Summary Leptin plays a pivotal role in regulation of energy balance. Via unknown central pathways leptin also affects peripheral glucose homeostasis and locomotor activity. We hypothesized that specifically Pro-opiomelanocortin (POMC) neurons mediate those actions. To examine this possibility we applied Cre-Lox technology to express leptin receptors (ObRb) exclusively in POMC neurons of the morbidly obese, profoundly diabetic, and severely hypoactive leptin receptor deficient Leprdb/db mice. We here show that expression of ObRb only in POMC neurons leads to a marked decrease in energy intake and a modest reduction in body weight in Leprdb/db mice. Remarkably, blood glucose levels are entirely normalized. This normalization occurs independently of changes in food intake and body weight. In addition, physical activity is greatly increased despite profound obesity. Our results suggest that leptin signaling exclusively in POMC neurons is sufficient to stimulate locomotion and prevent diabetes in the severely hypoactive and hyperglycemic obese Leprdb/db mice.

Vacuolar (H+)-ATPases (V-ATPases) are multisubunit complexes responsible for acidification of intracellular compartments in eukaryotic cells. V-ATPases possess a subunit of approximate molecular mass 100 kDa of unknown function that is composed of an amino-terminal hydrophilic domain and a carboxyl-terminal hydrophobic domain. To test whether the 100-kDa subunit plays a role in proton transport, site-directed mutagenesis of the VPH1 gene, which is one of two genes that encodes this subunit in yeast, has been carried out in a strain lacking both endogenous genes. Ten charged and twelve polar residues located in the seven putative transmembrane helices in the COOH-terminal domain of the molecule were individually changed, and the effects on proton transport, ATPase activity, and assembly of the yeast V-ATPase were measured. Two mutations (R735L and Q634L) in transmembrane helix 6 and at the border of transmembrane helix 5, respectively, showed greatly reduced levels of the 100-kDa subunit in the vacuolar membrane, suggesting that these mutations affected stability of the 100-kDa subunit. Two mutations, D425N and K538A, in transmembrane helix 1 and at the border of transmembrane helix 3, respectively, showed reduced assembly of the V-ATPase, with the D425N mutation also reducing the activity of V-ATPase complexes that did assemble. Two mutations, H743A and K593A, in transmembrane helix 6 and at the border of transmembrane helix 4, respectively, have significantly greater effects on activity than on assembly, with proton transport and ATPase activity inhibited 40-60%. One mutation, E789Q, in transmembrane helix 7, virtually completely abolished proton transport and ATPase activity while having no effect on assembly. These results suggest that the 100-kDa subunit may be required for activity as well as assembly of the V-ATPase complex and that several charged residues in the last four putative transmembrane helices of this subunit may play a role in proton transport.

The membrane topography of the yeast vacuolar proton-translocating ATPase a subunit (Vph1p) has been investigated using cysteine-scanning mutagenesis. A Cys-less form of Vph1p lacking the seven endogenous cysteines was constructed and shown to have 80% of wild type activity. Single cysteine residues were introduced at 13 sites within the Cys-less mutant, with 12 mutants showing greater than 70% of wild type activity. To evaluate their disposition with respect to the membrane, vacuoles were treated in the presence or absence of the impermeant sulfhydryl reagent 4-acetamido-4-maleimidylstilbene-2,2-disulfonic acid (AMS) followed by the membrane permeable sulfhydryl reagent 3-(Nmaleimidylpropionyl) biocytin (MPB). Three of the 12 active cysteine mutants were not labeled by MPB. The mutants E3C, D89C, T161C, S266C, N447C, K450C, and S703C were labeled by MPB in an AMS-protectable manner, suggesting a cytoplasmic orientation, whereas G602C and S840C showed minimal protection by AMS, suggesting a lumenal orientation. Factor Xa cleavage sites were introduced at His-499, Leu-560, and Pro-606. Cleavage at 560 was observed in the absence of detergent, suggesting a cytoplasmic orientation for this site. Based on these results, we propose a model of the a subunit containing nine transmembrane segments, with the amino terminus facing the cytoplasm and the carboxyl terminus facing the lumen.The vacuolar proton-translocating ATPases (or V-ATPases) 1 are multisubunit complexes found in a variety of intracellular compartments, such as clathrin-coated vesicles, endosomes, lysosomes, Golgi-derived vesicles, chromaffin granules, synaptic vesicles, and the central vacuoles of yeast, Neurospora, and plants (1-9). Acidification of these intracellular compartments is in turn essential for a variety of cellular processes, including receptor-mediated endocytosis, intracellular targeting, protein processing and degradation, and coupled transport. V-ATPases are also present in the plasma membrane of certain specialized cells, where they function in such processes as renal acidification (7), bone resorption (10), and pH homeostasis (11).The V-ATPases from fungi, plants, and animals are structurally very similar and are composed of two functional domains (1). The V 1 domain is a peripheral complex of molecular mass of 570 kDa composed of eight different subunits of molecular mass 70 -14 kDa (subunits A-H) that is responsible for ATP hydrolysis. The V 0 domain is a 260-kDa integral complex composed of five subunits of molecular mass 100 -17 kDa (subunits a, d, c, cЈ, and cЉ) that is responsible for proton translocation. This structure is similar to that of the ATP synthases (or F-ATPases) that function in ATP synthesis in mitochondria, chloroplasts and bacteria (12-17), and sequence homology between these classes of ATPase has been demonstrated for both the nucleotide binding subunits (A, B, ␣, and ␤) (18, 19) and the proteolipid subunits (subunits c, cЈ, and cЉ) (20,21). No sequence homology has been identified for any of the remaining subun...

