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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.

We have employed a combination of site-directed mutagenesis and covalent cross-linking to identify subunits in close proximity to subunit B in the vacuolar H ؉ -ATPase (V-ATPase) complex. Unique cysteine residues were introduced into a Cys-less form of subunit B, and the V-ATPase complex in isolated vacuolar membranes from each mutant strain was reacted with the bifunctional, photoactivable maleimide reagent 4-(N-maleimido)benzophenone. are predicted to reside near the bottom of V 1 , with all four residues predicted to be oriented toward the external surface of the complex. A model incorporating these and previous data is presented in which subunit E exists in an extended conformation on the outer surface of the A 3 B 3 hexamer that forms the core of the V 1 domain. This location for subunit E suggests that this subunit forms part of the peripheral stalk of the V-ATPase that links the V 1 and V 0 domains.The vacuolar H ϩ -ATPases (V-ATPases) 1 are a family of ATPdependent proton pumps that are responsible for acidification of intracellular compartments in eukaryotic cells. The VATPases are present in a variety of intracellular compartments, including clathrin-coated vesicles; endosomes; lysosomes; Golgi-derived vesicles; chromaffin granules; synaptic vesicles; and central vacuoles of yeast, Neurospora, and plants (1-8). Acidification of vacuolar compartments plays an important role in a variety of cellular processes, including receptormediated endocytosis, intracellular targeting, protein processing and degradation, and coupled transport. The V-ATPases are also present in the plasma membrane of various specialized cells, including osteoclasts (9), renal intercalated cells (10), and neutrophils (11), where they function in such processes as bone resorption, renal acidification, and pH homeostasis, respectively.The V-ATPases are composed of two functional domains, V 1 and V 0 (1-8). The V 1 domain is a 570-kDa peripheral complex containing eight different subunits with molecular masses of 70 to 14 kDa and is responsible for ATP hydrolysis. The nucleotide-binding sites are located on two subunits of the V 1 domain: the 69-kDa A subunit and the 57-kDa B subunit. The V 0 domain is a 260-kDa integral complex composed of five subunits with molecular masses of 100 to 17 kDa and is responsible for proton translocation.The V-ATPases are structurally and evolutionarily related to the ATP synthases (or F-ATPases) of mitochondria, chloroplasts, and bacteria (12-17). Thus, the nucleotide-binding subunits of the V-ATPase (A and B) are homologous to the corresponding ␤ and ␣ subunits of F 1 (18,19), and the proteolipid subunits of the two complexes are also homologous (20, 21). The structure of the peripheral F 1 domain of the mitochondrial F-ATPase has been determined by x-ray crystallography and shown to consist of a hexamer of alternating ␣ and ␤ subunits surrounding a central cavity containing the highly ␣-helical ␥ subunit (22-24). F 1 is attached to the F 0 domain via both a central stalk, which includes both the ␥ and ⑀...

The 32-kDa galectin (LEC-1 or N32) of the nematode Caenorhabditis elegans is the first example of a tandem repeat-type galectin and is composed of two domains, each of which is homologous to typical vertebrate 14-kDa-type galectins. To elucidate the biological meaning of this unique structure containing two probable sugar binding sites in one molecule, we analyzed in detail the sugar binding properties of the two domains by using a newly improved frontal affinity chromatography system. The whole molecule (LEC-1), the N-terminal lectin domain (Nh), and the C-terminal lectin domain (Ch) were expressed in Escherichia coli, purified, and immobilized on HiTrap gel agarose columns, and the extent of retardation of various sugars by the columns was measured. To raise the sensitivity of the system, we used 35 different fluorescence-labeled oligosaccharides (pyridylaminated (PA) sugars). All immobilized proteins showed affinity for N-acetyllactosamine-containing Nlinked complex-type sugar chains, and the binding was stronger for more branched sugars. Ch showed 2-5-fold stronger binding toward all complex-type sugars compared with Nh. Both Nh and Ch preferred Gal␤1-3GlcNAc to Gal␤1-4GlcNAc. Because the Fuc␣1-2Gal␤1-3GlcNAc (H antigen) structure was found to interact with all immobilized protein columns significantly, the K d value of pentasaccharide Fuc␣1-2Gal␤1-3GlcNAc␤1-3Gal␤1-4Glc-PA for each column was determined by analyzing the concentration dependence. Obtained values for immobilized LEC-1, Nh, and Ch were 6.0 ؋ 10 ؊5 , 1.3 ؋ 10 ؊4 , and 6.5 ؋ 10 ؊5 M, respectively. The most significant difference between Nh and Ch was in their affinity for GalNAc␣1-3(Fuc␣1-2)Gal␤1-3GlcNAc␤1-3Gal␤1-4Glc-PA, which contains the blood group A antigen; the K d value for immobilized Nh was 4.8 ؋ 10 ؊5 M, and that for Ch was 8.1 ؋ 10 ؊4 M. The present results clearly indicate that the two sugar binding sites of LEC-1 have different sugar binding properties.

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