Mutational Analysis of the Non-homologous Region of Subunit A of the Yeast V-ATPase

Abstract

Subunit A is the catalytic nucleotide binding subunit of the vacuolar proton-translocating ATPase (or V-ATPase) and is homologous to subunit ␤ of the F 1 F 0 ATP synthase (or F-ATPase). Amino acid sequence alignment of these subunits reveals a 90-amino acid insert in subunit A (termed the non-homologous region) that is absent from subunit ␤. To investigate the functional role of this region, site-directed mutagenesis has been performed on the VMA1 gene that encodes subunit A in yeast. Substitutions were performed on 13 amino acid residues within this region that are conserved in all available A subunit sequences. Most of the 18 mutations introduced showed normal assembly of the V-ATPase. Of these, one (R219K) greatly reduced both proton transport and ATPase activity. By contrast, the P217V mutant showed significantly reduced ATPase activity but higher than normal levels of proton transport, suggesting an increase in coupling efficiency. Two other mutations in the same region (P223V and P233V) showed decreased coupling efficiency, suggesting that changes in the non-homologous region can alter coupling of proton transport and ATP hydrolysis. It was previously shown that the V-ATPase must possess at least 5-10% activity relative to wild type to undergo in vivo dissociation in response to glucose withdrawal. However, four of the mutations studied (G150A, D157E, P177V, and P223V) were partially or completely blocked in dissociation despite having greater than 30% of wild type levels of activity. These results suggest that changes in the non-homologous region can also alter in vivo dissociation of the V-ATPase independent of effects on activity.The vacuolar proton-translocating ATPases (or V-ATPases) 1 are ATP-driven proton pumps present in both intracellular compartments and the plasma membrane of eukaryotic cells (1-8). They couple the energy released upon ATP hydrolysis to the active transport of protons from the cytoplasm to either the lumen of various intracellular compartments or to the extracellular environment. Acidification of intracellular compartments is important for such processes as receptor-mediated endocytosis, intracellular trafficking of lysosomal enzymes, degradation of macromolecules, uptake of neurotransmitters, and the entry of various envelope viruses and toxins (1-8). Plasma membrane V-ATPases have also been implicated in many normal and disease processes, including bone resorption, renal acidification, pH homeostasis, and tumor metastasis (9 -13). Defects in specific V-ATPase subunits have been shown to be responsible for a number of human genetic diseases, including autosomal recessive osteopetrosis and renal tubular acidosis (9, 14 -16).The V-ATPases are multi-subunit complexes composed of two functional domains (1-8). The 640-kDa peripheral V 1 domain is responsible of ATP hydrolysis and consists of 8 different subunits (subunits A-H) with molecular masses of 70 -14 kDa. The V 0 domain is a 260-kDa integral complex composed of five different subunits (subunits a, d, cЈЈ, cЈ, and c with mole...

PKA Regulates Vacuolar H+-ATPase Localization and Activity via Direct Phosphorylation of the A Subunit in Kidney Cells

“…Specifically, it is conceivable that PKA phosphorylation of the A subunit modulates protein-protein interactions within the multisubunit V-ATPase complex and/or with other interacting proteins involved with cellular trafficking of the pump. For example, the sequence around our identified PKA phosphorylation site is highly conserved in eukaryotes and lies within a so-called "non-homologous" region (63). This region received its name because it is not present in the F-ATPase ␤ subunit, which otherwise has high homology to the V-ATPase A subunit.…”

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

“…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). In this context, our observation that reversible salicylihalamide analog 14 at the lower dose did not induce the cellular V 1 redistribution response reminiscent of salicylihalamide A, but behaved more like bafilomycin, suggests that the unique response with salicylihalamide might be related to its unique inhibition characteristics.…”

Renal Vacuolar H+-ATPase

“…In addition, the yeast vacuolar H ϩ -ATPase A subunit possesses a stretch of an additional 90 amino acids compared with ␣-subunits. This additional segment may play a role in the coupling efficiency of ATP hydrolysis to proton transport and the dissociation of V 1 and V 0 complexes (461).…”