-translocating ATPases play an important role in the acidification of extracelluar compartments and organelles and are found in most eukaryotic cells. In contrast to the F-type ATPases, which generate ATP from a proton gradient across a membrane, the V-type enzymes use ATP to acidify compartments for receptor-mediated endocytosis, intracellular trafficking, and protein degradation (1, 2). The enzyme is composed of two functionally distinct complexes V 1 and V 0 (Fig. 1). V 0 is integral to the membrane and is thought to consist of at least five different subunits with a total molecular mass of about 260 kDa. This complex is involved in translocation of protons across the membrane. The V 1 complex is more hydrophilic and is composed of at least eight different subunits totaling a molecular mass of about 570 kDa. Because of the relatively high sequence identity between the catalytic subunits ␣ and ␤ of the F-type ATPases and subunits B and A of the V-type ATPases, the V 1 complex is thought to use ATP to drive the proton translocation across the membrane. The rotary catalytic mechanism of ATP hydrolysis and proton transportation (3, 4) is thought to be very similar among the two classes of enzymes, but the different subunit stoichiometry and architectural appearance in electron micrographs suggests that the V-type ATPases are more complex (5, 6).Because the V-type enzymes are involved in key biochemical processes, such as the regulation of pH, and also because they consume rather than generate energy, their function is likely to be under tight control. Previous studies have revealed that most components of the V-type ATPase are essential for assembly of the enzyme complex (1). Characterization of subunit H, also known as Vma13p, however, indicates that this polypeptide is essential for the activity of the enzyme but not for targeting or assembly (7). The regulatory function of subunit H recently was demonstrated by Parra et al. (8). Binding of the subunit to isolated, cytosolic V 1 particles inhibits CaATP hydro...

Subunit H of the V-ATPase Involved in Endocytosis Shows Homology to β-Adaptins

“…The monomeric GGAs are a multidomain protein family implicated in protein trafficking between the Golgi and endosomes. Previous structural analysis shows that the small 18-kDa VHS domain of the Hrs protein consists of three HEAT or ARM repeats (Mao et al, 2000), a protein fold that has been recently found also in the structure of the regulatory subunit H of the V-ATPase (Sagermann et al, 2001). On the basis of the sequence similarity found, we conclude that also ␤-adaptins and ␤-COP are HEAT or ARM repeat-containing proteins.…”

“…The crystal structure of the yeast homologue of subunit H, VMA13p, was published recently (Sagermann et al, 2001). Although the yeast homologue VMA13p contains only 21.7% sequence identity to human V1H, a structure-based alignment enabled us to transform the secondary structure elements of VMA13p to V1H ( Figure 1B).…”

“…Although the yeast homologue VMA13p contains only 21.7% sequence identity to human V1H, a structure-based alignment enabled us to transform the secondary structure elements of VMA13p to V1H ( Figure 1B). The all-helical structure contains eight so-called HEAT or armadillo (ARM) repeats that are also found in Importins (Sagermann et al, 2001). A single ARM repeat consists of ϳ42 residues that fold into three ␣-helices with an almost triangular cross section.…”

“…The degree of sequence identity is displayed as shaded area (average sequence identity is 58.5%), the sequence similarity is indicated by a solid line. (B) Secondary structure of V1H as derived from the structure-based sequence alignment to the yeast homologue VMA13p (Sagermann et al, 2001). The position of the eight armadillo repeats (ARM) is indicated.…”

“…identity 51.7%). The analysis of sequence insertion and deletion sites as well as consideration of putative proteolytic cleavage sites led to the determination of four cleavage sites in the protein of 483 residues ( Figure 1).The crystal structure of the yeast homologue of subunit H, VMA13p, was published recently (Sagermann et al, 2001). Although the yeast homologue VMA13p contains only 21.7% sequence identity to human V1H, a structure-based alignment enabled us to transform the secondary structure elements of VMA13p to V1H ( Figure 1B).…”