We have previously shown that mutations in buried charged residues in the last two transmembrane helices of Vph1p (the 100-kDa subunit of the yeast V-ATPase) inhibit proton transport and ATPase activity (Leng, X. H., Manolson, M., Liu, Q., and Forgac, M. (1996) J. Biol. Chem. 271, 22487-22493). In this report we have further explored the function of this region of Vph1p (residues 721-840) using a combination of site-directed and random mutagenesis. Effects of mutations on stability of Vph1p, assembly of the V-ATPase complex, 9-amino-6-chloro-2-methoxyacridine quenching (as a measure of proton transport), and ATPase activity were assessed. Additional mutations were analyzed to test the importance of Glu-789 in TM7 and His-743 in TM6. Although substitution of Asp for Glu at position 789 led to a 50% decrease in 9-amino-6-chloro-2-methoxyacridine quenching, substitution of Ala at this position gave a mutant with 40% quenching relative to wild type, suggesting that a negative charge at this position is not absolutely essential for proton transport. Similarly, a positive charge is not essential at position His-743, since the H743Y and H743A mutants retain 20 and 60% of wild-type quenching, respectively. Interestingly, H743A approaches wild-type ATPase activity at elevated pH while the E789D mutant shows a slightly lower pH optimum than wild type, suggesting that these residues are in a location to influence V-ATPase activity. The low pumping activity of the double mutant (E789H/H743E) suggests that these residues do not form a simple ion pair. Random mutagenesis identified a number of additional mutations both inside the membrane (L739S and L746S) as well as external to the membrane (H729R and V803D) which also significantly inhibited proton pumping and ATPase activity. By contrast, a cluster of five mutations were identified between residues 800 and 814 in the soluble segment just COOH-terminal to TM7 which affected either assembly or stability of the VATPase complex. Two mutations (F809L and G814D) may also affect targeting of the 100-kDa subunit. These results suggest that this segment of Vph1p plays a crucial role in organization of the V-ATPase complex.The vacuolar (H ϩ )-ATPases (or V-ATPases) 1 are a widely distributed family of ATP-driven proton pumps responsible for acidifying the interior of various intracellular organelles and providing the energy for numerous coupled transport processes (for reviews, see Refs. 1-9). The V-ATPases also play an important role in such processes as receptor-mediated endocytosis, intracellular membrane traffic, and protein processing and degradation. V-ATPases in the plasma membrane of specialized cells function in renal acidification, pH homeostasis, bone resorption, and tumor metastasis (7, 10 -12). The V-ATPases are multisubunit complexes composed of two structural domains (1-9). The peripheral V 1 domain is a 500-kDa complex with the structure A

Mutation of G250D in the glycine-rich loop also resulted in destabilization of the A subunit, whereas mutation of the lysine residue in this region (K263Q) gave a V-ATPase complex which showed normal levels of A subunit on the vacuolar membrane but was unstable to detergent solubilization and isolation and was totally lacking in V-ATPase activity. By contrast, mutation of the acidic residue, which has been postulated to play a direct catalytic role in the homologous F-ATPases (E286Q), had no effect on stability or assembly of the V-ATPase complex, but also led to complete loss of VATPase activity. The E286Q mutant showed labeling by 2-azido-[ 32 P]ATP that was approximately 60% of that observed for wild type, suggesting that mutation of this glutamic acid residue affected primarily ATP hydrolysis rather than nucleotide binding.The vacuolar (H ϩ )-ATPases (or V-ATPases) are a class of ATP-dependent proton pumps that play an important role in a variety of cellular processes, including receptor-mediated endocytosis, intracellular targeting, macromolecular processing and degradation, and coupled transport (1-9). The V-ATPases are located in both intracellular compartments and in the plasma membrane of certain specialized cells (7, 10 -12). In yeast, acidification of the central vacuole by the V-ATPase serves to activate degradative enzymes and to drive uptake of solutes such as Ca 2ϩ and amino acids (5). The V-ATPases are composed of two domains, a 500-kDa peripheral V 1 domain with the structure A 3 B 3 (54) 1 C 1 D 1 E 1 F 1 G 1 (13-17) which is responsible for ATP hydrolysis and a 250-kDa V 0 domain with the structure 100 1 36 1 19 1 c 6 that is responsible for proton translocation (13,18,19). In Saccharomyces cerevisiae, the V-ATPase subunits are encoded by at least 14 genes, including VMA1 (encoding the 69-kDa A subunit) (20, 21), VMA2 (the 60-kDa B subunit) (22), VMA13 (the 54-kDa subunit) (17), VMA5 (the 42-kDa C subunit) (23, 24), VMA8 (the 32-kDa D subunit) (25), VMA4 (the 27-kDa E subunit) (23, 26), VMA10 (the 16-kDa G subunit) (17), VMA7 (the 14-kDa F subunit) (27, 28), VPH1 and STV1 (encoding isoforms of the 100-kDa subunit) (29, 30), VMA6 (encoding the 36-kDa V 0 subunit) (31), ppa1 (encoding the 19-kDa V 0 subunit) (32), and VMA3 and VMA11 (encoding the 17-kDa c subunits) (33, 34). Disruption of the VMA genes leads to a conditional lethal phenotype in which cells are unable to grow at neutral pH and in the presence of elevated Ca 2ϩ concentrations but are able to grow at acidic pH (11,35).Both the A and B subunits participate in nucleotide binding by the V-ATPases (36 -40) and show approximately 25% sequence identity with the ␤ and ␣ subunits of the F-ATPases (20,(41)(42)(43)(44)(45)(46). The F-ATPases normally function in ATP synthesis in mitochondria, chloroplasts, and bacteria (47-51), and a recent x-ray crystal structure of the F 1 domain has revealed a hexameric arrangement of ␣ and ␤ subunits, with the catalytic sites located primarily on ␤ and the noncatalytic sites located primarily on ␣ (52)....