Photoaffinity Labeling of Wild-type and Mutant Forms of the Yeast V-ATPase A Subunit by 2-Azido-[32P]ADP

Abstract

To test the proximity of these aromatic residues to the adenine ring, the yeast V-ATPase containing wild-type and mutant forms of the A subunit was reacted with 2-azido-[ 32 P]ADP, a photoaffinity analog that stably modifies tyrosine but not phenylalanine residues. Mutant forms of the A subunit were constructed in which the two endogenous tyrosine residues were replaced with phenylalanine and in which a single tyrosine was introduced at each of the four positions. Strong ATP-protectable labeling of the A subunit was observed for the wild-type and the mutant containing tyrosine at 532, significant ATP-protectable labeling was observed for the mutants containing tyrosine at positions 452 and 538, and only very weak labeling was observed for the mutants containing tyrosine at 535 or in which all four residues were phenylalanine. These results suggest that Tyr 532 and possibly Phe 452 and Tyr 538 are in close proximity to the adenine ring of ATP bound to the A subunit. In addition, the effects of mutations at Phe 452 , Tyr 532 , Tyr 535 , and Glu 286 on dissociation of the peripheral V 1 and integral V 0 domains both in vivo and in vitro were examined. The results suggest that in vivo dissociation requires catalytic activity while in vitro dissociation requires nucleotide binding to the catalytic site.The vacuolar proton-translocating ATPases (or V-ATPases) 1 are a family of ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells (1-6). The V-ATPases function in a variety of cellular processes, including protein processing and degradation, receptormediated endocytosis, intracellular membrane traffic, and coupled transport (1-6). For certain specialized cells, including renal intercalated cells (7), macrophages and neutrophils (8), tumor cells (9), and osteoclasts (10), the V-ATPase has been identified at the plasma membrane and acidifies the extracellular space. In yeast, acidification of the central vacuole by the V-ATPase serves to activate protein degradation and to drive the uptake of solutes such as Ca 2ϩ and amino acids for storage (5).The V-ATPases are composed of two domains, a peripheral, cytoplasmic 570-kDa V 1 domain responsible for nucleotide binding and hydrolysis, and a membrane integral 260-kDa V 0 domain responsible for proton translocation across the membrane (1-6). The V 1 domain contains eight different subunits (subunits A-H, with molecular masses 70 -14 kDa) while the V 0 domain contains five different subunits (subunits a, d, cЉ, c, and cЈ, with molecular masses from 100 to 17 kDa). The nucleotide binding subunits have been identified as the A and B subunits, which are believed to form a hexameric structure containing three copies of each subunit (11).Previous studies have demonstrated that the catalytic sites reside on the A subunits and that a second class of sites, termed "noncatalytic" sites, reside on the B subunits, giving a total of six nucleotide binding sites per complex (12-15). The A and B subunits of the V-ATPase are approximately 2...

Salicylihalamide A Inhibits the V0 Sector of the V-ATPase through a Mechanism Distinct from Bafilomycin A1

“…Therefore the in vivo V 1 re-distribution that was induced by salicylihalamide cannot be explained by a simple pH effect alone. Although the salicylihalamide effect in vivo is consistent with previous observation that inhibition of V-ATPase activity in yeast through mutagenesis blocks dissociation (19,34), the correlation between the activity of V-ATPase and its reversible dissociation has not been clearly established. Mutagenesis studies with subunit D showed that a mutant with only 6% of wild-type level of proton pumping activity still demonstrated normal dissociation in response to glucose deprivation (35), whereas some mutations in subunit A resulted in a partial or complete blocking of dissociation despite possessing more than 30% of wild-type levels of activity (25).…”

Involvement of the Nonhomologous Region of Subunit A of the Yeast V-ATPase in Coupling and in Vivo Dissociation

“…This absorbance effect was eliminated by measuring the decrease in NADH absorbance at 360 nm instead of 341 nm, at which wavelength interference due to absorbance of light by chloroquine is negligible. Because 80% of control activity is far in excess of what is required to allow in vivo dissociation of the V-ATPase to occur (16,58), chloroquine does not appear to block glucose-dependent dissociation of V 1 V 0 by directly inhibiting V-ATPase activity.…”

“…Effect of Vacuolar Neutralization on in Vivo Glucose-dependent Dissociation of the V-ATPase-In yeast, dissociation of the V 1 and V 0 domains in response to glucose depletion has been shown to require catalytic activity of the V-ATPase (16,58). Consistent with this, the R219K mutation in the nonhomologous region that inhibits Ͼ90% of both proton transport and ATPase activity in isolated vacuolar membranes results in a block in glucose-dependent dissociation (29).…”

“…The 10-kDa subunit e, previously identified in bovine and insect (13,14), has recently been shown to be essential for function of the yeast V-ATPase (15). Both the 70-kDa A subunit and the 60-kDa B subunit of V 1 possess nucleotide binding sites (16,17), with the catalytic nucleotide binding sites located on subunit A (18). The V-ATPases structurally resemble the F-ATPases (proton-driven ATP synthases) of mitochondria, chloroplasts, and bacteria (19 -23).…”

Interacting Helical Surfaces of the Transmembrane Segments of Subunits a and c′ of the Yeast V-ATPase Defined by Disulfide-mediated Cross-linking

“…V 1 is composed of eight different subunits (A-H) of molecular mass 70 -13 kDa that are present in a stoichiometry of A 3 B 3 C 1 D 1 E 1 F 1 G 2 H 1-2 (14, 15) and that form a complex of ϳ640 kDa. Both the A and B subunits participate in nucleotide binding, with the catalytic sites located on the A subunits (16,17). The V 0 domain is a 240-kDa complex composed of five different subunits (a, d, c, cЈ, and cЉ) of molecular mass 100 -16 kDa that are present in a stoichiometry of a 1 d 1 c 4 c 1 Јc 1 Љ (14, 18).…”