Protein phosphorylation is a fundamental mechanism regulating nearly every aspect of cellular life. Several secreted proteins are phosphorylated, but the kinases responsible are unknown. We identified a family of atypical protein kinases that localize within the Golgi apparatus and are secreted. Fam20C appears to be the Golgi casein kinase that phosphorylates secretory pathway proteins within S-x-E motifs. Fam20C phosphorylates the caseins and several secreted proteins implicated in biomineralization, including the small integrin-binding ligand, N-linked glycoproteins (SIBLINGs). Consequently, mutations in Fam20C cause an osteosclerotic bone dysplasia in humans known as Raine syndrome. Fam20C is thus a protein kinase dedicated to the phosphorylation of extracellular proteins.

To gain a better understanding of the critical role of mitochondria in cell function, we have compiled an extensive catalogue of the mitochondrial proteome using highly purified mitochondria from normal human heart tissue. Sucrose gradient centrifugation was employed to partially resolve protein complexes whose individual protein components were separated by one-dimensional PAGE. Total in-gel processing and subsequent detection by mass spectrometry and rigorous bioinformatic analysis yielded a total of 615 distinct protein identifications. All protein pI values, molecular weight ranges, and hydrophobicities were represented. The coverage of the known subunits of the oxidative phosphorylation machinery within the inner mitochondrial membrane was >90%. A significant proportion of identified proteins are involved in signaling, RNA, DNA, and protein synthesis, ion transport, and lipid metabolism. The biochemical roles of 19% of the identified proteins have not been defined. This database of proteins provides a comprehensive resource for the discovery of novel mitochondrial functions and pathways.

Summary The existence of extracellular phosphoproteins has been acknowledged for over a century. However, research in this area has been undeveloped largely because the kinases that phosphorylate secreted proteins have escaped identification. Fam20C is a kinase that phosphorylates S-x-E/pS motifs on proteins in milk and in the extracellular matrix of bones and teeth. Here, we show that Fam20C generates the majority of the extracellular phosphoproteome. Using CRISPR/Cas9 genome editing, mass spectrometry, and biochemistry, we identify more than 100 secreted phosphoproteins as genuine Fam20C substrates. Further, we show that Fam20C exhibits broader substrate specificity than previously appreciated. Functional annotations of Fam20C substrates suggest roles for the kinase beyond biomineralization, including lipid homeostasis, wound healing, and cell migration and adhesion. Our results establish Fam20C as the major secretory pathway protein kinase and serve as a foundation for new areas of investigation into the role of secreted protein phosphorylation in human biology and disease.

Iron-sulfur (Fe-S) proteins are key players in vital processes involving energy homeostasis and metabolism from the simplest to most complex organisms. We report a 1.5 Å x-ray crystal structure of the first identified outer mitochondrial membrane Fe-S protein, mitoNEET. Two protomers intertwine to form a unique dimeric structure that constitutes a new fold to not only the Ϸ650 reported Fe-S protein structures but also to all known proteins. We name this motif the NEET fold. The protomers form a two-domain structure: a ␤-cap domain and a cluster-binding domain that coordinates two acid-labile 2Fe-2S clusters. Binding of pioglitazone, an insulin-sensitizing thiazolidinedione used in the treatment of type 2 diabetes, stabilizes the protein against 2Fe-2S cluster release. The biophysical properties of mitoNEET suggest that it may participate in a redox-sensitive signaling and/or in Fe-S cluster transfer. diabetes ͉ FeS cluster ͉ iron homeostasis ͉ thiazolidinedione ͉ oxidative stress I ron (Fe) is a vital trace element for virtually all organisms. Incorporation of this transition metal into iron-sulfur (Fe-S) clusters forms cofactors integral to diverse biological pathways involved in the capture and metabolism of light and chemical energy (1, 2). Because free iron can be highly toxic, an elaborate array of proteins has evolved to facilitate the transfer of iron through cell compartments, to insert iron into Fe-S clusters, and to incorporate Fe-S clusters into proteins. Fe-S cluster assembly takes place primarily, although not exclusively, within the mitochondrial matrix of eukaryotic cells, and defects in mitochondrial cluster assembly and export have profound consequences for rates of growth, iron accumulation, oxidative stress, and heme biosynthesis (1, 2).Mitochondrial dysfunction is associated with insulin resistance and the development of type 2 diabetes (3). Recent studies suggest that disease pathogenesis involves diminished mitochondrial oxidative capacity in insulin-sensitive tissues. Pharmacologic agents extensively used to treat insulin resistance such as the thiazolidinedione (TZD) pioglitazone are known to enhance oxidative capacity and normalize lipid metabolism (4, 5). Although TZDs are conventionally thought to operate through binding to peroxisome proliferator-activated receptors, a recent study by Colca and colleagues (6) identified an additional binding target within mitochondrial membranes that was named mitoNEET, on the basis of the subcellular localization (mito) and the presence of the amino acid sequence Asn-Glu-Glu-Thr (NEET).MitoNEET was determined to be an integral protein of the outer mitochondrial membrane (OMM) by a series of studies, including immuno-electron microscopy and detailed fractionation studies of highly purified rat liver mitochondria. An amino-terminal signal sequence within the first 32 residues, containing a predicted transmembrane domain, targets mitoNEET to the outer membrane. The orientation of this protein toward the cytoplasm was established by proteolytic digestion...

-X-C-X2-(S/T)-X3-P-X-C-D-G-(S/A/T)-H is a defining feature of this unique family of proteins and is likely involved in ironbinding. Localization studies demonstrate that mitoNEET is an integral protein present in the outer mitochondrial membrane. An amino-terminal anchor sequence tethers the protein to the outer membrane with the CDGSH domain oriented toward the cytoplasm. Cardiac mitochondria isolated from mitoNEET-null mice demonstrate a reduced oxidative capacity, suggesting that mito-NEET is an important iron-containing protein involved in the control of maximal mitochondrial respiratory rates. mitochondria ͉ oxidative phosphorylation ͉ pioglitazone