YAP is a multifunctional adapter protein and transcriptional coactivator with several binding partners well described in vitro and in cell culture. To explore in vivo requirements for YAP, we generated mice carrying a targeted disruption of the Yap gene. Homozygosity for the Yap tm1Smil allele (Yap ؊/؊ ) caused developmental arrest around E8.5. Phenotypic characterization revealed a requirement for YAP in yolk sac vasculogenesis. Yolk sac endothelial and erythrocyte precursors were specified as shown by histology, PECAM1 immunostaining, and alpha globin expression. Nonetheless, development of an organized yolk sac vascular plexus failed in Yap ؊/؊ embryos. In striking contrast, vasculogenesis proceeded in both the allantois and the embryo proper. Mutant embryos showed patterned gene expression domains along the anteroposterior neuraxis, midline, and streak/tailbud. Despite this evidence of proper patterning and tissue specification, Yap ؊/؊ embryos showed developmental perturbations that included a notably shortened body axis, convoluted anterior neuroepithelium, caudal dysgenesis, and failure of chorioallantoic fusion. These results reveal a vital requirement for YAP in the developmental processes of yolk sac vasculogenesis, chorioallantoic attachment, and embryonic axis elongation.

The Saccharomyces cerevisiae SAC1 gene was identified via independent analyses of mutations that modulate yeast actin function and alleviate the essential requirement for phosphatidylinositol transfer protein (Sec14p) activity in Golgi secretory function. The SAC1 gene product (Sac1p) is an integral membrane protein of the endoplasmic reticulum and the Golgi complex. Sac1p shares primary sequence homology with a subfamily of cytosolic/peripheral membrane phosphoinositide phosphatases, the synaptojanins, and these Sac1 domains define novel phosphoinositide phosphatase modules. We now report the characterization of a rat counterpart of Sac1p. Rat Sac1 is a ubiquitously expressed 65-kDa integral membrane protein of the endoplasmic reticulum that is found at particularly high levels in cerebellar Purkinje cells. Like Sac1p, rat Sac1 exhibits intrinsic phosphoinositide phosphatase activity directed toward phosphatidylinositol 3-phosphate, phosphatidylinositol 4-phosphate, and phosphatidylinositol 3,5-bisphosphate substrates, and we identify mutant rat sac1 alleles that evoke substrate-specific defects in this enzymatic activity. Finally, rat Sac1 expression in ⌬sac1 yeast strains complements a wide phenotypes associated with Sac1p insufficiency. Biochemical and in vivo data indicate that rat Sac1 phosphatidylinositol-4-phosphate phosphatase activity, but not its phosphatidylinositol-3-phosphate or phosphatidylinositol-3,5-bisphosphate phosphatase activities, is essential for the heterologous complementation of Sac1p defects in vivo. Thus, yeast Sac1p and rat Sac1 are integral membrane lipid phosphatases that play evolutionary conserved roles in eukaryotic cell physiology.Phosphoinositides regulate diverse cellular processes such as cell proliferation, signal transduction, organization of the cytoskeleton, and membrane trafficking. They do so by regulating the intracellular localization and biological properties of a variety of proteins involved in these processes. Such effector proteins recognize specific phosphoinositides via lipid binding modules such as C2 domains, pleckstrin homology domains, FYVE domains, and SH2 1 domains (reviewed in Refs.

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi secretory function. It is widely accepted, though unproven, that phosphatidylinositol transfer between membranes represents the physiological activity of phosphatidylinositol transfer proteins (PITPs). We report that Sec14pK66,239A is inactivated for phosphatidylinositol, but not phosphatidylcholine (PC), transfer activity. As expected, Sec14pK66,239A fails to meet established criteria for a PITP in vitro and fails to stimulate phosphoinositide production in vivo. However, its expression efficiently rescues the lethality and Golgi secretory defects associated with sec14-1ts and sec14 null mutations. This complementation requires neither phospholipase D activation nor the involvement of a novel class of minor yeast PITPs. These findings indicate that PI binding/transfer is remarkably dispensable for Sec14p function in vivo.

