The heat-stable polypeptide (APF-1) required for ATP-dependent proteolysis in reticulocytes enters into high molecular weight conjugates upon incubation with the fraction of reticulocytes that is retained by DEAE-cellulose. Conjugate formation requires ATP and Mg2+ and is inhibited by N-ethylmaleimide. UTP and GTP are inactive. These properties are identical to those of ATP-dependent protein breakdown in the same system, suggesting that the conjugates are intermediates in this process. The APF-1 conjugates are stable in sodium dodecyl sulfate/polyacrylamide gel electrophoresis and Sephadex C-75 isolation and are resistant to mild acid, alkali, heat denaturation, and reduction; the conjugates are therefore covalent.Although most cellular proteins turn over rapidly, the enzymic reactions of protein degradation have not yet been identified. A basic feature of intracellular protein breakdown is its absolute requirement for cellular energy. Inhibitors of ATP production inhibit the degradation of liver proteins (1, 2), of tyrosine aminotransferase in hepatoma cells (3), of normal proteins in bacteria (4, 5) and cultured cells (6, 7), and of abnormal proteins in Escherichia coli (8, 9) and reticulocytes (10, 11). Recently several reports have shown an ATP requirement for protein breakdown in cell-free systems. ATP stimulates the degradation of abnormal proteins in crude soluble extracts of reticulocytes (10, 11) and E. coli (12), and a requirement for ATP has been found in the cleavage of bacteriophage X repressor in vitro (13,14).We have shown that the ATP-dependent proteolytic system from rabbit reticulocytes is composed of several required components. A heat-stable polypeptide of a relatively small size, (Mr -8000) designated ATP-dependent proteolysis factor 1 (APF-1), has been resolved (15). APF-1 has no proteolytic activity itself but stimulates ATP-dependent protein breakdown by a crude protein fraction eluted from DEAE-cellulose, fraction 11 (15). Fraction II has been resolved into two subfractions, both required for protein breakdown by the ATP-APF-1 system (16).To gain a better insight into the roles of the different factors and of ATP in protein breakdown, we have now purified APF-1, radiolabeled it, and observed its association with other reticulocyte components. ATP is required for binding of APF-1 to reticulocyte proteins and the binding seems to be covalent in nature. METHODS Preparation of Enzyme Fractions and Purification of APF-1. Lysates from ATP-depleted rabbit reticulocytes were prepared and separated on DEAE-cellulose into fraction I (unadsorbed material) and fraction 11 (0.5 M KCI eluate), as described earlier (15, 16). Fraction I was subjected to heat treatment (90'C, 20 min) and gel filtration on a column (1.5 X 90 cm) of Sephadex G-75, as described (15). Then 10 mg of this material was adsorbed onto a column (1.5 X 30 cm) of CM-Sephadex equilibrated with 10 mM potassium phosphate and was eluted with a 0-150 mM linear gradient of KCl. The active peak, designated APF-1, eluted at about 6...

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SUMMARYIn vertebrates, canonical Wnt signaling controls posterior neural cell lineage specification. Although Wnt signaling to the neural plate is sufficient for posterior identity, the source and timing of this activity remain uncertain. Furthermore, crucial molecular targets of this activity have not been defined. Here, we identify the endogenous Wnt activity and its role in controlling an essential downstream transcription factor, Meis3. Wnt3a is expressed in a specialized mesodermal domain, the paraxial dorsolateral mesoderm, which signals to overlying neuroectoderm. Loss of zygotic Wnt3a in this region does not alter mesoderm cell fates, but blocks Meis3 expression in the neuroectoderm, triggering the loss of posterior neural fates. Ectopic Meis3 protein expression is sufficient to rescue this phenotype. Moreover, Wnt3a induction of the posterior nervous system requires functional Meis3 in the neural plate. Using ChIP and promoter analysis, we show that Meis3 is a direct target of Wnt/-catenin signaling. This suggests a new model for neural anteroposterior patterning, in which Wnt3a from the paraxial mesoderm induces posterior cell fates via direct activation of a crucial transcription factor in the overlying neural plate.

Neural crest induction is thought to occur by a two-step process. Axially fated mesoderm induces neural plate, which is then recruited to neural crest by nonneural epidermal ectoderm at the neural plate border. This model suggests a rather indirect role for mesoderm in inducing neural crest. We extensively examined the role of mesoderm in neural crest induction by determining which types of mesoderm induce neural crest cells in Xenopus embryos. We found that noggin-dorsalized ventral marginal zone (VMZ) explants differentiate as melanocytes in the absence of axial mesoderm. Dorsalized VMZ is also a potent inducer of melanocytes when juxtaposed to animal cap ectoderm in recombinant explants. Dorsalized VMZ is analogous to the dorsal-lateral marginal zone (DLMZ) region of the embryo. Neural crest-inducing activities of gastrula stage DLMZ and dorsal marginal zone (DMZ) were also compared in recombinant explants. DLMZ was a stronger inducer of neural crest than was DMZ; DLMZ induced high levels of XSlug expression and melanocyte formation in recombinants, whereas DMZ weakly induced neural crest. In whole embryos lacking DLMZ, XSlug expression and melanocyte formation were significantly reduced; in contrast, no significant reduction of XSlug expression or melanocyte formation was seen in embryos lacking a DMZ. These results suggest that paraxial-fated mesoderm plays a central role in neural crest formation by inducing a novel type of lateral neural plate. This lateral neural plate is then recruited to neural crest by adjacent nonneural epidermal ectoderm.