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The contribution of stem and progenitor cell dysfunction and depletion in normal aging remains incompletely understood. We explored this concept in the Klotho mouse model of accelerated aging. Analysis of various tissues and organs from young Klotho mice revealed a decrease in stem cell number and an increase in progenitor cell senescence. Because klotho is a secreted protein, we postulated that klotho might interact with other soluble mediators of stem cells. We found that klotho bound to various Wnt family members. In a cell culture model, the Wnt-klotho interaction resulted in the suppression of Wnt biological activity. Tissues and organs from klotho-deficient animals showed evidence of increased Wnt signaling, and ectopic expression of klotho antagonized the activity of endogenous and exogenous Wnt. Both in vitro and in vivo, continuous Wnt exposure triggered accelerated cellular senescence. Thus, klotho appears to be a secreted Wnt antagonist and Wnt proteins have an unexpected role in mammalian aging.

The membrane topology of GAT-1, a sodium-and chloride-coupled ␥-aminobutyric acid transporter from rat brain, has been probed using N-glycosylation scanning mutagenesis. Overall, the results support the theoretical 12-transmembrane segment model. This model (based on hydropathy analysis) was originally proposed for GAT-1 and adopted for all other members of the sodiumand chloride-dependent neurotransmitter transporter superfamily. However, our data indicate that the loop connecting putative transmembrane domains 2 and 3, which was predicted to be located intracellularly, can be glycosylated in vivo. Furthermore, studies with permeant and impermeant methanesulfonate reagents suggest that cysteine 74, located in the hydrophilic loop connecting transmembrane domains 1 and 2, is intracellular rather than extracellular. We present a model in which the topology deviates from the theoretical one in the amino-terminal third of the transporter. It also contains 12 transmembrane segments, but the highly conserved domain 1 does not form a conventional transmembrane ␣-helix.Sodium-coupled transporters of neurotransmitters, located in presynaptic and glial membranes, are thought to play a major role in maintaining low synaptic levels of the transmitter (for a review, see Ref. 1). Recently, this has been shown directly for the dopamine transporter using homozygous mice in which the transporter was disrupted (2). Transporters of many neurotransmitters, including GABA, 1 norepinephrine, serotonin, dopamine, and glycine, belong to a large superfamily of sodiumand chloride-dependent neurotransmitter transporters (see Refs. 3 and 4 for reviews). GAT-1 is a GABA transporter that was reconstituted, purified to homogeneity (5), and cloned (6). It is the first identified member of the superfamily and catalyzes the electrogenic transport of GABA with one chloride and two sodium ions (7-9).Knowledge of the membrane topology is one of the first steps toward the elucidation of the structural basis of transporter function. The topology of GAT-1 has been predicted using hydropathy plots (6), and this interpretation has been adopted for the other members of the superfamily. The theoretical model predicts that the transporter spans the membrane 12 times in ␣-helical stretches, with amino and carboxyl termini both at the intracellular side of the membrane. GAT-1 is modified by asparagine-linked glycosylation (5, 10, 11), and the model predicts a large extracellular loop between transmembrane helices 3 and 4 containing three N-linked glycosylation sites (see Fig. 1) (6). Even though experimental support has been obtained recently for a few aspects of this model and predictions by hydropathy plots usually provide a reasonable first approximation, it is imperative to verify the topology experimentally.In this report, we describe an experimental evaluation of the topology mainly by N-glycosylation scanning mutagenesis. Since glycosylation occurs on the luminal side of the endoplasmic reticulum only, this method can be used to determine which doma...