Phosphatidylinositol transfer proteins (PITPs) regulate the interface between lipid metabolism and cellular functions. We now report that ablation of PITP␣ function leads to aponecrotic spinocerebellar disease, hypoglycemia, and intestinal and hepatic steatosis in mice. The data indicate that hypoglycemia is in part associated with reduced proglucagon gene expression and glycogenolysis that result from pancreatic islet cell defects. The intestinal and hepatic steatosis results from the intracellular accumulation of neutral lipid and free fatty acid mass in these organs and suggests defective trafficking of triglycerides and diacylglycerols from the endoplasmic reticulum. We propose that deranged intestinal and hepatic lipid metabolism and defective proglucagon gene expression contribute to hypoglycemia in PITP␣ ؊/؊ mice, and that hypoglycemia is a significant contributing factor in the onset of spinocerebellar disease. Taken together, the data suggest an unanticipated role for PITP␣ in with glucose homeostasis and in mammalian endoplasmic reticulum functions that interface with transport of specific luminal lipid cargoes. PITPs1 mobilize PtdIns or PtdCho between membrane bilayers in vitro (1,2). In vivo studies demonstrate that PITPs control the interface between membrane trafficking and lipid metabolic pathways in yeast (3-6). By contrast, the physiological functions for mammalian PITPs, which are structurally unrelated to yeast PITPs (7, 8), are not understood at either the cellular or organismal levels.Mammals express at least three soluble PITPs: PITP␣, PITP␤, and rdgB␤ (9 -11). PITP␣ and PITP␤ share 77% primary sequence identity, are encoded by distinct genes, and exhibit biochemical differences. Yet both PITP␣ and PITP␤ (and even yeast PITPs) function as soluble factors that stimulate various reconstitutions of PIP-dependent functions in permeabilized mammalian cells. These functions include regulated and constitutive membrane trafficking and phospholipase Cdependent signaling through G-protein-coupled receptors (12)(13)(14). Given the lack of PITP specificity in these assays, it remains unclear how faithful such reconstitutions are in reporting physiological functions for mammalian PITPs. Genetic studies are providing initial clues regarding PITP function in metazoans. An inherited form of light-enhanced retinal degeneration in Drosophila results from inactivation of a membrane-bound PITP (15). In mice, reduction of PITP␣ to 18% of wild-type levels is the basis for the vibrator neurodegenerative disorder (16,26). Gene ablation approaches suggest PITP␤ plays an essential housekeeping function, whereas PITP␣ is nonessential for ES cell viability and is not a quantitatively significant factor in membrane trafficking, PIP metabolism, or growth factor signaling in ES cells (17).In this report, we describe the consequences associated with ablation of PITP␣ function in the mouse. We find that PITP␣, although dispensable for prenatal development, is required for neonatal survival. PITP␣ Ϫ/Ϫ neonates suffer from a...

The Drosophila retinal degeneration B (rdgB) gene encodes an integral membrane protein involved in phototransduction and prevention of retinal degeneration. RdgB represents a nonclassical phosphatidylinositol transfer protein (PITP) as all other known PITPs are soluble polypeptides. Our data demonstrate roles for RdgB in proper termination of the phototransduction light response and dark recovery of the photoreceptor cells. Expression of RdgB's PITP domain as a soluble protein (RdgB-PITP) in rdgB2 mutant flies is sufficient to completely restore the wild-type electrophysiological light response and prevent the degeneration. However, introduction of the T59E mutation, which does not affect RdgB-PITP's phosphatidylinositol (PI) and phosphatidycholine (PC) transfer in vitro, into the soluble (RdgB-PITP-T59E) or full-length (RdgB-T59E) proteins eliminated rescue of retinal degeneration in rdgB2 flies, while the light response was partially maintained. Substitution of the rat brain PITPα, a classical PI transfer protein, for RdgB's PITP domain (PITPα or PITPα-RdgB chimeric protein) neither restored the light response nor maintained retinal integrity when expressed in rdgB2 flies. Therefore, the complete repertoire of essential RdgB functions resides in RdgB's PITP domain, but other PITPs possessing PI and/or PC transfer activity in vitro cannot supplant RdgB function in vivo. Expression of either RdgB-T59E or PITPα-RdgB in rdgB + flies produced a dominant retinal degeneration phenotype. Whereas RdgB-T59E functioned in a dominant manner to significantly reduce steady-state levels of rhodopsin, PITPα-RdgB was defective in the ability to recover from prolonged light stimulation and caused photoreceptor degeneration through an unknown mechanism. This in vivo analysis of PITP function in a metazoan system provides further insights into the links between PITP dysfunction and an inherited disease in a higher eukaryote.