Previously we identified MIR16 (membrane interacting protein of RGS16) as an integral membrane glycoprotein that interacts with regulator of G protein signaling proteins and shares significant sequence homology with bacterial glycerophosphodiester phosphodiesterases (GDEs), suggesting that it is a putative mammalian GDE. Here we show that MIR16 belongs to a large, evolutionarily conserved family of GDEs with a characteristic putative catalytic domain that shares a common motif (amino acids 92-116) with the catalytic domains of mammalian phosphoinositide phospholipases C. Expression of wild-type MIR16 (renamed GDE1), but not two catalytic domain mutants (E97A/D99A and H112A), leads to a dramatic increase in glycerophosphoinositol phosphodiesterase (GPI-PDE) activity in HEK 293T cells. Analysis of substrate specificity shows that GDE1, MIR16 selectively hydrolyzes GPI over glycerophosphocholine. The GPI-PDE activity of GDE1/MIR16 expressed in HEK 293T cells can be regulated by stimulation of G protein-coupled, a alpha/beta-adrenergic, and lysophospholipid receptors. Membrane topology studies suggest a model in which the catalytic GDE domain faces the lumen/extracellular space and the C terminus faces the cytoplasm. Our results suggest that by serving as a PDE for GPI with its activity regulated by G protein signaling, GDE1/MIR16 provides a link between phosphoinositide metabolism and G protein signal transduction.

Mitochondria are dynamic organelles that play key roles in metabolism, energy production, and apoptosis. Coordination of these processes is essential to maintain normal cellular functions. Here we characterized hNOA1, the human homologue of AtNOA1 (Arabidopsis thaliana nitric oxide-associated protein 1), a large mitochondrial GTPase. By immunofluorescence, immunoelectron microscopy, and mitochondrial subfractionation, endogenous hNOA1 is localized within mitochondria where it is peripherally associated with the inner mitochondrial membrane facing the mitochondrial matrix. Overexpression and knockdown of hNOA1 led to changes in mitochondrial shape implying effects on mitochondrial dynamics. To identify the interaction partners of hNOA1 and to further understand its cellular functions, we performed immunoprecipitation-mass spectrometry analysis of endogenous hNOA1 from enriched mitochondrial fractions and found that hNOA1 interacts with both Complex I of the electron transport chain and DAP3 (death-associated protein 3), a positive regulator of apoptosis. Knockdown of hNOA1 reduces mitochondrial O(2) consumption approximately 20% in a Complex I-dependent manner, supporting a functional link between hNOA1 and Complex I. Moreover, knockdown of hNOA1 renders cells more resistant to apoptotic stimuli such as gamma-interferon and staurosporine, supporting a role for hNOA1 in regulating apoptosis. Thus, based on its interactions with both Complex I and DAP3, hNOA1 may play a role in mitochondrial respiration and apoptosis.

RGS-PX1 (also known as sorting nexin 13) is a member of both the regulator of G protein signaling (RGS) and sorting nexin (SNX) protein families. Biochemical and cell culture studies have shown that RGS-PX1/SNX13 attenuates Galphas-mediated signaling through its RGS domain and regulates endocytic trafficking and degradation of the epidermal growth factor receptor. To understand the functions of RGS-PX1/SNX13 in vivo, we generated mice carrying targeted mutations of Snx13 and found that systemic Snx13-null mice were embryonic lethal around midgestation. Snx13-null embryos had significant overall growth retardation and defects in neural tube closure, blood vessel formation, and the formation of the placental labyrinthine layer. Moreover, the Snx13-null visceral yolk sac endoderm cells showed dramatic changes in the organization of endocytic compartments, abundant autophagic vacuoles, and abnormal localization of several endocytic markers, including megalin, a receptor for nutrients and proteins; ARH, a coat protein that binds megalin; LAMP2; and LC3. These changes suggest that Snx13-null embryos are defective in nutrient uptake and transport, which may contribute to the other developmental abnormalities observed. Taken together, our findings demonstrate an essential role for RGS-PX1/SNX13 in mouse development and provide previously undescribed insights into its cellular function in the regulation of endocytosis dynamics.