Localization of Subunits D, E, and G in the Yeast V-ATPase Complex Using Cysteine-Mediated Cross-Linking to Subunit B

Abstract

Using a combination of cysteine mutagenesis and covalent cross-linking, we have identified subunits in close proximity to specific sites within subunit B of the vacuolar (H(+))-ATPase (V-ATPase) of yeast. Unique cysteine residues were introduced into subunit B by site-directed mutagenesis, and the resultant V-ATPase complexes were reacted with the bifunctional, photoactivatable maleimide reagent 4-(N-maleimido)benzophenone (MBP) followed by irradiation. Cross-linked products were identified by Western blot using subunit-specific antibodies. Introduction of cysteine residues at positions Glu(106) and Asp(199) led to cross-linking of subunits B and E, at positions Asp(341) and Ala(424) to cross-linking of subunits B and D, and at positions Ala(15) and Lys(45) to cross-linking of subunits B and G. Using a molecular model of subunit B constructed on the basis of sequence homology between the V- and F-ATPases, the X-ray coordinates of the F(1)-ATPase, and energy minimization, Glu(106), Asp(199), Ala(15), and Lys(45) are all predicted to be located on the outer surface of the complex, with Ala(15) and Lys(45) located near the top of the complex furthest from the membrane. By contrast, Asp(341) and Ala(424) are predicted to face the interior of the A(3)B(3) hexamer. These results suggest that subunits E and G form part of a peripheral stalk connecting the V(1) and V(0) domains whereas subunit D forms part of a central stalk. Subunit D is thus the most likely homologue to the gamma subunit of F(1), which undergoes rotation during ATP hydrolysis and serves an essential function in rotary catalysis.

Building the Stator of the Yeast Vacuolar-ATPase

“…This shows that the interaction between subunits E and G is necessary to elongate subunit G in order for it to act as a stator. The positioning of the EG complex at the periphery of the V 0 -V 1 complex and its extended character are in accordance with cross-linking data of cysteine mutants that have shown that subunits E and G are in close contact to the outer surface of subunit-B of V 1 , reaching almost to the top of the V 0 -V 1 complex (19).…”

“…Based on the alignment of sequences of subunits G from several species, a region mapping to a single face of a predicted helix (26,43,44) has been proposed to be involved in protein-protein interaction. Cross-linking studies and native complex isolation also indicated that subunits E and G could form part of the peripheral stator (16,19). The data presented in this study clearly show that subunit E is a partner for subunit G. The fact that these subunits spontaneously associate after cleavage from their respective fusion partner and purify as a single entity when co-expressed is a clear indication of a tight and specific interaction.…”

“…Early data obtained from yeast two-and three-hybrid experiments and native subcomplex isolation in subunit deficient mutant strains, as well as co-immunoprecipitation, have helped identify important interactions between the different subunits of yeast V-ATPase (16,38,48,49). In addition, neighboring subunits have been identified by chemical cross-linking experiments (17,19) in native complexes. These data paved the way to the establishment of a consensus on the topology of the ATPase, especially of the peripheral subunits.…”

Subunit Arrangement in V-ATPase from Thermus thermophilus

“…The D subunit had been the most probable candidate of rotor subunit in V 1 portion (2). Cross-linking studies have suggested that the D subunit was adjacent to B subunit at central cavity region of A 3 B 3 hexamer, and the F subunit was associated with the D subunit (29,30). In contrast, studies on the V-ATPase from Manduca sexta suggested that subunit E, rather than subunit D, was the rotor subunit (31).…”

Topological Characterization of the c, c′, and c″ Subunits of the Vacuolar ATPase from the Yeast Saccharomyces cerevisiae

“…The role of the other V 1 subunits is not so clear. It is thought that the central stalk may be composed of D (32 kDa) and F (14 kDa) subunits (8,9). The stator region is thought to be composed of E (27 kDa), G (13 kDa), C (42 kDa), and H (54 kDa) subunits (9 -13).…”