Subunit a of the Yeast V-ATPase Participates in Binding of Bafilomycin

Abstract

The vacuolar (H ϩ )-ATPases (or V-ATPases) 2 are ATP-dependent proton pumps widely distributed among intracellular and plasma membranes of eukaryotic cells (1-11). They carry out ATP-driven transport of protons from the cytoplasm to either the lumen of internal compartments or to the extracellular space. V-ATPases present in intracellular membranes are important for membrane traffic processes such as receptor-mediated endocytosis and intracellular targeting of newly synthesized lysosomal enzymes (1). They also provide the acidic environment required for processing and degradation of macromolecules and the entry of certain envelope viruses and bacterial toxins, including anthrax toxin (12), as well as the driving force for coupled transport of small molecules, such as neurotransmitters. Plasma membrane V-ATPases function in a wide variety of normal and disease processes, including renal acidification, bone resorption, K ϩ secretion, sperm maturation, and tumor cell invasion (1, 7-11). V-ATPase inhibitors are thus potentially useful therapeutic agents for the treatment of a number of human diseases, including viral infection, osteoporosis, and cancer. V-ATPases are multisubunit complexes composed of two domains (1-9). The peripheral V 1 domain is composed of eight subunits (A-H) and functions to hydrolyze ATP. The integral V 0 domain is composed of six subunits (in yeast these are subunits a, c, cЈ, cЉ, d, and e) and is responsible for proton transport. The V-ATPases thus structurally resemble the ATP synthases (or F-ATPases) of mitochondria, chloroplasts, and bacteria that function in the synthesis of ATP (13-16).Like the F-ATPases (13-16), the V-ATPases have been shown to operate by a rotary mechanism (17, 18). ATP hydrolysis in the V 1 domain drives rotation of a central stalk composed of subunits D and F (17, 19). These subunits are linked to a ring of proteolipid subunits (c, cЈ, and cЉ) via subunit d (20). Each proteolipid subunit contains a single buried acidic residue that undergoes reversible protonation and deprotonation during the rotational catalysis (21). As the ring of proteolipid subunits rotates relative to subunit a, each buried carboxyl group on the ring is thought to pick up a proton from the cytoplasmic compartment via a hemi-channel located in subunit a (1, 15, 22, 23). Rotation of the ring brings this protonated carboxyl into contact with a second a subunit hemi-channel leading to the luminal or extracellular side of the membrane. Interaction between the carboxyl group and the positively charged guanidinium group of Arg 735 of subunit a (23) then causes release of the proton into the luminal hemi-channel.Subunit a is a 100-kDa integral membrane protein containing two domains (24). The N-terminal hydrophilic domain has a molecular mass of 50 kDa and is oriented toward the cytoplasmic side of the membrane (25). Together with subunits C, E, G, H, and the non-homologous domain of subunit A (19, 26 -29), the N-terminal domain forms a peripheral stalk or stator that keeps the catalytic domain of t...

Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance

“…Other than these differences, comparison of the two states reveals only minor conformational changes in individual subunits, suggesting little flexibility in the enzyme as a whole (Movie S3). The T. thermophilus V/A-ATPase thus seems to be relatively rigid compared with the S. cerevisiae V-ATPase (8) Numerous studies, many by Forgac and coworkers, have investigated the topology, function, and subunit arrangement of the S. cerevisiae V-ATPase V O region (31,34,(38)(39)(40)(41)(42)(43)(44). The model of the a subunit presented here is in excellent agreement with these studies.…”

“…Ten residues were proposed to participate in bafilomycin binding, all of which are conserved in S. cerevisiae. In addition, three a subunit residues were identified in S. cerevisiae where mutation conferred bafilomycin resistance (44). Visualizing these residues together suggests a binding site for bafilomycin between two c subunits accessible from the cytoplasm through the a subunit (Fig.…”

“…(D) Residues that, when mutated (blue spheres) in the c subunit (T32, T39, L50, I54, V57, G61, L131, F135, L139, and Y142) and a subunit (E721, L724, and N725), give partial resistance to bafilomycin (43,44). The positions of these residues suggest a binding site for bafilomycin between two adjacent c subunits (pink and magenta) that is accessed via the a subunit (green ribbon).…”

Identification of the fnx1+ and fnx2+ genes for vacuolar amino acid transporters in Schizosaccharomyces pombe

“…Fig. 3 shows the effect of concanamycin A, a specific inhibitor of the vacuolar H + -ATPase [23,24], on the uptake of 2-DG, histidine, and isoleucine by S. pombe cells. 2-deoxy-D D-glucose (2-DG) is known to be a substrate for the glucose transporter Ght1p on the plasma membrane of S. pombe [16,25].…”

Bafilomycin K, a new antifungal macrolide from Streptomyces flavotricini Y12-26

“…Bafilomycins have antibacterial, antifungal, antineoplastic and immunosuppressive activities. 5 Some bafilomycins are specific inhibitors of vacuolar-type H + -ATPase (V-ATPase), [8][9][10][11] in which bafilomycin A 1 is the most used. Being a specific inhibitor of V-ATPase, bafilomycin A 1 can prevent the re-acidification of synaptic vesicles once they have undergone exocytosis.…”